Crimped helical peptides for force dependent applications

Abstract

Force-sensing peptides that can be used as sensor molecules or that can be used for signal transduction by detecting forces and converting the forces into biological signals, and methods of using the force-sensing peptides, are described. The force-sensing peptide comprises two alpha-helical domains linked by a linker sequence wherein the two alpha-helical domains form a coiled helical domain, referred to as a closed conformation, in the absence of an external force, and further wherein the two alpha-helical domains form a coiled helical domain, referred to as a closed conformation, in the presence of a force above the unhelicizing threshold level of the coiled helical domain. The coiled-coil domain undergoes a conformational change to an open conformation.

Description

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under R21GM132661 and R35GM131714 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 357,272, filed on June 30, 2022, each of which is incorporated herein by reference in its entirety. Background Art

[0005] Mechanical forces are ubiquitous in biological processes. They are central to numerous intracellular (e.g., cell division, cell motility, organelle morphogenesis, etc.), intercellular (e.g., adhesion, T-cell antigen recognition, etc.), and developmental (e.g., stem cell differentiation, organ development, regeneration, etc.) processes. Despite the ubiquity of forces in biology, there is a lack of universally applicable molecular tools that can detect and quantify forces at the molecular level in vivo. The only tools currently available are difficult to use because they are very large and therefore potentially damaging, and rely on The measurement of resonance energy transfer (FRET) efficiency, which is difficult and tedious to perform and control. Furthermore, there are no tools that can detect mechanical signals and use them to trigger a biochemical response.

[0006] Therefore, there is a need in the art for tools for detecting and utilizing mechanical forces in biological processes. The present invention satisfies this unmet need. Summary of the invention

[0007] In some embodiments, the present invention provides a force sensing peptide comprising two α-helical domains connected by a linker sequence, wherein, in the absence of external force, the two α-helical domains form a coiled-coil domain, referred to as a closed conformation, and further, wherein, in the presence of a force above a threshold level of uncoiling of the coiled-coil domain, the coiled-coil domain undergoes a conformational change to an open conformation.

[0008] In some embodiments, the force-sensing peptide is operably linked to a target molecule of interest such that when a force is applied to the target molecule of interest, the force-sensing peptide undergoes a conformational change.

[0009] In some embodiments, the linker sequence comprises at least one functional domain or portion for generating a signal when the force sensing peptide is in an open conformation. In some embodiments, at least one functional domain or portion is a fragment of a fluorescent protein, a circularly permuted fluorescent protein, a protein cleavage recognition domain, an RNA binding molecule, a DNA binding domain, an epitope for recognition by a binding molecule, an ion binding domain, a lipid binding domain, a peptide comprising residues for post-translational modification, a peptide comprising unnatural amino acids, a peptide that is a toxin to cells, a peptide with antibacterial activity, a peptide with antiviral activity, a peptide with antifungal activity, a peptide with enzymatic activity, a peptide that modifies the enzymatic activity of other proteins, a peptide comprising a localization signal for a cellular compartment, a peptide comprising a secretion signal, or a peptide that binds to another peptide or protein construct.

[0010] In some embodiments, the coiled-coil domain has a threshold level of uncoiling of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or greater than 25 piconewtons (pN).

[0011] In some embodiments, the force-sensing peptide comprises at least one alpha-helical domain of SEQ ID NO: 1-SEQ ID NO:40.

[0012] In some embodiments, the force-sensing peptide comprises at least one set of alpha-helical domains from:

[0013] a) SEQ ID NO: 1 and SEQ ID NO: 21;

[0014] b) SEQ ID NO:2 and SEQ ID NO:21;

[0015] c) SEQ ID NO: 2 and SEQ ID NO: 22;

[0016] d) SEQ ID NO:3 and SEQ ID NO:23;

[0017] e) SEQ ID NO:4 and SEQ ID NO:23;

[0018] f) SEQ ID NO:4 and SEQ ID NO:24;

[0019] g) SEQ ID NO:5 and SEQ ID NO:25;

[0020] h) SEQ ID NO:6 and SEQ ID NO:26;

[0021] i) SEQ ID NO:7 and SEQ ID NO:27;

[0022] j) SEQ ID NO: 8 and SEQ ID NO: 28;

[0023] k) SEQ ID NO:9 and SEQ ID NO:29;

[0024] 1) SEQ ID NO: 10 and SEQ ID NO: 30;

[0025] m) SEQ ID NO:11 and SEQ ID NO:31;

[0026] n) SEQ ID NO:12 and SEQ ID NO:32;

[0027] o) SEQ ID NO: 13 and SEQ ID NO: 33;

[0028] p) SEQ ID NO: 14 and SEQ ID NO: 34;

[0029] q) SEQ ID NO:15 and SEQ ID NO:35;

[0030] r) SEQ ID NO:16 and SEQ ID NO:36;

[0031] s) SEQ ID NO:17 and SEQ ID NO:37;

[0032] t) SEQ ID NO: 18 and SEQ ID NO: 38;

[0033] u) SEQ ID NO: 19 and SEQ ID NO: 39; or

[0034] v) SEQ ID NO: 20 and SEQ ID NO: 40.

[0035] In some embodiments, the set of alpha-helical domains are connected by a linker sequence.In some embodiments, the linker sequence is one of SEQ ID NO:41-SEQ ID NO:56.

[0036] In some embodiments, the force sensing peptide comprises one of SEQ ID NO:57-SEQ ID NO:80.

[0037] In some embodiments, the force sensing peptide is linked to a target molecule of interest. In some embodiments, the target molecule of interest is a detectable moiety, a fluorescent protein, a purification tag, a target domain, a cellular localization signal, a DNA molecule, an RNA molecule, cAMP, cGMP, eicosapentaenoic acid, eicosatetraenoic acid, phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, an aminomethyl polystyrene linker, a chloromethyl polystyrene linker, a PEG linker, a solid phase protein synthesis resin, a C-terminal fragment of End4p, a C-terminal fragment of actin, a C-terminal fragment of clathrin, a C-terminal fragment of vinculin, a C-terminal fragment of talin, a C-terminal fragment of integrin, glycogen, cellulose, a therapeutic agent, an antibiotic, an antiviral, an anti-fungal, an anti-helminthic, an anti-inflammatory molecule, or a chemotherapeutic agent.

[0038] In some embodiments, the force-sensing peptide is attached to a molecule to tether the force-sensing peptide to a surface or substrate.

[0039] In some embodiments, the present invention provides a system for determining the level of force exerted on a target of interest, the system comprising at least two force-sensing peptides, wherein each of the at least two force-sensing peptides has a different unwinding threshold level.

[0040] In some embodiments, the present invention provides nucleic acid molecules encoding force-sensing peptides.

[0041] In some embodiments, the present invention provides a genetically modified host cell comprising a nucleic acid molecule encoding a force-sensing peptide.

[0042] In some embodiments, the present invention provides a method for detecting the presence or level of force applied to a target molecule of interest, the method comprising contacting a target molecule of interest operably linked to a force-sensing peptide with a sufficient level of force to induce a conformational change in the force-sensing peptide, and detecting the presence of an open conformation of the force-sensing peptide.

[0043] In some embodiments, the force-sensing peptide comprises a linker sequence comprising at least one functional domain or portion for generating a signal when the force-sensing peptide is in an open conformation.

[0044] In some embodiments, at least one functional domain or portion is a fragment of a fluorescent protein, a circular fluorescent protein, a bioluminescent protein, a protein cleavage domain, an RNA binding molecule, a DNA binding domain, an epitope recognized by a binding molecule, an ion binding domain, a lipid binding domain, a peptide comprising residues for post-translational modification, a peptide comprising unnatural amino acids, a peptide that is a toxin to cells, a peptide with antibacterial activity, a peptide with antiviral activity, a peptide with antifungal activity, a peptide with enzymatic activity, a peptide that modifies the enzymatic activity of other proteins, a peptide comprising a localization signal for a cellular compartment, or a peptide comprising a secretion signal.

[0045] In some embodiments, methods for detecting the presence or level of force include detecting a fluorescent signal produced in the presence of an open conformation of a force-sensing peptide.

[0046] In some embodiments, methods for detecting the presence or level of force include detecting a bioluminescent signal produced in the presence of an open conformation of a force-sensing peptide.

[0047] In some embodiments, methods for detecting the presence or level of force include detecting differential levels of protein or mRNA molecules when the force-sensing peptide is in an open conformation.

[0048] In some embodiments, methods for detecting the presence or level of force include detecting changes in the localization of a target molecule due to cleavage of a peptide cleavage domain that is exposed when the force-sensing peptide is in an open conformation.

[0049] In some embodiments, the force-sensing peptide comprises at least one alpha-helical domain of SEQ ID NO: 1-SEQ ID NO:40.

[0050] In some embodiments, the force-sensing peptide comprises at least one set of alpha-helical domains from:

[0051] a) SEQ ID NO: 1 and SEQ ID NO: 21;

[0052] b) SEQ ID NO:2 and SEQ ID NO:21;

[0053] c) SEQ ID NO: 2 and SEQ ID NO: 22;

[0054] d) SEQ ID NO:3 and SEQ ID NO:23;

[0055] e) SEQ ID NO:4 and SEQ ID NO:23;

[0056] f) SEQ ID NO:4 and SEQ ID NO:24;

[0057] g) SEQ ID NO:5 and SEQ ID NO:25;

[0058] h) SEQ ID NO:6 and SEQ ID NO:26;

[0059] i) SEQ ID NO:7 and SEQ ID NO:27;

[0060] j) SEQ ID NO: 8 and SEQ ID NO: 28;

[0061] k) SEQ ID NO:9 and SEQ ID NO:29;

[0062] 1) SEQ ID NO: 10 and SEQ ID NO: 30;

[0063] m) SEQ ID NO:11 and SEQ ID NO:31;

[0064] n) SEQ ID NO:12 and SEQ ID NO:32;

[0065] o) SEQ ID NO: 13 and SEQ ID NO: 33;

[0066] p) SEQ ID NO: 14 and SEQ ID NO: 34;

[0067] q) SEQ ID NO:15 and SEQ ID NO:35;

[0068] r) SEQ ID NO:16 and SEQ ID NO:36;

[0069] s) SEQ ID NO:17 and SEQ ID NO:37;

[0070] t) SEQ ID NO: 18 and SEQ ID NO: 38;

[0071] u) SEQ ID NO: 19 and SEQ ID NO: 39; or

[0072] v) SEQ ID NO: 20 and SEQ ID NO: 40.

[0073] In some embodiments, the set of alpha-helical domains are connected by a linker sequence. In some embodiments, the linker sequence is one of SEQ ID NO:41-SEQ ID NO:56. In some embodiments, the force sensing peptide comprises one of SEQ ID NO:57-SEQ ID NO:80. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] The following detailed description of the preferred embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, presently preferred embodiments are shown in the accompanying drawings. However, it should be understood that the present invention is not limited to the precise arrangements and means of the embodiments shown in the accompanying drawings.

[0075] Figure 1 ,include Figure 1 A to Figure 1 H, Schematic diagram and representative data depicting the insertion of a calibrated coiled-coil force sensor within End4p. Figure 1 A depicts a schematic diagram of End4p connecting lipid membranes to F-actin at sites of clathrin-mediated endocytosis. Upper left, cross-section of a fission yeast cell with endocytic pits at different stages. Lower left, side view of an endocytic pit with an invaginated membrane. Right, End4p dimers pass through the clathrin coat (grey dashed curve) to transmit forces generated by the assembly of actin filaments (magenta sticks) to deform the plasma membrane. Cyan: End4p lipid-binding AP180 N-terminal homology (ANTH) domain; yellow: End4p proline-rich domain; magenta: End4p F-actin binding talin-HIP1 / R / Sla2p actin-tethering C-terminal homology (THATCH) domain. The figures are not to scale. Figure 1 B depicts a schematic representation of an End4p dimer with a coiled-coil force sensor inserted before the THATCH domain. Figure 1 C depicts a schematic diagram of the construct used to calibrate the coiled-coil force sensor using optical tweezers. The coiled-coil is connected to two beads captured by the optical tweezers via a 2260-bp DNA handle. Figure 1 D depicts a representative force-extension curve obtained by stretching (black curve) and then relaxing (red curve) a coiled-coil force sensor. Figure 1 E depicts a representative distribution of unfolding forces for the coiled-coil force sensor cc-11 pN (N=46). Average unfolding forces are presented as mean±SEM. Figure 1F depicts a representative fission yeast cell expressing fluorescently labeled wild-type End4p. End4p is primarily present in endocytic patches enriched at the cell tip and division plane. See also Figure 2 . Figure 1 G depicts the expression Figure 1 Representative fission yeast cells with fluorescently labeled End4p with cc-11pN inserted before the THATCH domain in B. Multiple large persistent spherical condensates of the End4p construct (arrows) can be distinguished from transient diffraction-limited endocytic patches. Figure 1 H depicts representative imaging of End4p condensates displaying liquid-like behavior. Top, fusion of two End4p condensates (arrows). Bottom, fission of an End4p condensate into two smaller condensates (arrows). See Figure 3 The complete dynamics of End4p condensates in . Figure 1 F and Figure 1 G, Scale bar: 5 μm. Figure 1 H, Scale bar: 2 μm. The schematic diagram below each image shows the modifications on End4p, and, for clarity, the fluorescent label is omitted.

[0076] Figure 2 ,include Figure 2 A and Figure 2 B, Schematic diagram depicting End4p. Figure 2 A depicts a schematic diagram of the End4p dimer. Numbers indicate residues where the domains begin and end. Coiled-coil force sensors are inserted after D155, P337 or V856. Figures are not to scale. Figure 2 B depicts a schematic diagram of the beginning of the End4p dimerization coiled coil, which contains a poly-Q region predicted to be prion-like based on Prion Like Amino Acid Composition (PLAAC) (Lancaster et al., 2014, Bioinformatics, Sep 1; 30(17): 2501-2502). Glutamine (Q) is highlighted in red.

[0077] Figure 3 ,include Figure 3 A to Figure 3 C, Depicted are representative data demonstrating that End4p constructs in condensates have longer lifetimes than End4p in endocytic sites. Figure 3A depicts a representative fission yeast cell with fluorescently labeled wild-type End4p. Right: The evolution of End4p in the boxed area over time. A typical End4p patch lasts for less than 50 seconds. Figure 3 B and Figure 3 C depicts a representative fission yeast cell with fluorescently labeled End4p, where cc-11pN is inserted before the THATCH domain. Right: Time evolution of End4p constructs in the boxed area. End4p condensates undergo rapid movement through the cell and occasionally fuse with each other ( Figure 3 B) or split ( Figure 3 C)(arrow). Figure 1 A subset of this montage is shown in G. Scale bar in left panel, 5 μm. Scale bar in right panel, 2 μm. End4p is tagged with mEGFP at the C-terminus. Images are inverted phase contrast images of maximum projection whole-cell sections. Note that Figure 3 In A, the contrast was enhanced to better visualize individual endocytic events.

[0078] Figure 4 ,include Figure 4 A to Figure 4 F, Schematic diagram depicting the coiled-coil force sensor in different conformations. Figure 4 A depicts a ribbon diagram of cc-11pN in a folded state. Two α-helices (blue and magenta) are connected by a 30-amino acid linker (black line). The dimensions of the coiled coil are shown according to the structure of PDB entry 2zta. Figure 4 B depicts the structure of folded cc-11pN with residues in the hydrophobic core highlighted. These hydrophobic residues become exposed when the coiled-coil unfolds under sufficient force. Figure 4 C depicts cc-11pN with a 30-amino acid linker properly folded by forming a parallel coiled coil. Figure 4 D depicts that the 10-amino acid cc-11pN is unable to form a parallel coiled coil and adopts an open conformation. Figure 5 A- Figure 5 This open conformation promotes the formation of protein aggregates, shown in B. Figure 4 E depicts the formation of intermolecular connections between two open cc-11pN during the refolding process. Each intermolecular connection generates two exposed α-helices that can mediate further intermolecular connections. Figure 4 F depicts cleavage at the junction of the two α-helices of cc-11pN preventing the formation of an intermolecular junction. See also Figure 5 F. Yes Figure 4 A- Figure 4 The N- and C-termini of the α-helix in F were labeled for orientation.

[0079] Figure 5 ,include Figure 5 A to Figure 5 K , Depicts force-induced unfolding of the coiled-coil force sensor promoting the formation of End4p condensates. Figure 5 A depicts a schematic diagram of End4p condensate formation. When the magnitude of the force on End4p exceeds the unfolding force threshold of the coiled-coil force sensor, the coiled-coil force sensor unfolds. During the refolding process, α-helices from different End4p molecules mediate the entanglement of End4p molecules into condensates. See also Figure 4 E. Figure 5 B- Figure 5 I depicts representative localization of different End4p constructs in fission yeast cells. Figure 5 E- Figure 5 In F, both the N-terminal and C-terminal End4p fragments are fluorescently labeled. Figure 5 B depicts the insertion of cc-11pN into End4p in the open conformation, resulting in larger End4p condensates. See also Figure 4 C- Figure 4 D. Figure 6 . Figure 5 C- Figure 5 E depicts that in the absence of force, cc-11pN insertion into End4p does not lead to condensate formation. By inserting after the THATCH domain ( Figure 5 C) Deletion of the THATCH domain ( Figure 5 D) or disconnecting the THATCH domain from the rest of End4p ( Figure 5 E) to remove the force on cc-11 pN. Figure 5 F depicts the formation of End4p condensates that depends on the connection between the two α-helices of cc-11pN. Cleavage of the linker of cc-11pN prevents condensate formation. See also Figure 4 E- Figure 4 F. Figure 5 G- Figure 5 H depicts representative data demonstrating that End4p does not form condensates when cc-11pN is inserted before the THATCH domain and RVK1010DDD ( Figure 5 G) or R1093G( Figure 5 H) Mutations were introduced into the THATCH domain to abolish its binding to F-actin. Figure 5 I depicts representative snapshots of fission yeast cells with End4p condensates before and after treatment with 100 μM LatA. The size of End4p condensates ( Figure 5 J) and quantity ( Figure 5 K) decreased. Data in 5J-5K are presented as mean ± SD (>300 aggregates from n = 3 independent replicates). Asterisks indicate significant differences. Figure 5 J, P = 0.0003, two-tailed t test. Figure 5 K, P = 0.01, two-tailed t test. Figure 5 B- Figure 5 H, Figure 5 Scale bar in B applies to all images, 5 μm. Figure 5 B- Figure 5 I, End4p is tagged with mEGFP at the C-terminus and, in Figure 5 E- Figure 5 In F, End4p is also tagged with mScarlet-I at the N-terminus. Figure 5 E- Figure 5 In vivo protein cleavage in F is achieved by insertion of the 2A peptide. The schematic diagram below each image shows the modifications on End4p, and the fluorescent label has been omitted for clarity. See Figure 7 Localization of End4p without cc-11pN.

[0080] Figure 6 ,include Figure 6 A to Figure 6 I, Representative quantification of protein aggregates is depicted. End4p aggregates were distinguished from End4p plaques by a custom ImageJ plugin and analyzed to compare sizes. Figure 6 A- Figure 6 C depicts quantification when End4p was tagged with mEGFP at the N-terminus. Figure 6 D- Figure 6 F depicts quantification when End4p was tagged with mEGFP at the N-terminus and cc-11pN was inserted before the THATCH domain. Figure 6 G- Figure 6 I depicts quantification when End4p was tagged with mEGFP at the N-terminus and cc-11pN in the open conformation was inserted before the THATCH domain. Figure 6 B. Figure 6 E and Figure 6 H depicts a representative image of a fission yeast cell with fluorescently labeled End4p or End4p constructs shown on the left and identified End4p condensates shown on the right. The distribution of condensate diameters is shown in Figure 6 C. Figure 6 F and Figure 6 I shows, each from >500 cells. Figure 6 B- Figure 6In C, a negligible population of End4p patches (usually arising from spatially overlapping endocytic events) was mistaken for End4p condensates. Figure 6 Scale bar in B applies to all images, 10 μm. Figure 6 B. Figure 6 E and Figure 6 The images in H were acquired and shown with the same settings.

[0081] Figure 7 ,include Figure 7 A to Figure 7 E, Representative data for control of End4p localization in mutant strains are depicted. Figure 7 A depicts a representative fission yeast cell expressing End4p in which the self-cleaving 2A peptide is inserted before the THATCH domain and is fluorescently labeled at its N- and C-termini. The End4p N-terminus abnormally accumulates at the cell ends and division plane, while the End4p C-terminus diffuses in the cytoplasm. The localization of the End4p fragments has not been determined. Figure 5 Insertion changes of cc-11pN shown in E. Figure 7 B depicts a representative fission yeast cell expressing fluorescently labeled End4p in which the THATCH domain was deleted. End4p without the THATCH domain abnormally accumulates at the cell ends and division plane. The localization of this End4p construct has not been determined. Figure 5 Insertion changes of cc-11pN shown in D. Figure 7 C depicts a representative fission yeast cell expressing fluorescently labeled End4p in which the self-cleaving 2A peptide is inserted after the proline-rich domain. The End4p N terminus diffuses in the cytoplasm, while its C terminus localizes to endocytic patches. Fig. 9 B comparison. Figure 7 D depicts a representative fission yeast cell expressing fluorescently labeled End4p in which the RVK1010DDD mutation was introduced into the THATCH domain to abolish binding to actin filaments. This mutant abnormally accumulates at cell ends and division planes. Figure 7 E depicts a representative fission yeast cell expressing fluorescently labeled End4p in which the R1093G mutation was introduced into the THATCH domain to abolish binding to actin filaments. This mutant abnormally accumulates at cell ends and division planes. Figure 7 D- Figure 7 The localization of End4p in E has not been Figure 5 G and Figure 5 Insertion changes of cc-11pN shown in H. Figure 7 A- Figure 7 E, Figure 7Scale bar in A applies to all images, 5 μm. Images are inverted phase contrast images of maximum projection whole-cell sections. Figure 7 A- Figure 7 In E, End4p is tagged with mScarlet-I at the N-terminus and, Figure 7 A and Figure 7 C- Figure 7 End4p is also tagged with mEGFP at the C-terminus in E. The schematic diagram below each image shows the modifications on End4p, and, for clarity, the fluorescent tag is omitted.

[0082] Figure 8 ,include Figure 8 A to Figure 8 B, Depicts representative data demonstrating that forces in End4p can be measured by inserting calibrated coiled-coils between different domains. Figure 8 A depicts End4p localization when a calibrated coiled-coil force sensor that unfolds at 14 pN (cc-14pN) is inserted before the Rend domain. The formation of End4p condensates indicates that the forces before Rend are higher than 14 pN. Figure 8 B depicts End4p localization when a calibrated coiled-coil force sensor that unfolds at 18 pN (cc-18pN) is inserted before the THATCH domain. The formation of End4p condensates indicates that the forces before THATCH are higher than 18 pN.

[0083] Fig. 9 ,include Fig. 9 A to Fig. 9 E , Depicted are representative data demonstrating that End4p is exposed to a force gradient. Fig. 9 A depicts End4p localization when the calibrated coiled-coil force sensor is inserted after the ANTH domain (first column), after the proline-rich domain (second column), or before the THATCH domain (third column). Coiled-coil unfolding thresholds are 20 pN (first row), 11 pN (second row), 10 pN (third row), and 8 pN (fourth row). Images containing End4p condensates are framed in blue. Fig. 9 B depicts the positioning of the End4p N- and C-termini when cc-11pN is inserted before the THATCH domain and the self-cleaving 2A peptide is introduced after the proline-rich domain. The End4p C-terminal fragment also forms condensates despite being disconnected from the lipid-binding ANTH domain, demonstrating that at least the force of 11pN is transmitted through the End4p C-terminus (after the proline-rich domain) to other components of the endocytic machinery. Figure 7 C Localization of the End4p fragment without cc-11pN. Fig. 9C depicts End4p localization when cc-8pN is inserted after the ANTH domain and the R1093G mutation is introduced into the THATCH domain to abolish its binding to actin filaments. This End4p construct forms condensates despite being unable to bind actin, demonstrating that at least 8 pN of force is transmitted to the End4p ANTH domain by other components of the endocytic machinery. Fig. 9 D depicts End4p localization when cc-8pN was inserted after the proline-rich domain and the R1093G mutation was introduced into the THATCH domain to abolish its binding to F-actin. This construct did not form condensates. This result and Fig. 9 The results of C demonstrate that most of the force not transmitted through the THATCH domain is transmitted through the proline-rich domain. Fig. 9 E depicts a schematic model of force transmission during CME. Top: Distribution of forces on End4p. The new coiled-coil library allowed the determination that forces are different between different domains of End4p. Bottom: Putative organization of adaptor proteins across the clathrin coat. End4p and Ent1p interact directly with the plasma membrane, the clathrin lattice and actin filaments, and are known mechanical adapters for transmitting forces generated by the actin meshwork to deform the membrane. Other endocytic proteins or protein complexes also transmit forces from F-actin to the membrane through their binding to domains in End4p or Ent1p. Double arrows show known protein-protein interactions. The figures are not to scale. Fig. 9 Scale bar in A applies to all images, 5 μm. Fig. 9 A- Fig. 9 In D, End4p is tagged with mEGFP at the C-terminus and Fig. 9 In B, End4p is also labeled at the N-terminus with mScarlet-I. The schematic diagram below each image shows the modifications on End4p, and, for clarity, the fluorescent label is omitted.

[0084] Fig.10 ,include Fig.10 A to Fig.10 D , The temporal evolution of End4p patches at endocytic sites is depicted. Fig.10 A- Fig.10 D depicts the time evolution of End4p recorded with or without a coiled-coil force sensor at an endocytic site. Insertion of a coiled-coil force sensor with an unfolding force threshold above the local force did not affect the assembly and disassembly of End4p molecules. Scale bar, 2 μm. Fig.10 A- Fig.10In D, End4p is tagged with mEGFP at the C-terminus. Images are inverted phase-contrast images of maximum projection whole-cell sections. Schematic diagrams above each image show the location of the coiled-coil force sensor in End4p, and, for clarity, the fluorescent label is omitted.

[0085] Fig.11 A representative comparison of End4p protein expression levels in different strains is depicted. The protein expression level of End4p was quantified by summing the whole-cell fluorescence of End4p C-terminally tagged with mEGFP after background subtraction. WT: No coiled-coil force sensor inserted ( Figure 1 Representative images in F). cc-11pN: 11pN coiled-coil force sensor inserted before THATCH ( Figure 1 Representative images in G). cc-11pN open: Open conformation cc-11pN before insertion of THATCH ( Figure 5 Representative images in B). Dunn's multiple comparison. ****: P < 0.0001. Each dot represents one field of view. >200 cells per group from at least three independent replicates.

[0086] Fig.12 Representative imaging demonstrating that force transmission to the membrane is robust to perturbations is depicted. Mutant backgrounds were created by removing the actin cytoskeleton binding (ACB) domain of Ent1p, and calibrated coiled-coil force sensors were inserted after the ANTH domain (first column), after the proline-rich domain (second column), or before the THATCH domain (third column). The force before the THATCH domain increased to greater than 20 pN, while the force after the ANTH domain or after the proline-rich domain remained unchanged. Scale bar, 5 μm. Images containing End4p condensates are framed in blue boxes. End4p is labeled with mEGFP at the C-terminus, and the fluorescent label in the schematic is omitted for clarity.

[0087] Fig.13 ,include Fig.13 A to Fig.13 E, A representative library of calibrated coiled-coil force sensors is depicted. Fig.13 A- Fig.13 D depicts the schematic sequence of the coiled-coil force sensors and the pulling direction (indicated by the black arrows). Among the four force sensors, cc-8pN, cc-11pN, and cc-20pN (respectively Fig.13 A. Fig.13 C and Fig.13 D) is a parallel coiled coil, while cc-10pN ( Fig.13B) Antiparallel coiled coil. The flexible linker (blue and magenta) between the two α-helices is drawn as a black line. Fig.13 E depicts representative average unfolding forces of coiled-coil force sensors, expressed as mean ± SEM. For cc-8 pN, N = 29. For cc-10 pN, N = 45. For cc-20 pN, N = 48. See Fig.14 Unfolding force distribution and comparison of the force sensor.

[0088] Fig.14 ,include Fig.14 A to Fig.14 G , Representative average unfolding forces for coiled-coils are depicted. Fig.14 A-14C depicts representative distributions of unfolding forces for cc-8pN, cc-10pN, and cc-20pN. The data were used to calculate Fig.13 Average unfolding forces shown in E. Fig.14 D- Fig.14 F depicts representative fission yeast cells expressing fluorescently labeled End4p in which cc-8pN, cc-10pN or cc-20pN was inserted before the THATCH domain and the R1093G mutation was introduced into the THATCH domain to abolish its binding to actin filaments. End4p showed abnormal accumulation at cell ends and division planes without forming condensates. Fig.14 G depicts a representative fission yeast cell expressing fluorescently labeled End4p in which a variant of cc-20pN that is always in an open conformation is inserted before the THATCH domain. Insertion of this "always open" coiled-coil into End4p results in End4p condensates. Fig.14 D- Fig.14 G: Fig.14 Scale bar in D applies to all images, 5 μm. Images are inverted phase contrast images of maximum projection whole cell sections. Schematic diagrams below each image show modifications on End4p, and, for clarity, fluorescent labels have been omitted.

[0089] Fig.15 Depicted are representative summaries of coiled-coil design libraries and corresponding force thresholds. Depicted are five template families (dynein stalk, peptide Oakley, peptide Velcro, VBP coiled-coil, and GCN4 coiled-coil). The predicted force ranges for each family are indicated in square brackets. The number of variants in the representative library corresponding to each family is in parentheses. Red circles represent coiled-coils within representative libraries that have been calibrated in vitro using optical tweezers and are located at positions corresponding to their average unfolding force thresholds.

[0090] Fig.16 ,include Fig.16 A to Fig.16 D, Schematic and representative images of force sensors with fluorescent reporters are depicted. Fig.16 A depicts a schematic representation of how the GFP11 peptide can be introduced into the loop between the two α-helices of the coiled coil. GFP1-10 is expressed in the cytoplasm and is non-fluorescent when it is not bound to GFP11. When the force is low, the coiled coil remains closed and the GFP11 peptide is inaccessible. When the force is high, the coiled coil opens and exposes GFP11 which can bind to GFP1-10, reconstituting fluorescent GFP. Fig.16 B depicts a coiled-coil force sensor combined with cpGFP. At lower forces, the coiled-coil folds; at higher forces, the coiled-coil unfolds and structural changes destroy cpGFP fluorescence. Fig.16 C depicts representative data illustrating the split-GFP strategy in vivo. GFP11 was inserted into the sensor ring that opened at 3pN and the force on End4p was probed. When the sensor was not under force, no fluorescence was detected. When force was present, fluorescence was detected and the fluorescence showed the localization of the End4p subset that was under force within the cell. Fig.16 D depicts fluorescent force sensors characterized by changes in the fluorescence ratio when excited at 400 nm and 490 nm (change in the 400 / 490 ratio, left) or changes in their peak fluorescence (ΔF / Fmin ratio, right) in the folded state (black) and unfolded state (red). These changes can be used to identify whether the force applied to the protein construct in the cell is greater than the threshold of the force sensor.

[0091] Fig.17 ,include Fig.17 A to Fig.17 G , A representative method for detecting unfolded coiled-coils is depicted. Fig.17 A depicts expression of 3pN coiled-coil-GFP inserted into Ent1p 11 Representative images of GFP fluorescence in fission yeast cells of the sensor showing fluorescence after GFP reassembly, indicating a peak force of at least 3 pN on Ent1p. Fig.17 B depicts expression of 5pN coiled-coil-GFP inserted into Ent1p 11 Representative images of GFP fluorescence in fission yeast cells of the sensor showing fluorescence after GFP reassembly, indicating that the peak force on Ent1p is less than 5 pN. Fig.17 C depicts the expression of 7pN coiled-coil-GFP inserted into Ent1p. 11 Representative images of GFP fluorescence in fission yeast cells of the sensor showing fluorescence after GFP reassembly, verifying that the peak force on Ent1p is less than 7 pN. Fig.17D depicts a schematic representation of a coiled-coil sensor with a binder linker that unfolds upon application of force, leading to a real-time enrichment of fluorescence. Fig.17 E depicts a representative kymograph of a 3pN coiled-coil-IAAL-K3 peptide sensor inserted into Ent1p, with images taken every two seconds. GFP-IAAL-E3 is recruited to endocytic sites only when the force on Ent1p exceeds 3pN. Fig.17 F depicts a schematic representation of a coiled-coil sensor with a HiBiT linker that is inaccessible until sufficient force is applied, upon which LgBiT and the exposed HiBiT form enzymatically active NanoLuc. Fig.17 G depicts fluorescence measurements of fission yeast cells expressing a 3pN coiled-coil-HiBiT linker inserted into Ent1p. 1: negative control, no HiBiT; 2-4: Ent1p variants with a 3pN coiled-coil HiBiT linker inserted; 5: positive control, HiBiT attached to the C-terminus of Ent1p. Fig.17 A to Fig.17 C, Scale bar = 5 μm.

[0092] Fig.18 ,include Fig.18 A to Fig.18 B, Schematic representation of focal adhesion proteins is depicted. Fig.18 A depicts a schematic diagram of focal adhesion components in mammalian cells. Fig.18 B depicts talin and vinculin constructs with force sensors, which replaced the previously reported FRET-based tension sensing module (TS module) for comparison of measurements by each method.

[0093] Fig.19 Schematic representation of a strategy for identifying proteins that regulate force in subcellular processes is depicted. Once the force on a protein of interest (POI) is known, mutations on other proteins can characterize their role in force generation. In this figure, the force on the POI is 7 pN, as demonstrated using a split-GFP sensor; the POI is visible (green dot) only if the force applied to the POI is greater than the sensor's threshold force. For a given force sensor, if the POI is no longer visible when protein X is mutated, then the force is reduced and the protein enhances force generation or transmission to the POI in the wild type.

[0094] Fig. 20A schematic diagram of a method for identifying force-bearing sites within a protein of interest is depicted. Force-bearing domains are identified by inserting weak force sensors (e.g., 3pN unfolding threshold) at different sites between domains of the protein of interest and observing which insertions unfold. Insertion of a always-closed variant of the coiled coil (or a coiled coil with very large unfolding force) will control that the observed phenotype is not an artifact.

[0095] Fig.21 Depicted is a schematic of a strategy to determine the force on a protein of interest (POI) using a library of force sensors. By testing a series of force sensors with increasing unfolding thresholds, the force on the POI can be determined by a dichotomy, i.e., a force greater than the threshold force of the sensor that allows a positive readout (here, reconstructed fluorescence of split-GFP) and less than the threshold force of the sensor that keeps the protein construct non-fluorescent (between 6pN and 8pN in this sketch).

[0096] Fig. 22 ,include Fig. 22 A to Fig. 22 D, Representative results depicting coiled-coil sensor insertion into C. elegans proteins. Fig. 22 A depicts a representative DIC image of a nematode, where the location of the sensory PLM neurons (solid lines) is shown in a DIC image of the nematode. The coordinates on the left show the anterior-posterior (A, P) and ventral-dorsal (V, D) axes. The PLM cell body (*) is located near the tail of the nematode, where it projects a short neurite posteriorly toward the tail and a long neurite anteriorly toward the center of the animal. Along this anterior neurite, the PLM forms short branches to innervate interneurons in the ventral nerve cord. The dashed line along the entire length of the neurite indicates Fig. 22 Analysis area of ​​D. Scale bar = 100 μm. Fig. 22B depicts a schematic representation of the membrane-associated periodic skeleton (MPS, left) and UNC-70 construct (right). The schematic representation on the left shows the spectrins that form the central building block of the membrane-associated periodic skeleton (MPS), which is composed of actin rings separated by spectrin tetramers and forming a periodic lattice below the plasma membrane throughout the entire length of the axon. Each spectrin tetramer is separated by a length of about 200nm and consists of two heterodimers of α-spectrin (SPC-1 in nematodes) and β-spectrin (UNC-70 in nematodes) subunits assembled head to head. The schematic representation on the right depicts the domain organization in UNC-70 / β-spectrin and the location of force sensor probe insertion. The force sensor or GFP11 repeat (control) is inserted into the genomic unc-70 locus at the location of the joint region encoding spectrin repeats 8 and 9. CH: calmodulin homology domain, SR: spectrin-like repeat, PH: pleckstrin homology domain. Fig. 22 C depicts expression of endogenous force sensor-GFP under a PLM-specific promoter (mec-17p) in a wild-type or unc-115(ky275) mutant background. 11 GFP 11 Representative confocal fluorescence images of animals labeled for UNC-70 and GFP1-10 (control). Unc-115 (ky275) causes loss of the periodic MPS lattice in the PLM. Signal intensity is color coded according to the LUT. Scale bar = 50 μm. The location of the cell body (*) and rectum (#) are shown. Note that the bright spots in the rectum and animal body represent background fluorescence. Fig. 22 D depicts Fig. 22 Quantification of fluorescence depicted in C. The p-values ​​shown are derived from statistical comparisons based on the Kruskal Wallis test followed by Dunn's test for multiple comparisons. GFP11 =11,n 3pN传感器 =12,n 10pN传感器 =17,n GFP11；unc-115(ky275) =11, and n 3pN；unc-115(ky275) ==11 animals. DETAILED DESCRIPTION

[0097] The present invention relates to compositions and methods for utilizing force-dependent protein activity. In certain aspects, the compositions and methods of the present invention relate to force sensor constructs. The force sensor can be used in vivo or in vitro. In some embodiments, the sensor construct can be expressed or inserted into a cell. The cell can be any organism that can express the sensor construct. In some embodiments, the composition comprises a coiled coil domain containing a first helical element, a linker domain, and a second helical element. In certain embodiments, the first protein domain is conjugated to the first helical element and the second protein domain is conjugated to the second helical element. In some embodiments, the composition comprises a protein, wherein the first helical element, the linker domain, and the second helical element are inserted into the amino acid sequence of the protein of interest at a position that is not harmful to the function and positioning of the protein of interest.

[0098] In some embodiments, the composition comprises a joint domain containing a functional domain or part. In some embodiments, the joint comprises a peptide that induces the cohesion of the composition. In some embodiments, the joint comprises a peptide that is directly or indirectly bound to another peptide or protein domain. In some embodiments, the joint comprises an epitope for being recognized by a binding molecule. In some embodiments, the joint comprises a domain for interacting with a small molecule or an intermediate of a small molecule. In some embodiments, the joint comprises a GFP11 peptide. In some embodiments, the joint comprises a circularly permuted fluorescent protein. In some embodiments, the joint comprises an RNA binding domain. In some embodiments, the joint comprises an IAAL-K3 / IAAL-E3 binding peptide. In some embodiments, the joint comprises a fragment of luciferase.

[0099] definition

[0100] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described.

[0101] As used herein, each of the following terms has the meaning associated with it in this section.

[0102] In the context of the present invention, the following abbreviations are used for common nucleic acid bases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0103] Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns, to the extent that a nucleotide sequence encoding a protein may contain introns in some versions.

[0104] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "element" means one element or more than one element.

[0105] As used herein, "about" when referring to measurable values ​​such as amounts and durations, etc., is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% relative to the specified value, as such variations are suitable for performing the disclosed methods.

[0106] As used herein, the term "activation" means inducing or increasing the expression, stability, function or activity of a molecule, reaction, interaction, gene, mRNA and / or protein in a measurable amount relative to a control comparator. An "activator" is a compound that, for example, binds, partially or completely induces stimulation, increases, promotes, induces activation, activates, sensitizes or upregulates protein, gene and mRNA stability, expression, function and activity, e.g., an agonist.

[0107] The term "assessment" includes any form of measurement, and also includes determining whether an element is present. The terms "determine," "measure," "evaluate," "assess," and "determine" are used interchangeably and may include quantitative and / or qualitative determinations. Assessments may be relative or absolute. "Assessing binding" includes determining the amount of binding and / or determining whether binding occurs (i.e., whether binding exists or does not exist). "Assessing activity" includes determining the amount of activity and / or determining whether activity exists (i.e., whether activity exists or does not exist).

[0108] As used herein, the term "bind" or "binding" refers to specific binding or other specific interactions between two molecular species, such as but not limited to protein-DNA / RNA interactions and protein-protein interactions, for example, specific binding between proteins and their DNA / RNA targets, between receptors and their ligands, between enzymes and their substrates, etc. Such binding can be specific or non-specific and can involve various non-covalent interactions, including, for example, hydrogen bonds, metal coordination, hydrophobic forces, van der Waals forces, π-π interactions and / or electrostatic effects.

[0109] As used herein, the term "coil" refers to a peptide α-helix structure, or a structure similar to a peptide α-helix in properties or appearance. As used herein, the term "coil" is not limited to 3.6 13 -spiral.

[0110] As used herein, the term "coiled-coil" refers to a structural protein motif comprising two or more alpha-helices that are wound around each other to form a supercoil. They typically contain one heptad repeat per two turns of the helix, called (abcdefg) n "a" and "d" generally represent non-polar, hydrophobic residues found at the interface of two helices, "e" and "g" are polar residues exposed to the solvent for electrostatic interactions, and "b", "c" and "f" are hydrophilic and exposed to the solvent. Different amino acids at positions "ag" determine the oligomeric state, specificity, helical orientation and stability. As used herein, the term "coiled coil" can also refer to a non-3.6 13 - made of spirals and having different n The term coiled-coil domain in the specification refers to a naturally occurring or designed coiled-coil protein structural motif, which comprises at least two heptads and may be parallel or antiparallel.

[0111] As used herein, the terms "complementary" or "complementarity" are used to refer to polynucleotides (i.e., sequences of nucleotides) that are related by the base pairing rules. For example, the sequence "AGT" is complementary to the sequence "TCA". Complementarity can be "partial", in which only the bases of some polynucleotides match according to the base pairing rules. Alternatively, there can be "complete" or "total" complementarity between polynucleotides. The degree of complementarity between polynucleotide chains has a significant effect on the efficiency and intensity of hybridization between polynucleotide chains. This is particularly important in amplification reactions and detection methods that rely on binding between polynucleotides.

[0112] "Contact" refers to the physical proximity of two or more molecules or the same molecule or two or more components of different molecules so that they can experience the process of interaction. Molecule or its components can be contacted by combining two or more different components comprising molecules, for example, by mixing two or more solution components, preparing a solution comprising two or more molecules such as target molecules, candidate molecules or competitive binding reference molecules and / or combining two or more mobile components. Alternatively, the fluid component can be contacted with a molecular combination fixed on a cell or in a cell or on or in a substrate, and the substrate is, for example, derivatized to provide a fixed polymer bead, film, polymer glass substrate or substrate surface of any combination of target molecules, candidate molecules, competitive binding reference molecules or these molecules. Molecule or its components can be contacted by selectively adjusting the composition, ionic strength, pH or temperature of solution conditions such as solutions. Molecule or its components can be contacted in static containers such as the micropores of a microarray system or in circulation systems such as microfluids or nanofluid systems. The molecules or components thereof can be contacted in or on a variety of cells, culture media, liquids, solutions, colloids, suspensions, emulsions, gels, solids, membrane surfaces, glass surfaces, polymer surfaces, vesicle samples, bilayer samples, micellar samples, and other types of cell models, or any combination of these.

[0113] As used herein, the terms "downstream" or "upstream" with respect to a signaling pathway are based on a superordinate relationship in a linear signaling cascade: if "A" activates "B" and "B" activates "C," then "A" is upstream of "B" and "B" is upstream of "C." Similarly, "B" is downstream of "A" and "C" is downstream of "B."

[0114] "Encoding" refers to the inherent properties of a specific nucleotide sequence in a polynucleotide such as a gene, cDNA or mRNA, which is used as a template for other polymers and macromolecules with a determined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a determined amino acid sequence and the resulting biological properties in a synthetic biology process. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, the gene encodes a protein. The coding strand that the nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence table and the non-coding strand used as a template for transcription of a gene or cDNA can both be referred to as encoding a protein or other product of the gene or cDNA.

[0115] The term "expression" as used herein is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0116] "Expression vector" refers to a vector containing a recombinant polynucleotide containing an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides.

[0117] A "fragment" of a polynucleotide sequence encoding an antigen may be 100% identical to the full length except for the deletion of at least one nucleotide from the 5' and / or 3' end, in each case with or without a sequence encoding a signal peptide and / or a methionine at position 1. A fragment may comprise more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% of the length of a particular full-length coding sequence, excluding any added heterologous signal peptide. Fragments may comprise fragments encoding a polypeptide having greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity to the polypeptide and optionally further comprise a sequence encoding an N-terminal methionine or a heterologous signal peptide which is not included in calculating the percent identity.

[0118] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When one position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if one position in each of the two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percentage homology between the two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, if 6 of the 10 positions in the two sequences are matched or homologous, the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC have a total of 50% homology. Typically, the comparison is performed when the two sequences are aligned to obtain maximum homology.

[0119] As used herein, "identical" or "identity" in the context of two or more nucleic acid or polypeptide sequences means that the sequence has a specified percentage of identical residues in a specified region. The percentage can be calculated as follows: optimally align the two sequences, compare the two sequences in a specified region, determine the number of positions where identical residues occur in the two sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the specified region, and multiply the result by 100 to obtain the percentage of sequence identity. In the case where the two sequences are of different lengths or the alignment produces more than one staggered ends and the specified comparison region contains only a single sequence, the residues of the single sequence are included in the denominator of the calculation, but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered to be equivalent. Can be identified manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0120] As used herein, the phrase "inhibit" means to reduce by a measurable amount or completely prevent the expression, stability, function or activity of a molecule, reaction, interaction, gene, mRNA and / or protein. Inhibitors are compounds that, for example, bind, partially or completely block stimulation, reduce, prevent, delay activation, inactivate, desensitize or downregulate protein, gene and mRNA stability, expression, function and activity, e.g., antagonists.

[0121] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated". An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-native environment, such as, for example, a host cell.

[0122] As used herein, the term "modulate" or "modulator" refers to the activation or inhibition of a molecule as described in the corresponding definitions herein.

[0123] The term "overexpressed" or "overexpression" is intended to refer to the abnormal expression level of a particular gene in cells from a disease area (such as a solid tumor within a particular tissue or organ of a patient) relative to the expression level in normal cells from that tissue or organ. Patients with solid tumors or hematological malignancies characterized by overexpression of a particular gene can be determined by standard assays known in the art.

[0124] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids that can constitute a protein sequence or a peptide sequence. Polypeptides include any peptide or protein comprising more than two amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, such as those commonly referred to in the art as peptides, oligopeptides and oligomers, and long chains, such as those commonly referred to in the art as proteins (of which there are many types). "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0125] As used herein, "polynucleotide" includes cDNA, RNA, DNA / RNA hybrids, antisense RNA, ribozymes, genomic DNA, synthetic forms and mixed polymers, both sense and antisense strands, and can be chemically or biochemically modified to contain non-natural or derived, synthetic or semi-synthetic nucleotide bases. In addition, changes in wild-type or synthetic genes are contemplated, including but not limited to the disappearance, insertion, substitution or fusion of one or more nucleotides with other polynucleotide sequences.

[0126] "Variant" of a peptide according to the present invention can be (i) a variant in which one or more amino acid residues are replaced by conservative or non-conservative amino acid residues and such replaced amino acid residues may or may not be encoded by the genetic code, (ii) a variant in which one or more modified amino acid residues (e.g., residues modified by the connection of a substituent) are present, (iii) a variant in which the peptide is an alternative splice variant of a peptide of the present invention, (iv) a fragment of a peptide, and / or (v) a variant in which the peptide is fused to another peptide such as a leader sequence or secretory sequence or a sequence for purification (e.g., a His tag) or a sequence for detection (e.g., an Sv5 epitope tag or an immunoglobulin Fc region). Fragments include peptides or original sequences produced via proteolytic cleavage (including multisite proteolysis). Variants may be post-translationally modified or chemically modified. Such variants are considered to be within the scope of those skilled in the art according to the teachings herein.

[0127] "Variants" of coiled coils may include peptides with extensions of coiled coils, peptides with additional heptapeptides from the same coiled coil, peptides with additional heptapeptides from other coiled coils, modifications of residues within the coiled coil (including substitutions, additions or deletions of residues) and chemical modifications of residues, or any combination thereof, as long as the variant is able to form a coiled coil structure.

[0128] A "vector" is a composition of matter that contains an isolated polynucleotide and can be used to deliver the isolated polynucleotide to the interior of a cell. Various vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic compounds or amphipathic compounds, plasmids and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted as including non-plasmid compounds and non-viral compounds that facilitate the transfer of polynucleotides into cells, such as, for example, polylysine compounds and liposomes. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, and reverse transcription vectors, etc.

[0129] Throughout this disclosure, various aspects of the present invention can be presented in the form of ranges. It should be understood that the description in the form of ranges is only for convenience and brevity and should not be interpreted as an unchangeable limitation on the scope of the present invention. Therefore, it should be considered that the description of the range has specifically disclosed all possible sub-ranges and the single numerical values ​​within the range. For example, it should be considered that the description of the range, such as 1 to 6, has specifically disclosed sub-ranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. and the single numerical values ​​within the range, for example, 1, 2, 2.7, 3, 4, 5, 5.3 and 6. Regardless of the width of the range, this is applicable.

[0130] describe

[0131] The present invention is based in part on the development of force sensing peptides. In certain embodiments, force sensing peptides can be used as sensor molecules or can be used for signal transduction by detecting force and translating the force into a chemical, physical or biological signal. Force sensing peptides are based on the unfolding of structured peptides under the action of force. Peptide unfolding exposes residues that are inaccessible when the peptide is folded. These exposed residues of the sensor can be tuned to react with other molecules, which can be used for detection or actuation.

[0132] In some embodiments, the invention relates to compositions that utilize forces acting on proteins. In some embodiments, the compositions measure forces acting on proteins. In some embodiments, the compositions detect protein interactions. In some embodiments, the compositions render domains accessible under the application of sufficient force.

[0133] In some embodiments, the force sensing peptides of the present invention comprise a domain containing a structure that can undergo a change in response to force. In some embodiments, the structure is a three-dimensional fold. In some embodiments, the structure comprises a folded domain that becomes unfolded or opened in response to force. Exemplary structures that can be incorporated into the force sensor peptides of the present invention include, but are not limited to, coiled coils, chimeric SNARE complexes, R domains of talin, collagen fibers, spectrin repeats, amyloid proteins, glycoproteins, or other structures that can undergo conformational changes in response to biologically relevant levels of force (e.g., forces in the range of 0.1-100 piconewtons).

[0134] In various embodiments, the force-sensing peptides of the present invention comprise at least one coiled coil containing at least two α-helical domains or helical elements connected by a flexible linker that allows the α-helical domains or helical elements to coil around each other. In some embodiments, the force-sensing peptides of the present invention comprise two, three, four, or more than four α-helical domains that coil around each other to form a coiled coil having two, three, four, or more than four strands.

[0135] In some embodiments, the first α-helical domain or helical element is conjugated to the first protein domain. In some embodiments, the second α-helical domain or helical element is conjugated to the second protein domain. In some embodiments, the composition comprises one or more peptide linkers between two or more α-helical domains. In some embodiments, the peptide linker is conjugated to the first helical element at its C-terminus. In some embodiments, the peptide linker is conjugated to the second helical element at its N-terminus. In some embodiments, the peptide linker comprises a functional peptide.

[0136] In some embodiments, applying force to the coiled-coil sensor peptide of the present invention induces a conformational change in the coiled-coil domain, wherein the two α-helical domains become separated from each other (referred to herein as an open conformation), exposing amino acid residues protected for interaction in the coiled-coil structure (referred to herein as a closed conformation). In some embodiments, the open conformation of the sensor peptide of the present invention forms an aggregate with one or more additional sensor peptides in the open conformation.

[0137] In some embodiments, the force sensor peptide comprises a folded domain that undergoes multiple changes (e.g., multi-step unfolding). In such embodiments, a first change or unfolding may occur at a first force level or force occurrence, and a second change or unfolding may occur when a second force level or force occurrence is encountered. For example, a coiled-coil domain comprising three or four coiled chains may undergo a first unwinding of the first chain when the sensor is in contact with a first instance of force or a first level of force, and may undergo a second unwinding of the second chain when the sensor is in contact with a second instance of force or a second level of force. In such embodiments, the first unwinding and the second or more unwinding events may be detected separately by different functional domains on the linker exposed at each unwinding event.

[0138] In some embodiments, the force required to induce a conformational change of the force sensor peptide from a closed conformation to an open conformation is at least 3 piconewtons (pN). In some embodiments, the force required to induce a conformational change of the force sensor peptide from a closed conformation to an open conformation is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more piconewtons (pN).

[0139] In some embodiments, the present invention provides a series of force sensor peptides that undergo a conformational change from a closed conformation to an open conformation in the presence of different levels of force. For example, in some embodiments, the present invention provides a series of at least two, three, four, five, six, seven, eight, nine, ten, or more than ten force sensor peptides, wherein the force sensor peptides in the series have been adjusted to undergo a conformational change under at least two of the following forces: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more than 25 piconewtons (pN).

[0140] In some embodiments, the present invention provides methods for characterizing proteins using force sensing peptides. In some embodiments, the methods are used to measure forces acting on a protein of interest. In some embodiments, the methods are used to detect protein interactions.

[0141] In some embodiments, the present invention provides methods of using force sensing peptides to induce a desired effect when subjected to sufficient force. For example, in some embodiments, force sensing peptides can be used to provide a detectable signal, activate a target molecule, or inhibit a target molecule in the presence of a sufficient level of force to induce a conformational change in the force sensing peptide.

[0142] In various embodiments, the present invention provides compositions utilizing forces acting on proteins. In various embodiments, the forces acting on proteins are measured. In some embodiments, the forces acting on proteins are used to activate the compositions. In some embodiments, the compositions are used to deliver targets in a force magnitude dependent manner. In some embodiments, the compositions are used to deliver targets in a force frequency dependent manner. In some embodiments, the compositions are used to deliver targets in a force direction dependent manner. In some embodiments, the compositions are used to deliver targets in a cell specific manner. In some embodiments, the compositions are used to deliver targets in an extracellular matrix dependent manner.

[0143] In some embodiments, the present invention provides methods for measuring forces acting on a protein of interest. In some embodiments, the present invention provides methods for studying protein interactions. In some embodiments, the present invention provides methods for inducing a desired effect. In some embodiments, the composition induces a desired effect in a spatially or temporally controlled manner, for example, in some embodiments, the effect is produced in a specific environment, under specified conditions (i.e., a force sufficient to induce a conformational change in a force sensor peptide of the present invention).

[0144] Composition

[0145] In some embodiments, the present invention provides force sensing molecules that provide a detectable output in the presence of a threshold level of force. In some embodiments, the force sensor molecules of the present invention include peptides, glycopeptides, proteins, protein fragments, protein domains, sugars, polysaccharides, glycosides, nucleic acids, lipids, glycolipids, phospholipids, resins, metabolites, or small molecules.

[0146] In one aspect, the force sensing molecules of the present invention are force-dependent peptides or proteins. In some embodiments, the force-dependent peptides or proteins include peptides that undergo changes when force is applied. The changes that the force sensor peptides of the present invention undergo can be changes in protein stability, conformation, localization, or activation, but are not limited to these types of changes, and can be any type of change that can be detected directly or indirectly. In some embodiments, the force-dependent peptides or proteins include peptides that are inactivated when force is applied. In some embodiments, the peptides or proteins include peptides that are exposed to the external environment when force is applied. In some embodiments, the force peptide sensor undergoes a conformational change that results in the dissociation of the coiled-coil domain to produce an open conformation.

[0147] In various embodiments, the composition comprises a force-sensing peptide comprising two α-helical domains or helical elements connected by a flexible linker in such a manner that the two α-helical domains can coil around each other to form a coiled coil.

[0148] In some embodiments, the first α-helical domain comprises a peptide sequence of SEQ ID NO: 1-20 or a variant thereof. In some embodiments, the second α-helical domain comprises a peptide sequence of SEQ ID NO: 21-40 or a variant thereof. In some embodiments, the linker comprises a peptide sequence of SEQ ID NO: 41-56 or a variant thereof.

[0149] In some embodiments, the first α-helical domain and the second α-helical domain are:

[0150] a) SEQ ID NO: 1 and SEQ ID NO: 21;

[0151] b) SEQ ID NO:2 and SEQ ID NO:21;

[0152] c) SEQ ID NO: 2 and SEQ ID NO: 22;

[0153] d) SEQ ID NO:3 and SEQ ID NO:23;

[0154] e) SEQ ID NO:4 and SEQ ID NO:23;

[0155] f) SEQ ID NO:4 and SEQ ID NO:24;

[0156] g) SEQ ID NO:5 and SEQ ID NO:25;

[0157] h) SEQ ID NO:6 and SEQ ID NO:26;

[0158] i) SEQ ID NO:7 and SEQ ID NO:27;

[0159] j) SEQ ID NO: 8 and SEQ ID NO: 28;

[0160] k) SEQ ID NO:9 and SEQ ID NO:29;

[0161] 1) SEQ ID NO: 10 and SEQ ID NO: 30;

[0162] m) SEQ ID NO:11 and SEQ ID NO:31;

[0163] n) SEQ ID NO:12 and SEQ ID NO:32;

[0164] o) SEQ ID NO: 13 and SEQ ID NO: 33;

[0165] p) SEQ ID NO: 14 and SEQ ID NO: 34;

[0166] q) SEQ ID NO:15 and SEQ ID NO:35;

[0167] r) SEQ ID NO:16 and SEQ ID NO:36;

[0168] s) SEQ ID NO:17 and SEQ ID NO:37;

[0169] t) SEQ ID NO: 18 and SEQ ID NO: 38;

[0170] u) SEQ ID NO: 19 and SEQ ID NO: 39; or

[0171] v) SEQ ID NO: 20 and SEQ ID NO: 40.

[0172] Exemplary sensor peptides include, but are not limited to, peptide sequences shown in SEQ ID NOs: 57-80 or variants or fragments thereof. In some embodiments, the sensor peptide fragment lacks a linker sequence (e.g., GGGG, SEQ ID NO: 91) at the C-terminus, N-terminus, or both ends of SEQ ID NOs: 57-80.

[0173] In some embodiments, the force sensing peptides of the present invention are operably linked to one or more functional domains or functional moieties. In some embodiments, the functional domains or functional moieties become active when the force sensing peptide is in an open conformation. For example, in some embodiments, the functional domains or functional moieties are incorporated into the linker of the sensor peptide and are unavailable or inactive when the coiled-coil domain is in a closed conformation, but become exposed (e.g., activated) when the sensor peptide is in an open conformation. For example, in some embodiments, the linker comprises at least one peptide binding domain, an RNA binding domain, a peptide cleavage domain, a small molecule or protein interaction domain, a small molecule or protein recognition domain (e.g., a domain comprising FRB / FKBP that forms oligomers only in the presence of rapamycin), an epitope bound by a binding molecule (e.g., spyTag / spyCatcher, antigen / nanobody), a DNA binding domain, an ion binding domain, a lipid binding domain, a peptide comprising residues for post-translational modification, a peptide comprising unnatural amino acids, a peptide that is a toxin to cells, a peptide with antibacterial activity, a peptide with antiviral activity, a peptide with antifungal activity, a peptide with enzymatic activity, a peptide that mediates or modifies the enzymatic activity of other proteins, a peptide comprising a localization signal to a cellular compartment, a peptide comprising a secretion signal, a GFP11 peptide (SEQ ID NO: 46), a circular permutation fluorescent protein (cpFP), a HiBiT peptide (SEQ ID NO: 52), a SsrA peptide (SEQ ID NO: 53), a TRAP peptide (SEQ ID NO: 54 and 55), a TEV linker (SEQ ID NO: 56), ... CPFP (SEQ ID NO ID NO: 56), IAAL-K3 (SEQ ID NO: 50) / IAAL-E3 (SEQ ID NO: 51) binding peptides, transcription factors, enzymes (e.g., TurboID), or any combination thereof. In some embodiments, the functional domain or peptide included in the linker becomes active / available / accessible when the force-sensing peptide is in an open conformation, but inactive / inaccessible when the force-sensing peptide is in a closed conformation.

[0174] In some embodiments, the force sensing peptides of the present invention are operably linked to at least one target molecule of interest. Examples of target molecules that can be operably linked to or incorporated into the force sensing peptides of the present invention include, but are not limited to, detectable moieties, fluorescent proteins, bioluminescent proteins, purification tags, target domains, cellular localization signals, DNA molecules, RNA molecules, cAMP, cGMP, eicosapentaenoic acid, eicosatetraenoic acid, phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, aminomethyl polystyrene linkers, chloromethyl polystyrene linkers, PEG linkers, solid phase protein synthesis resins, C-terminal fragments of End4p, C-terminal fragments of actin, C-terminal fragments of clathrin, C-terminal fragments of focal adhesion proteins, C-terminal fragments of talin, C-terminal fragments of integrins, glycogen, cellulose, therapeutic agents, antibiotics, antiviral agents, antifungal agents, antihelmintics, anti-inflammatory molecules, and chemotherapeutic agents.

[0175] In some embodiments, the force sensing peptides of the present invention are operably linked to at least one target molecule of interest at one end and further linked to at least one molecule at the other end to tether the force sensing peptide to a surface or substrate. Exemplary molecules for tethering the force sensing peptide include, but are not limited to, transmembrane domains or proteins comprising transmembrane domains, biotin, streptavidin, antibodies, peptides or epitopes for recognition and binding by binding molecules, or any type of protein or molecule that can be used to tether a force sensor peptide to a surface or substrate.

[0176] In some embodiments, the target molecule of interest becomes activated or inactivated when the force sensing peptide is in an open conformation. For example, in some embodiments, the target molecule of interest is operably linked to the N-terminus or C-terminus of the force sensing peptide and is active when the force sensing peptide is in a closed conformation, but is sequestered when the force sensing peptide is in an open conformation due to aggregation of the open conformation of the force sensing peptide. In some embodiments, the target molecule of interest is operably linked to the N-terminus or C-terminus of the force sensing peptide and is tethered when the force sensing peptide is in a closed conformation, but is released when the force sensing peptide is in an open conformation due to exposure of a peptide cleavage sequence in the linker, which releases the bound target molecule in the open conformation of the force sensing peptide. In some embodiments, the linker comprises a functional domain surrounded by a cleavage site that can be cleaved by a protease only when the sensor is in an open conformation. In such embodiments, the functional domain is released into the cell when the protease cleavage site is cleaved. The functional domain includes, but is not limited to, transcription factors, signaling proteins, enzymes, toxins, and therapeutic agents. The above exemplary embodiments are provided as examples and are not intended to limit the application of the force sensing peptide or to restrict its use.

[0177] In some embodiments, the force sensing peptides of the invention are inserted internally into a target molecule of interest such that when the force sensing peptide is in a closed conformation, the insertion does not alter the function or localization of the target protein. In some embodiments, the conformational change of the force sensing peptide to an open conformation in the presence of a force level above the unfolding threshold subsequently disrupts the localization or activity of the target protein, providing a detectable change.

[0178] In some embodiments, the force sensing peptides of the present invention are flanked by a pair of molecules that are used to generate a signal when the force sensing peptide is in an open conformation, but not when the force sensing peptide is in a closed conformation. Exemplary molecules that can be used to flank the force sensing peptide and provide a signal in an open conformation include, but are not limited to, fluorescent quencher / reporter systems.

[0179] In some embodiments, the force sensing peptides of the present invention are flanked by a pair of molecules that are used to generate a signal when the force sensing peptide is in a closed conformation, but not to generate a signal when the force sensing peptide is in an open conformation. Exemplary molecules that can be used to flank the force sensing peptide and provide a signal in a closed conformation include, but are not limited to, FRET donor / acceptor systems.

[0180] In various embodiments, the compositions of the invention comprise a coiled coil comprising a first helical element and a second helical element. In some embodiments, the coiled coil comprises the first helical element and the second helical element in parallel. In some embodiments, the first helical element in the parallel coiled coil comprises a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12 and variants thereof. In some embodiments, the second helical element in the parallel coiled coil comprises a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-32 and variants thereof. For example, the linker between the parallel coiled coil and the first helical element and the second helical element can comprise the amino acid sequence of any of SEQ ID NOs: 57-70.

[0181] In some embodiments, the coiled coil comprises a first helical element and a second helical element in antiparallel. In some embodiments, the first helical element in the antiparallel coiled coil comprises a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-20 and variants thereof. In some embodiments, the second helical element in the antiparallel coiled coil comprises a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-40 and variants thereof. For example, the linker between the antiparallel coiled coil and the first helical element and the second helical element can comprise the amino acid sequence of any of SEQ ID NOs: 71-80.

[0182] In some embodiments, the coiled coil opens when sufficient force is applied, and is expressed as a required force. Example 6 provides exemplary force sensing peptides and their unfolding force thresholds. For example, in some embodiments, cc-11pN refers to a coiled coil that opens when a force of 11pN or greater is applied. A force sensing peptide comprising the peptide sequence of SEQ ID NO: 57 comprises cc-11pN.

[0183] The peptides of the present invention can be made and combined using chemical methods. For example, the peptides can be synthesized by solid phase techniques (Roberge, JY et al., 1995, Science, 269: 202-204), cut from a resin, and purified by high performance liquid chromatography. Automatic synthesis can be achieved, for example, using an ABI 431A peptide synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer.

[0184] The peptides of the present invention can be made by biological means and combined using chemical methods.

[0185] The peptides of the present invention can be made by biological means, already assembled in tandem into a complete final composition.

[0186] The peptides of the present invention can be made by biological means, already assembled in tandem as complete proteins and loaded with the desired cargo to obtain the final composition.

[0187] The peptides of the present invention can be made by recombinant means or by cleavage from longer peptides. The composition of the peptides can be confirmed by amino acid analysis or sequencing.

[0188] In some embodiments, the composition comprises variants of the provided sequences. Such variants include fragments, including but not limited to fragments of SEQ ID NOs: 1-80, comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 and 81 consecutive amino acids.

[0189] In some embodiments, the present invention comprises any form of peptides containing amino acids derived from the amino acid sequences of SEQ ID NOs: 1-80. In certain embodiments, the present invention comprises amino acid sequences having substantial homology to the peptides of the amino acid sequences of SEQ ID NOs: 1-80. In some embodiments, the peptides "substantially homologous" are about 50% homologous, about 70% homologous, about 80% homologous, about 85% homologous, about 90% homologous, about 91% homologous, about 92% homologous, about 93% homologous, about 94% homologous, about 95% homologous, about 96% homologous, about 97% homologous, about 98% homologous, about 99% homologous to the amino acid sequences of SEQ ID NOs: 1-80.

[0190] As known in the art, the "similarity" between two peptides is determined by comparing the amino acid sequence of one polypeptide and its conservative amino acid substitutions with the sequence of a second polypeptide. Variants are defined as peptide sequences that include residues that are different from the original sequence, preferably less than 40% of the residues of each fragment of interest are different from the peptide sequence of the original sequence, more preferably less than 25% of the residues of each fragment of interest are different from the peptide sequence of the original sequence, more preferably less than 10% of the residues of each fragment of interest are different, and most preferably only a few residues of each fragment of interest are different from the original protein sequence and are homologous enough to retain the functionality of the original sequence. The present invention includes but is not limited to amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similar or identical to the amino acid sequence. The degree of identity between two peptides is determined using computer algorithms and methods well known to those skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, MD 20894, Altschul, S. et al., 1990, J. Mol. Biol., 215: 403-410).

[0191] The peptides of the present invention may be post-translationally modified. For example, post-translation modifications falling within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, phosphorylation, protein folding and proteolytic processing, etc. Some modifications or processing events require the introduction of additional biological mechanisms. For example, processing events such as signal peptide cleavage and core glycosylation are investigated by adding canine microsomal membranes or African clawed frog egg extracts (U.S. Patent No. 6,103,489) to standard translation reactions.

[0192] The peptides of the invention may include unnatural amino acids formed by post-translational modification or by the introduction of unnatural amino acids during translation. A variety of methods can be used to introduce unnatural amino acids during protein translation or synthesis.

[0193] The peptides or proteins of the present invention can be conjugated with other molecules such as proteins to prepare fusion proteins. This can be achieved, for example, by synthesizing N-terminal or C-terminal fusion proteins, provided that the resulting fusion protein retains the functionality of the force-activating composition.

[0194] The peptides or proteins of the present invention can be phosphorylated using conventional methods, such as the method described by Reedjik et al. (1992, The EMBO Journal, 11: 1365).

[0195] In some embodiments, the present invention provides a composition comprising an isolated nucleic acid encoding a force-dependent peptide or protein. In various embodiments, the isolated nucleic acid sequence encodes as follows: a first helical element, the first helical element is conjugated to a peptide linker at its N-terminus, and the peptide linker is in turn conjugated to a second helical element at its N-terminus. In some embodiments, the isolated nucleic acid sequence encodes as follows: a first part, the first part is conjugated to a first helical element at its N-terminus, the first helical element is conjugated to a peptide linker at its N-terminus, and the peptide linker is in turn conjugated to a second helical element at its N-terminus. In some embodiments, the isolated nucleic acid sequence encodes as follows: a first part, the first part is conjugated to a first helical element at its N-terminus, the first helical element is conjugated to a peptide linker at its N-terminus, the peptide linker is in turn conjugated to a second helical element at its N-terminus, and the second helical element is conjugated to a second part at its N-terminus.

[0196] In some embodiments, the isolated nucleic acid sequence encodes a peptide or protein comprising a first helical element comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20 and variants thereof. In some embodiments, the isolated nucleic acid sequence encodes a peptide or protein comprising a linker comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 41-56. In some embodiments, the isolated nucleic acid sequence encodes a peptide or protein comprising a second helical element comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21-40. In some embodiments, the isolated nucleic acid sequence encodes a peptide or protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 57-80.

[0197] In addition, the present invention encompasses isolated nucleic acids encoding peptides having substantial homology to peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-80. In certain embodiments, the isolated nucleic acid sequence encodes a peptide having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to an amino acid sequence selected from SEQ ID NOs: 1-80.

[0198] The isolated nucleic acid sequence can comprise any type of nucleic acid, including but not limited to DNA and RNA. For example, in some embodiments, the composition comprises an isolated DNA molecule (including, for example, an isolated cDNA molecule) encoding the coiled coil of SEQ ID NO: 1-80. In some embodiments, the composition comprises an isolated RNA molecule encoding the coiled coil of SEQ ID NO: 1-80.

[0199] Nucleic acid molecules of the present invention can be modified to improve stability in serum or in a growth medium for cell culture. Modifications can be added to enhance the stability, functionality and / or specificity of nucleic acid molecules of the present invention and to minimize the immunostimulatory properties of nucleic acid molecules of the present invention. For example, in order to enhance stability, 3'-residues can be stabilized to prevent degradation, for example, they can be selected so that they are composed of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, pyrimidine nucleotides are replaced by modified analogs (for example, uridine is replaced by 2'-deoxythymidine) and are acceptable and do not affect the function of the molecule.

[0200] In some embodiments of the invention, the nucleic acid molecule may comprise at least one modified nucleotide analog. For example, the end may be stabilized by incorporating a modified nucleotide analog.

[0201] Non-limiting examples of nucleotide analogs include sugar-modified ribonucleotides and / or backbone-modified ribonucleotides (i.e., including modifications to the phosphate sugar backbone). For example, the phosphodiester bond of a natural RNA can be modified to include at least one of a nitrogen heteroatom or a sulfur heteroatom. In preferred backbone-modified ribonucleotides, the phosphate groups attached to adjacent ribonucleotides are replaced by modifying groups such as thiophosphate groups. In preferred sugar-modified ribonucleotides, the 2'OH group is selected from H, OR, R, halogen, SH, SR, NH 2 、HNR、NR 2 or ON, where R is C 1 -C 6 alkyl, alkenyl or alkynyl and halogen is F, Cl, Br or I.

[0202] Other examples of modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides comprising at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. The bases may be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at position 5, e.g., 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at position 8, e.g., 8-bromoguanosine; deazanucleotides, e.g., 7-deaza-d adenosine; O- and N-alkylated nucleotides, e.g., N6-methyladenosine are suitable. It should be noted that the above modifications may be combined.

[0203] In some cases, nucleic acid molecules include at least one of the following chemical modifications: 2'H, 2'-O-methyl or 2'-OH of one or more nucleotides are modified. In certain embodiments, nucleic acid molecules of the present invention can have enhanced resistance to nucleases. In order to increase nuclease resistance, nucleic acid molecules can include, for example, ribose units and / or thiophosphate bonds (phosphorothioate linkage) modified at position 2. For example, 2' hydroxyl (OH) can be modified or replaced with many different "oxygen (oxy)" or "deoxy (deoxy)" substituents. In order to increase nuclease resistance, nucleic acid molecules of the present invention can include 2'-O-methyl, 2'-fluoro, 2'-O methoxyethyl, 2'-O-aminopropyl, 2'-amino and / or thiophosphate bonds. Including locked nucleic acid (LNA), ethylene nucleic acid (ENA) such as 2'-4'-ethylene bridged nucleic acid and some core base modifications such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp (G-clamp) modification can also improve the binding affinity to the target. In some embodiments, the nucleic acid molecule includes a 2'-modified nucleotide, for example, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-ODMAEOE), or 2'-ON-methylacetamido (2'-O-NMA). In some embodiments, the nucleic acid molecule includes at least one 2'-O-methyl modified nucleotide, and in some embodiments, all nucleotides of the nucleic acid molecule include a 2'-O-methyl modification.

[0204] In certain embodiments, the nucleic acid molecules of the present invention preferably have one or more of the following properties:

[0205] Nucleic acid agents discussed herein also include unmodified RNA and DNA and modified (e.g., to improve efficacy) RNA and DNA, and polymers of nucleoside surrogate. Unmodified RNA refers to a molecule in which the components of nucleic acid (i.e., sugar, base and phosphate moiety) are identical or substantially identical to the components present in nature, preferably identical or substantially identical to the components naturally present in the human body. Rare or unusual but naturally occurring RNA is referred to as modified RNA in the art, see, e.g., Limbach et al. (Nucleic Acids Res., 1994, 22: 2183-2196). Such rare or unusual RNA (often referred to as modified RNA) is generally the result of post-transcriptional modification, and is within the scope of unmodified RNA as used herein. Modified RNA as used herein refers to a molecule in which one or more components of nucleic acid (i.e., sugar, base and phosphate moiety) are different from the components present in nature, preferably different from the components present in the human body. Although they are referred to as "modified RNAs", they will of course include molecules that are not strictly RNA due to the modification. Nucleoside surrogates are molecules in which the ribose phosphate backbone is replaced with a non-ribose phosphate construct that allows the bases to be presented in the correct spatial relationship to allow hybridization substantially similar to that seen when the ribose phosphate backbone is used, for example, a non-charged mimetic of the ribose phosphate backbone.

[0206] Modifications of the nucleic acids of the invention may occur at one or more of the following: phosphate group, sugar group, backbone, 5'-end, 3'-end or nucleobase.

[0207] The present invention also includes vectors having an isolated nucleic acid of the present invention inserted therein. The art is rich in suitable vectors for use in the present invention.

[0208] Briefly, expression of natural or synthetic nucleic acids encoding coiled-coil peptides is generally achieved by operably linking the nucleic acid encoding the coiled-coil peptide or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector to be used is suitable for replication and optionally integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating expression of the desired nucleic acid sequence.

[0209] The vectors of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides gene therapy vectors.

[0210] The isolated nucleic acid of the present invention can be cloned into various types of vectors. For example, the nucleic acid can be cloned into vectors including but not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids.

[0211] Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0212] In addition, the vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Typically, a suitable vector is included in a replication origin, a promoter sequence, a convenient restriction endonuclease site, and one or more selective markers that are functional in at least one organism (e.g., WO 01 / 96584, WO 01 / 29058, and U.S. Patent No. 6,326,193).

[0213] Many virus-based systems have been developed for gene transfer in mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells (in vivo or ex vivo). Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used.

[0214] For example, vectors derived from retroviruses such as lentiviruses are suitable tools for realizing long-term gene transfer, because they allow long-term stable integration of transgenics and their propagation in daughter cells. Lentiviral vectors have additional advantages compared to vectors derived from oncogenic retroviruses such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. In some embodiments, compositions include vectors derived from adeno-associated viruses (AAV). Adeno-associated viruses (AAV) vectors have become powerful gene delivery tools for treating various diseases. AAV vectors have many features that make them ideally suitable for gene therapy, including lack of pathogenicity, minimal immunogenicity, and the ability to transduce post-mitotic cells in a stable and effective manner. By selecting an appropriate combination of AAV serotypes, promoters, and delivery methods, the expression of the specific genes contained in the AAV vectors can specifically target one or more types of cells.

[0215] In some embodiments, a replication-deficient adenovirus may be used. In some embodiments, a replication-deficient serotype 5 adenovirus may be used.

[0216] In certain embodiments, the vector further comprises conventional control elements, which are operably connected to the transgene in a manner that allows the transgene to be transcribed, translated and / or expressed in cells transfected with a plasmid vector or infected with the virus produced by the present invention. As used herein, "operably connected" sequences include expression control sequences adjacent to the gene of interest and expression control sequences that act in trans or remotely to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance secretion of the encoded product when necessary. A large number of expression control sequences (including natural, constitutive, inducible and / or tissue-specific promoters) are known in the art and can be used.

[0217] Additional promoter elements such as enhancers regulate the frequency of transcription initiation. Usually, they are located in the region of 30-110bp upstream of the start site, although recently many promoters have been shown to also include functional elements downstream of the start site. The spacing between the promoter elements is usually flexible, so when the elements are inverted or moved relative to each other, the promoter function is retained. In the thymidine kinase (tk) promoter, before the activity begins to decline, the spacing between the promoter elements can be increased to 50bp apart. Depending on the promoter, it seems that each element can play a role in synergistically or independently to activate transcription.

[0218] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably connected thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered as part of the present invention. Using inducible promoters provides a molecular switch that can turn on the expression of the polynucleotide sequence operably connected thereto when such expression is needed, or turn off expression when expression is not needed. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0219] The enhancer sequence found on the vector also regulates the expression of the gene contained therein. Generally, the enhancer is combined with a protein factor to enhance the transcription of the gene. The enhancer can be located upstream or downstream of the gene it regulates. The enhancer can also be tissue-specific, to enhance the transcription in a specific cell or tissue type. In some embodiments, the vector of the present invention comprises one or more enhancers to strengthen the transcription of the gene present in the vector.

[0220] In order to assess the expression of peptides or proteins, the expression vector to be introduced into the cell may also include a selective marker gene or a reporter gene or both, so as to identify and select expressing cells from a cell population attempted to be transfected or infected by a viral vector. In other aspects, the selective marker may be carried on a separate DNA fragment and used for cotransfection procedures. Both the selective marker and the reporter gene may have appropriate regulatory sequences on both sides to enable them to be expressed in host cells. Useful selective markers include, for example, antibiotic resistance genes, such as neo, etc.

[0221] Reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of regulatory sequences. Generally, reporter gene is a gene that does not exist or does not express and encodes the following polypeptide in a receptor organism or tissue, and the expression of the polypeptide is shown as some properties that are easy to detect, such as enzymatic activity. After DNA is introduced into the receptor cell, the expression of reporter gene is measured at the appropriate time. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein gene (for example, Ui-Tei et al., 2000FEBS Letters 479:79-82). Suitable expression system is well-known, and known technology can be used to prepare or commercially obtain. Generally, the construct with the minimum 5' flanking region showing the highest level reporter gene expression is identified as a promoter. Such promoter region can be connected to a reporter gene and used to evaluate the ability of the transcription driven by the promoter of the reagent regulation promoter.

[0222] Genetically modified cells

[0223] Exogenous genetic material (e.g., nucleic acid molecules encoding force sensor peptides) can be introduced into host cells in vivo by gene transfer methods such as transfection or transduction to provide genetically modified cells. Various expression vectors (i.e., vehicles that facilitate the delivery of exogenous genetic material into target cells) are known to those of ordinary skill in the art.

[0224] As used herein, "transfection of cells" refers to the acquisition of new genetic material by cells by incorporating added DNA. Thus, transfection refers to the insertion of nucleic acids into cells using physical or chemical methods. Several transfection techniques are known to those of ordinary skill in the art, including calcium phosphate DNA coprecipitation, DEAE-dextran, electroporation, cationic liposome-mediated transfection, and microparticle bombardment promoted by tungsten particles. Strontium phosphate DNA coprecipitation is another possible transfection method. In contrast, "transduction of cells" refers to the process of transferring nucleic acids into cells using DNA (e.g., adenovirus) or RNA viruses (e.g., retroviruses).

[0225] In some embodiments, a host cell that has been transfected or transduced with an expression vector to express a force sensor peptide of the invention will not incorporate exogenous genetic material into its genome, but will be able to express exogenous genetic material that remains outside the chromosome within the cell. In some embodiments, a host cell that has been transfected or transduced with an expression vector to express a force sensor peptide of the invention will incorporate exogenous genetic material into its genome. The incorporation of genetic material into the genome of the host cell can be non-specific (e.g., retroviral-based recombination) or can be directed by site-specific recombination methods (e.g., homology-directed recombination or CRISPR / Cas9 recombination).

[0226] In some embodiments, the exogenous genetic material includes a nucleotide sequence encoding the force sensor peptide construct of the present invention and a promoter that controls the transcription of the force sensor peptide construct. The promoter characteristically has a specific nucleotide sequence necessary for initiating transcription. Optionally, the exogenous genetic material further includes additional sequences (i.e., enhancers) required to obtain the desired gene transcription activity. For the purpose of this discussion, an "enhancer" is simply any non-translated DNA sequence that acts adjacent to the coding sequence (in cis) to change the basic transcription level indicated by the promoter. Exogenous genetic material can be introduced into the cell genome immediately downstream of the promoter so that the promoter and the coding sequence are operably connected, thereby allowing transcription of the coding sequence. The expression vector may further include an exogenous promoter element to control the transcription of the force sensor peptide construct after the construct is inserted into the host cell genome. Such exogenous promoters include both constitutive promoters and inducible promoters.

[0227] Naturally occurring constitutive promoters control the expression of basic cellular functions. As a result, genes controlled by constitutive promoters are expressed under all cell growth conditions. Exemplary constitutive promoters include promoters of genes encoding certain constitutive or "housekeeping" functions: hypoxanthine phosphoribosyltransferase (HPRT), dihydrofolate reductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, actin promoter, and other constitutive promoters known to those skilled in the art. In addition, many viral promoters work constitutively in eukaryotic cells. These include the early and late promoters of SV40, the long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus, etc. Therefore, any of the above-mentioned constitutive promoters can be used to control the transcription of heterologous gene inserts.

[0228] Genes controlled by inducible promoters are expressed only in the presence of an inducer or expressed to a higher degree (e.g., in the presence of certain metal ions, transcription controlled by metallothionein promoters is greatly increased). Inducible promoters include response elements (REs) that stimulate transcription when the inducing factor of the response element is bound. For example, serum factors, steroid hormones, retinoic acid, and cyclic AMP all have REs. Promoters containing specific REs can be selected to obtain an induction response, and, in some cases, the REs themselves can be connected to different promoters, thereby conferring inducibility to the recombinant gene. Therefore, by selecting appropriate promoters (constitutive vs. inducible; strong vs. weak), both the presence and expression levels of the force sensor construct in the genetically modified cells can be controlled. If the gene encoding the therapeutic agent is controlled by an inducible promoter, the delivery of the therapeutic agent in situ is initiated by, for example, exposing the genetically modified cells in situ to conditions that allow transcription of the force sensor construct by intraperitoneal injection of a specific inducer of the inducible promoter that controls the transcription of the force sensor construct. For example, in situ expression of a force sensor construct encoded by a gene under the control of a metallothionein promoter by genetically modified cells is enhanced by contacting the genetically modified cells in situ with a solution containing appropriate (ie, inducing) metal ions.

[0229] In addition to at least one promoter and at least one heterologous nucleic acid encoding a force sensor construct, the expression vector may also include a selection gene, e.g., a neomycin resistance gene, to facilitate selection of cells that have been transfected or transduced with the expression vector. Alternatively, the cells are transfected with two or more expression vectors, at least one vector comprising a gene encoding a therapeutic agent and another vector comprising a selection gene. The selection of appropriate promoters, enhancers, selection genes, and / or signal sequences is considered to be within the scope of one of ordinary skill in the art without undue experimentation.

[0230] The force sensor constructs of the present invention can be inserted into any type of target cell or host cell. In the context of an expression vector, the vector can be readily introduced into a host cell, such as a mammalian cell, a bacterial cell, a yeast cell, or an insect cell, by any method known in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0231] Methods for introducing genes into cells and expressing them are known in the art. In the context of expression vectors, the vectors can be easily introduced into host cells, such as mammalian cells, bacterial cells, yeast cells, or insect cells, by any method in the art. For example, the expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0232] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New 20 York). The preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0233] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used methods for inserting genes into mammalian, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0234] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0235] In the case of utilizing a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using lipid formulations to introduce nucleic acid into a host cell (in vitro, in vitro or in vivo). On the other hand, nucleic acid can be associated with lipid. Nucleic acid associated with lipid can be encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, connected to the liposome via a connecting molecule associated with both liposome and oligonucleotide, embedded in the liposome, compounded with the liposome, dispersed in a solution comprising lipid, mixed with lipid, combined with lipid, included in lipid as a suspension, containing or compounded with micelle, or otherwise associated with lipid. Lipid, lipid / DNA or lipid / expression vector related compositions are not limited to any specific structure in the solution. For example, they can exist with a double-layer structure, as micelle or with a "collapse" structure. They can also be simply dispersed in the solution, and may form an aggregate with uneven size or shape. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets naturally occurring in the cytoplasm as well as the class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0236] Lipids suitable for use can be obtained from commercial sources. For example, dimyristoylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristoylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a general term covering a wide variety of monolayer and multilayer lipid carriers formed by generating closed lipid bilayers or aggregates. Liposomes can be characterized by having a vesicle structure having a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They are formed spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure and trap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions having structures different from normal vesicle structures in solution are also contemplated. For example, lipids may present micellar structures or exist only as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0237] Regardless of the method used to introduce exogenous nucleic acid into the host cell, a variety of assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern and Northern blots, RT-PCR, RT-qPCR and PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blots) or by assays described herein to identify agents falling within the scope of the present invention.

[0238] Methods of measuring force

[0239] In some embodiments, the force sensing molecules of the present invention are operably linked to a target protein such that when a force is applied to the target protein of interest, the force sensing molecule undergoes a change or generates a signal or a combination thereof. In some embodiments, the signal is a detectable signal that can be detected directly or indirectly. Exemplary detectable signals include, but are not limited to, aggregation, fluorescent signals, and electrochemical signals. Detection of a change or signal in the sensor protein is a sign that the force sensor protein is subjected to sufficient force to induce a change or signal. For example, in some embodiments, if subjected to a force of at least 3pN, the sensor protein undergoes a change or generates a signal, so detecting a change or signal would indicate that the sensor protein has been subjected to a force of at least 3pN. In some embodiments, if subjected to a force of at least 5pN, the sensor protein undergoes a change or generates a signal, so detecting a change or signal would indicate that the sensor protein has been subjected to a force of at least 5pN. In some embodiments, if subjected to a force of at least 7pN, the sensor protein undergoes a change or generates a signal, so detecting a change or signal would indicate that the sensor protein has been subjected to a force of at least 7pN. In some embodiments, if subjected to a force of at least 10 pN, the sensor protein undergoes a change or generates a signal, and thus detecting a change or signal indicates that the sensor protein has been subjected to a force of at least 10 pN. In some embodiments, if subjected to a force of at least 11 pN, the sensor protein undergoes a change or generates a signal, and thus detecting a change or signal indicates that the sensor protein has been subjected to a force of at least 11 pN. In some embodiments, if subjected to a force of at least 14 pN, the sensor protein undergoes a change or generates a signal, and thus detecting a change or signal indicates that the sensor protein has been subjected to a force of at least 14 pN. In some embodiments, if subjected to a force of at least 18 pN, the sensor protein undergoes a change or generates a signal, and thus detecting a change or signal indicates that the sensor protein has been subjected to a force of at least 18 pN. In some embodiments, if subjected to a force of at least 20 pN, the sensor protein undergoes a change or generates a signal, and thus detecting a change or signal indicates that the sensor protein has been subjected to a force of at least 20 pN.

[0240] In some embodiments, the present invention provides a method for measuring the force applied to a protein. In some embodiments, when sufficient force is applied to a peptide or protein of the present invention, the force-responsive domain is open, exposing the joint to the environment. In some embodiments, when the joint is exposed to the environment, the protein or peptide aggregates to form a visible condensate. In some embodiments, when the joint is exposed to the environment, the GFP11 domain is exposed, which interacts with soluble GFP1-10 to provide a visible reading. In some embodiments, when the force-responsive domain is open, the joint is confirmed to change, so that it changes the photophysical properties of the circular arrangement fluorescent protein inserted into the joint, allowing absorption and / or emission at different wavelengths when the coiled coil domain is in a closed conformation. In some embodiments, when the joint is exposed to the environment, the HiBiT domain is exposed, which interacts with soluble LgBiT to provide a visible reading. In some embodiments, when the joint is exposed to the environment, the small stable RNAA (SsrA) domain is exposed, which interacts with strict hunger protein B (SspB) to recruit molecules marked with SspB. In some embodiments, when the linker is exposed to the environment, a triangular tetrapeptide (tetratricopeptide) repeat affinity protein (TRAP) peptide is exposed, which interacts with fluorescently labeled TRAP, providing a visible readout. In some embodiments, when the linker is exposed to the environment, a tobacco etch virus (TEV) recognition linker is exposed, which is cleaved by a TEV protease.

[0241] In some embodiments, two or more force sensing peptides are used in parallel, or, if two or more force sensing peptides each produce a different signal, they are optionally used in series, wherein the two or more force sensing peptides each have a different unwinding threshold, allowing the force applied to the target to be determined to be determined within a narrow range. For example, when the force to which the protein of interest is subjected is insufficient, the force-responsive domain will not open, the linker will not be exposed, and there is no detectable signal. For other force-responsive domains, the force is large enough, the force-responsive domain opens, the linker is exposed, and there is a positive detectable signal. The force applied to the protein is then determined to be greater than the highest open conformational composition and less than the lowest closed conformational composition. For example, if the highest open conformational composition comprises a coiled-coil domain that responds (unwinds) at 10 pN (cc-10 pN) and the lowest closed conformational composition comprises a coiled-coil domain that responds (unwinds) at 11 pN (cc-11 pN), the force applied to the protein of interest is determined to be between 10 pN and 11 pN.

[0242] Methods for inducing effects in a force-dependent manner

[0243] In some embodiments, when force is applied to protein, the composition of the present invention produces a desired effect. In some embodiments, the present invention provides a method for producing a desired effect when sufficient force is applied to protein. In some embodiments, when sufficient force is applied to the peptide or protein of the present invention, the force responsiveness domain is open, and the joint is exposed to the environment. In some embodiments, the joint comprises an RNA binding domain, which is exposed to the environment when sufficient force is applied. In some embodiments, the RNA molecule becomes accessible when exposed to the RNA binding domain. In some embodiments, the RNA molecule is a ribozyme. In some embodiments, the RNA molecule is a shRNA. In some embodiments, the RNA molecule is a miRNA. In some embodiments, the RNA molecule is a siRNA. For example, when the composition of the present invention is exposed to sufficient force, the coiled coil is open, exposing the RNA binding domain, which is combined with the target mRNA molecule, thereby reducing the mRNA level of the protein of interest.

[0244] Experimental example

[0245] The present invention is further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only, and, unless otherwise indicated, are not intended to be limiting. Therefore, the present invention should never be interpreted as being limited to the following examples, but should be interpreted as covering any and all changes that become apparent due to the teachings provided herein.

[0246] Without further description, it is believed that one of ordinary skill in the art can use the foregoing description and the following illustrative examples to make and utilize the present invention and to implement the claimed methods. Therefore, the following working examples specifically point out certain embodiments of the present invention and should not be construed in any way to limit the remainder of the present disclosure.

[0247] Example 1: Force redistribution in clathrin-mediated endocytosis

[0248] Extracellular substances are transported into cells via endocytosis. In eukaryotic cells, clathrin-mediated endocytosis (CME) is the main internalization pathway for nutrients, signaling molecules, and pathogens (Mettlen, M. et al., 2018, Annual Review of Biochemistry, 87: 871-896; Kaksonen, M. et al., 2018, Nat Rev Mol Cell Biol, 19: 313-326). It is involved in a variety of diseases, including cancer, neurological disorders, and viral invasion, and is therefore the focus of both basic and translational research (McMahon, HT et al., 2011, Nat Rev Mol Cell Biol, 12: 517-533). The distinctive feature of CME is a layer of protein coat, of which clathrin is the main member. The coat contains more than 20 evolutionarily conserved proteins that assemble on the intracellular side of endocytic sites, bend as the endocytic pits mature, and disassemble after budding of the endocytic vesicles (Kukulski, W. et al., 2012, Cell, 150:508-520; Sun, Y. et al., 2019, eLife, 8:e50749). Endocytic coat proteins interact extensively with each other, with lipids of the plasma membrane, and with the network of actin filaments that surround the endocytic coat (Skruzny, M. et al., 2020, Molecular Systems Biology, 16: e9009; Legendre-Guillemin, V., 2004, Journal of Cell Science, 117: 9-18; Baggett, JJ et al., 2003, Genetics, 165: 1661-1674; Maldonado-Báez, L. et al., 2006, Trends in Cell Biology, 16: 505-513). The force generated by actin polymerization is transmitted by adaptor proteins of the endocytic coat to help convert flat membrane sheets into cargo-filled vesicles that enter the cytoplasm (Skruzny, M. et al., 2012, Proc Natl Acad Sci USA, 109: E2533-E2542; Skruzny, M., 2015, Developmental Cell, 33: 150-162; Lacy, M. et al., 2018, FEBS Letters, 592: 3586-3605) ( Figure 1 A).

[0249] The deformation of the membrane during CME consumes a lot of energy (Lacy, M. et al., 2018, FEBS Letters, 592: 3586-3605; Ma, R. et al., 2021, Biophys J, 120: 1625-1640; Basu, R. et al., 2014, Mol Biol Cell, 25: 679-687). In mammalian cells with elevated membrane tension and in yeast cells with turgor pressures as high as 1 MPa, successful CME requires actin polymerization (Kukulski, W. et al., 2012, Cell, 150: 508-520; Basu, R. et al., 2014, Mol Biol Cell, 25: 679-687; Aghamohammadzadeh, S. et al., 2009, Nat Cell Biol, 11: 1039-1042). Epsins and Hip1R are thought to transmit the forces generated by actin assembly to the plasma membrane because their removal delays invagination of endocytic pits, and their C-terminal domains (ACB and THATCH, respectively) bind actin filaments and their N-terminal domains (ENTH and ANTH, respectively) bind PIP2 on the membrane (Baggett, JJ et al., 2003, Genetics, 165:1661-1674; Skruzny, M. et al., 2012, Proc Natl Acad Sci USA, 109:E2533-E2542; Skruzny, M., 2015, Developmental Cell, 33:150-162; Messa, M. et al., 2013, eLife. 3:e03311) ( Figure 1 B). Since both proteins also bind clathrin and other endocytic coat proteins, it is possible that forces can be transmitted to the membrane via multiple pathways (Skruzny, M. et al., 2020, Molecular Systems Biology, 16: e9009; Aguilar, RC et al., 2003, J. Biol. Chem, 278: 10737-10743; Engqvist-Goldstein, EY et al., 2001, J Cell Biol, 154:1209-1224; Wilbur, JD et al., 2008, J Biol Chem, 283:32870-32879). Compared to the rich knowledge gleaned from biochemical and genetic approaches, a quantitative understanding of force generation and distribution at the molecular level during CME is lacking because tools to measure forces in living cells in the context of small (~100 nm in diameter) and transient (~10 s) endocytic pits are scarce, difficult to use, and are often too large to be inserted into proteins without causing side effects.

[0250] The assembly of the endocytic coat relies on weak and multivalent interactions, which facilitate the rapid exchange of binding partners during the dynamic rearrangement of the endocytic coat and the continuous changes in membrane shape during CME (Smith, SM et al., 2017, Front. Mol. Biosci, 4:e00072; Garcia-Alai, MM et al., 2018, Nature Communications, 9:328; Zhuo, Y. et al., 2015, Biochemistry, 54:2571-2580). Many endocytic proteins contain promiscuous binding sites, multiple copies of short peptide motifs, and intrinsically disordered regions (IDRs) (Maldonado-Báez, L. et al., 2006, Trends in Cell Biology, 16:505-513; Smith, SM et al., 2017, Front. Mol. Biosci, 4:e00072; Garcia-Alai, MM et al., 2018, Nature Communications, 9:328; Miao, Y. et al., 2018, The FEBS Journal, 285:2762-2784). Recent developments in protein engineering have shown that dual incorporation of IDRs and oligomerization domains (controlled by light or small molecules) promotes phase separation of proteins in vivo (Bracha, D. et al., 2019, Nature Biotechnology, 37: 1435-1445; Bracha, D. et al., 2018, Cell, 175, 1467-1480.e13; Nakamura, H. et al., 2018, Nat Mater, 17: 79-89). If a mechanically driven oligomerization domain is introduced into an endocytic adaptor protein containing an IDR, protein phase separation can be induced in a force-dependent manner.

[0251] Dimeric coiled coils are excellent candidates for such mechanically driven oligomerization domains because they are small, have well-characterized shapes and mechanical properties, and can be easily introduced into proteins without affecting their normal function (Tanenbaum, M. E. et al., 2014, Cell. 159, 635-646; Truebestein, L. et al., 2016, Bioessays, 38:903-916; Lebar, T. et al., 2020, Nat Chem Biol, 16:513-519) ( Figure 4 A. Fig.10 ). The unfolding forces and energies of dimeric coiled coils have been measured previously, and they were found to unfold at a tension threshold in the range of 2-14 piconewtons (pN) (Goktas, M. et al., 2018, Chem. Sci., 9:4610-4621; Gao, Y. et al., 2011, J. Am. Chem. Soc., 133:12749-12757; Xi, Z. et al., 2012, PNAS, 109:5711-5716). The unfolding of coiled coils exposes two hydrophobic interaction surfaces that can mediate higher-order oligomerization (Pandya, MJ et al., 2000, Biochemistry, 39:8728-8734) ( Figure 4 B and Figure 4 E), while the folded coiled-coil places an upper limit on the local force magnitude and serves as a control for insertion.

[0252] The force requirements in CME have been the subject of multiple theoretical works, but direct force measurements in the densely interwoven shell of CME have been lacking (Lacy, M. et al., 2018, FEBS Letters, 592: 3586-3605; Dmitrieff, S. et al., 2015, PLOS Computational Biology, 11: e1004538; Nickaeen, M. et al., 2019, bioRxiv, 518423). Only one recent study in Saccharomyces cerevisiae used a FRET-based force sensor to measure forces on Sla2, a budding yeast homolog of End4p, in a mutant background (Abella, M. et al., 2021, Developmental Cell, 56: 2419-2426.e4). The study measured forces greater than 8 pN near the THATCH actin-binding domain. We developed a calibrated coiled-coil as a novel force sensor to induce force-dependent phase separation of the endocytic adaptor End4p. Furthermore, we found that the force transmitted directly to the C-terminus of End4p through the actin network was between 11 pN and 20 pN in a wild-type background. A force gradient existed along End4p, demonstrating that force is transmitted and redistributed across the endocytic coat.

[0253] This strategy for measuring forces in living cells exploits pre-existing multivalent interactions in protein complexes and avoids the need to insert two large fluorescent proteins in the middle of the target protein as specified in the method using FRET force sensors. These force sensors are also significantly smaller because the effector of the force (i.e., the calibrated coiled coil) and the fluorescent reporter are physically separated. The self-propagating intermolecular interactions of the coiled coil force sensor are triggered by the transient force on End4p, and the effect of the force is amplified both spatially and temporally by producing condensates with a micron diameter and a longer life than the endocytic shell. The combination of small size and clear readings makes this coiled coil-based strategy a useful and novel tool for easy in vivo force measurement at locations that were previously inaccessible using standard fluorescence microscopes. Another advantage of this strategy is that it embeds internal controls and redundancy because all sensors have very similar folding. After insertion into the same position in the protein, the folded coiled coil force sensor detects the upper limit of the local force size without changing the protein function, while the unfolded coiled coil force sensor reports the lower limit of the force and drives the formation of protein condensates for easy detection. The paired application of coiled coil force sensors defines the force range. These calibrated coiled-coils are compatible with FRET methods (Ringer, P. et al., 2017, Nature Methods, 14: 1090-1096) and complement FRET-based strategies well, especially when insertion of large FRET pairs cannot be tolerated.

[0254] It is expected that the coiled-coil force thresholds measured in vitro will not be significantly different from those in vivo. First, coiled-coils are derived from prokaryotic proteins or are artificially designed, and no proteins are known to interact with them, so their unfolding properties are unlikely to change in vivo. Second, the in vivo phenotype is always consistent with the order of the calibrated coiled coil (for example, if the cc-11pN construct aggregates, the cc-10pN and cc-8pN constructs also aggregate). Third, they are small and they are inserted into unstructured regions that are unlikely to interfere with the function of the protein. Fourth, even though the pulling rates on the protein in a subcellular context and in vitro may be different and may affect the opening force threshold, the rates used in vitro are in the same range as those expected during most cellular processes (including clathrin-mediated endocytosis in yeast), and previous data and theory strongly suggest that the pulling rate has a small effect on the force threshold (less than 30% for a 10-fold faster rate).

[0255] It is estimated that the forces on each End4p molecule are in the range of 8–10 pN after the lipid-binding ANTH domain, 10–11 pN between the proline-rich domain and the dimerization domain, and 11–20 pN before the THATCH domain ( Fig. 9E). The force gradient along End4p and mutant data show that even if End4p cannot bind actin, force can still be transmitted to the membrane, which strongly suggests that endocytic forces are integrated along the endocytic shell according to a "collection and redistribution" mechanism. Since previous quantitative microscopy studies have shown that there are up to 120 End4p molecules per endocytic shell (Sun, Y. et al., 2019, eLife, 8: e50749), the data indicate that forces in the range of 1320-2400pN are generated by the actin meshwork on End4p at the periphery of the endocytic shell, and at least 960-1200pN are directly transmitted to the lipid membrane through the End4pANTH domain. These forces on End4p are smaller, but are of the same order of magnitude as the total endocytic force predicted by theory. Since force transmission in the endocytic shell is transmitted by binding to other adapter proteins, the total force on the plasma membrane is likely to be larger, because fragments of End4p that do not bind actin are still subject to tension in the endocytic shell ( Figure 8 B-8C). The Sla1p / Pan1p / End3p protein complex, which arrives later than End4p in the assembly of the endocytic coat and has been shown to interact with both End4p and Ent1p, bridges the transmission of force from F-actin to End4p ( Fig. 9 E) (Sun, Y. et al., 2015, Molecular Biology of the Cell, 26:3841-3856; Tang, H.-Y. et al., 2000, Molecular and Cellular Biology, 20:12-25). The clathrin lattice is likely to be a hub for integrating the transmission of forces in the endocytic coat, because End4p, Ent1p, Sla1p and many other endocytic adaptor proteins bind to the clathrin lattice, and changes in the magnitude of forces on End4p have been detected before and after the dimerization domain of End4p, which contains a putative clathrin binding site (Engqvist-Goldstein, EY et al., 2001, J Cell Biol, 154: 1209-1224; Wilbur, JD et al., 2008, J Biol Chem, 283: 32870-32879). Despite peripheral perturbations closer to the F-actin binding side, the solidly interconnected endocytic shell ensures redundancy to robustly transmit forces deep into the endocytic shell and to the membrane ( Fig.12 ). The magnitude of the forces during CME is slightly smaller in budding yeast because of the slightly lower turgor pressure there, and the forces are even smaller in mammalian cells where turgor pressure is several orders of magnitude lower. However, the redundancy in force transmission through the binding of endocytic coat proteins is likely to be preserved.

[0256] It is increasingly recognized that protein phase separation is actively involved during natural cellular processes. Forces generated by the cytoskeleton or motor proteins may change the folding of protein domains to promote or inhibit protein phase separation. The phase separation of LIMD1 recruits focal adhesion proteins in a force-dependent manner (Wang, Y. et al., 2021, Dev Cell, 56: 1313-1325.e7). The prevalence of low-affinity interactions in endocytic shells suggests that protein phase separation may be involved in normal CME (Kozak, M. et al., Preprint 2019, eLife, 11: e72865; Day, KJ et al., 2021, Nat Cell Biol, 23: 366-376; Bergeron-Sandoval, L.-P. et al., 2021, bioRxiv, 145664), which may be more transient in nature, but appears to be enhanced by the insertion of coiled-coil force sensors within End4p.

[0257] Although the focus was not on in vitro characterization of the phase separation properties of End4p proteins with coiled-coil insertions, the in vivo data provide a clear mechanism for phase separation of End4p constructs. The increase in valency from 0 to 2 upon coiled-coil unfolding is critical for the generation of End4p condensates ( Figure 6 , Fig.14 G), and the disconnection of the two α-helices that prevent intermolecular interactions disrupts End4p condensates. Therefore, these coiled-coil force sensors represent a new way to induce protein condensates in vivo in a force-dependent manner, combining currently existing methods using light or small molecules. The rich knowledge associated with the design and engineering of coiled-coils paves the way for other functions based on this simple protein motif.

[0258] Design and calibration of cc-11pN

[0259] First, the two parallel α-helices in the heterodimeric GCN4 leucine zipper were connected with a 30-amino acid flexible linker to form a single-chain polypeptide that can be genetically encoded ( Figure 4 A). To measure its unfolding force using optical tweezers, a single GCN4 coiled-coil was tethered between two beads held in two optical traps ( Figure 1 C) and pulled to higher forces by separating the two wells at a speed of 10 nm / s (Gao, Y. et al., 2011, J. Am. Chem. Soc., 133:12749-12757), which is a typical predicted speed for actin polymerization during endocytosis. Unfolding of the coiled coil is manifested as a sudden increase in extension at ~11 pN ( Figure 1 D, black trace). The unfolded coiled-coil refolds during relaxation, but at lower forces ( Figure 1 D, red trace). Repeated pulling and relaxation explained the distribution of unfolding forces, with a single peak at 10.8 ± 0.4 pN (mean ± SEM) ( Figure 1 E). Hereinafter, this coiled-coil is referred to as cc-11pN.

[0260] Force applied to End4p induces aggregation

[0261] CRISPR / Cas9 was used to insert cc-11pN into the genomic locus of the fission yeast homologue of Hip1R (End4p) so that the expression level of End4p was not perturbed ( Fig.11 End4p functions as a dimer in vivo (Skruzny, M. et al., 2012, Proc Natl Acad Sci USA, 109: E2533-E2542; Abella, M. et al., 2021, Developmental Cell, 56: 2419-2426.e4) and contains an IDR between its proline-rich domain and its dimerization domain ( Figure 2 In wild-type cells, fluorescently labeled End4p is present at endocytic sites and appears as diffraction-limited puncta (hereinafter referred to as End4p puncta) that are enriched at cell ends during interphase and around the division plane during mitosis ( Figure 1 F. Figure 3 A. Figure 6 B- Figure 6 C) When cc-11pN is inserted in front of the actin-binding THATCH domain of End4p, many large spherical End4p aggregates appear among the small plaques ( Figure 1 G. Figure 6 E- Figure 6 F). Condensates remained visible in the cell for the entire time period exceeding 10 min, whereas End4p remained at endocytic patches for a much shorter time (~50 sec). Interestingly, the condensates displayed liquid-like features such as fusion and fission ( Figure 1 G. Figure 3 B- Figure 3 C).

[0262] Condensate assembly of the End4p construct is mediated by force-induced unfolding of cc-11pN, which in turn leads to the formation of intermolecular associations of unfolded α-helices ( Figure 5 A. Figure 4E). To test this, a construct with a shortened linker between the two α-helices of cc-11pN was used to force the coiled-coil to unfold (Figure). Indeed, larger condensates were formed ( Figure 5 B. Figure 6 H-6I), supporting the fact that the unfolded coiled-coil mediates condensate assembly.

[0263] To further verify that End4p condensate formation is indeed force-dependent, several controls were generated. To eliminate the force on cc-11pN, it was inserted at the C-terminus of End4p ( Figure 5 C), delete the THATCH domain ( Figure 5 D), or using a self-cleaving 2A peptide to split the construct after the dimerization domain of End4p ( Figure 5 E). No condensates were observed in any of the three cases, indicating that incorporation of cc-11pN itself does not induce End4p condensation and that force is required to generate End4p condensates. In all of these experiments, cellular localization of these End4p constructs was identical with or without cc-11pN insertion ( Figure 7 A- Figure 7 B). Splitting cc-11pN between two α-helices prevents intermolecular coiled-coil formation and thereby prevents the formation of End4p aggregates ( Figure 5 F. Figure 4 F). Mutations in the THATCH domain that abolish End4p binding to F-actin ( Figure 5 RVK1010DDD in G, Figure 5 H) also prevents the formation of End4p condensates (McCann, RO et al., 1997, Proc Natl Acad Sci USA, 94:5679-5684; Brett, TJ et al., 2006, Nat Struct Mol Biol, 13:121-130). It was also confirmed here that the insertion of cc-11pN did not change the localization of End4p ( Figure 7 D- Figure 7 E). Finally, inhibition of F-actin polymerization by Latrunculin A (LatA) resulted in a decrease in both the size and number of End4p condensates ( Figure 5 I-5K), demonstrating that the formation of End4p condensates depends on F-actin polymerization. Taken together, the results suggest that force-induced unfolding of cc-11pN in End4p promotes phase separation of End4p constructs, and that the magnitude of the forces between the dimerization and THATCH domains of End4p is higher than that of 11pN.

[0264] Further investigation of the forces involved was accomplished by measuring the forces preceding a newly discovered domain in End4p that resembles R12 of talin (called Rend for the R domain in End4p). cc-14pN was subsequently inserted into End4p preceding the Rend domain. Examination of cells expressing this construct produced condensates, indicating that peak forces exerted preceding Rend were greater than 14 pN ( Figure 8 A). Returning to End4p containing the THATCH domain, the additional coiled-coil component cc-18pN is inserted before THATCH. Under the same conditions as above, it is formed again, indicating that a peak force greater than 18pN is applied before THATCH ( Figure 8 B).

[0265] Fine force measurement and measurement at alternative positions

[0266] To further assess the forces involved in condensate assembly, three additional force sensors (based on artificial coiled-coil (O'Shea, EK et al., 1993, Current Biology, 3:658-667; Gurnon, DG et al., 2003, J. Am. Chem. Soc., 125:7518-7519) and B-ZIP protein (Moll, JR et al., 2001, Protein Sci, 10:649-655) were designed and tested. Fig.12 A- Fig.12 D), the average unfolding force thresholds were 8.2±0.2 pN, 10.0±0.1 pN, and 20.0±0.7 pN, hereinafter referred to as cc-8 pN, cc-10 pN, and cc-20 pN, respectively ( Fig.13 E. Fig.14 A- Fig.13 C). Insertion of these three coiled-coils into actin-binding-deficient End4p does not result in the formation of End4p condensates ( Fig.13 D- Fig.13 F), and these force sensors inserted in the same position as cc-11pN showed that the force magnitude before the THATCH domain was 11-20pN ( Fig. 9 A, right column).

[0267] This library of calibrated coiled-coil force sensors not only allowed for force measurements prior to the refinement of the THATCH domain, but also allowed for force measurements at two other locations on End4p ( Fig. 9 A. Figure 2 A) The coiled-coil force sensor is inserted after the lipid-binding ANTH domain ( Fig. 9 A, left column), between the proline-rich domain and the dimerization domain ( Fig. 9A, middle column) or before the THATCH domain ( Fig. 9 A, right column). Insertion of the same coiled-coil at different positions resulted in End4p condensates in some but not all cases (compare Fig. 9 Each row in A). Since the formation of End4p condensates reflects the local presence of forces above the unfolding force threshold of the coiled-coil force sensor, the results demonstrate a gradient in the magnitude of the forces along the End4p molecule: between 8 pN and 10 pN after the ANTH domain, between 10 pN and 11 pN between the proline-rich domain and the dimerization domain, and between 11 pN and 20 pN before the THATCH domain. These data also provide an internal control for the full functionality of the constructs when forces are below the force threshold of the sensor, since the timing of endocytosis is unchanged for all uncondensed constructs ( Fig.10 ).

[0268] Force from F-actin is transmitted and redistributed by End4p

[0269] The force gradient on End4p suggests that binding of other endocytic coat proteins to End4p dissipates the forces transmitted from F-actin to the lipid membrane. This was demonstrated by splitting End4p after its proline-rich domain, thereby disconnecting the End4p N-terminus from its C-terminus, but preserving the localization of the End4p C-terminus at the endocytic patch ( Figure 7 C). Although the End4p C terminus cannot bind lipids, the forces on the End4p C terminus are still large enough to cause cc-11pN to unfold, confirming that forces are also transmitted to End4p via its binding partners in the endocytic shell ( Fig. 9 B). Similarly, when the THATCH domain was mutated to prevent direct binding between End4p and F-actin, forces above 8 pN were still measured between the ANTH and proline-rich domains of End4p, whereas forces behind the proline-rich domain were now less than 8 pN (whereas they were greater than 8 pN in wild-type cells) ( Fig. 9 C-9D). This result clearly shows that even when End4p itself is unable to bind F-actin, other endocytic coat proteins mediate force transmission to the N terminus of End4p. Due to the extensive interconnectivity of the endocytic coat, deletion of the F-actin binding domain of another putative force-transmitting endocytic protein, Ent1p (homologous to human epsin-1), only resulted in an increase in tension on End4p before the THATCH domain, while forces after the ANTH domain or after the proline-rich domain remained unchanged ( Fig.12Together, these results demonstrate that, contrary to popular belief, forces are not directly transmitted via adaptor proteins such as End4p, but are instead transported and redistributed along different layers of the endocytic envelope by End4p and its binding partners.

[0270] The materials and methods used are described herein.

[0271] Protein constructs and purification

[0272] Codon optimized DNA (Invitrogen) of synthetic coiled coil is cloned into pGEX-6P-1 vector (Sigma-Aldrich) after PCR amplification and Gibson cloning (New England BioLabs).Construct is confirmed by DNA sequencing and introduced into BL21 (DE3) (New England BioLabs) for protein expression.GST fusion protein is purified by glutathione sepharose 4B beads (GE Healthcare) binding, and GST tag is removed by cutting with PreScission protease (Sigma-Aldrich).Purified protein is exchanged into the biotinylation buffer containing 25mM HEPES, 200mM potassium glutamate at pH 7.7 and is biotinylated with Avi tag at 4°C overnight in the presence of 50 μg / mL BirA, 50mM bicine buffer (pH 8.3), 10mM ATP, 10mM magnesium acetate and 50 μm d-biotin (Avidity).

[0273] Protein-DNA handle crosslinks

[0274] The PCR-generated DNA handle used in the single-molecule experiment was 2,260 bp in length and contained a sulfhydryl group (-SH) at one end and two digoxigenin moieties at the other end. The DNA handle was cross-linked to the coiled-coil protein construct as previously described (Gao, Y. et al., 2011, J. Am. Chem. Soc., 133: 12749-12757). Briefly, the protein construct was mixed with the DTDP-treated DNA handle at a molar ratio of 50:1 in 100 mM phosphate buffer, 500 mM NaCl, pH 8.5 and incubated overnight at room temperature.

[0275] Single molecule manipulation experiments

[0276] As previously described, all pulling experiments were performed using double-well high-resolution optical tweezers (Gao, Y. et al., 2011, J. Am. Chem. Soc., 133: 12749-12757; Xi, Z. et al., 2012, PNAS, 109: 5711-5716). Briefly, aliquots of the cross-linked protein-DNA mixture were mixed with 5 μL of 2.1 μm diameter anti-digoxigenin antibody-coated polystyrene beads (Spherotech) and incubated at room temperature for 15 min. Then, the anti-digoxigenin-coated beads and 0.5 μL of 1.7 μm diameter streptavidin-coated beads were mixed in 1 mL PBS buffer (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4) and injected into the top and bottom channels of the homemade microfluidic chamber respectively. Both channels are connected to the central channel with glass tubes. The two beads entering the central channel are captured in two optical traps and close to each other so that a single protein is tethered between them. All operation experiments are carried out in the PBS buffer supplemented with an aerobic scavenging system (400 mg / mL glucose (Sigma-Aldrich), 0.02 units / mL glucose oxidase (Sigma-Aldrich) and 0.06 units / mL catalase). Pull and relax all single molecules at a speed of 10 nm / s by increasing and reducing the trap separation respectively. This speed was chosen because during clathrin-mediated endocytosis in yeast, ~100 nm long actin filaments assemble in ~10 seconds (Sirotkin, V. et al., 2010, Mol Biol Cell, 21:2894-2904; Berro, J. et al., 2010, MBoC, 21:2905-2915), corresponding to an average growth rate of 10 nm / s. Since actin dynamics are the main source of force during endocytosis, endocytosed proteins involved in force transmission are expected to be subject to loads moving at ~10 nm / s. As described elsewhere, the data were processed by MATLAB code (Gao, Y. et al., 2011, J. Am. Chem. Soc., 133:12749-12757; Xi, Z. et al., 2012, PNAS, 109:5711-5716), and the unfolding force was determined from the force-extension curve.

[0277] Yeast strains and culture media

[0278] The pombe (S. pombe) strains used in this study are shown in Supplementary Table S1. The strains were constructed using methods described in previous publications (Fernandez, R. et al., 2016, Yeast, 33: 549-557) and verified by sequencing of colony PCR products. Fission yeast cells were grown in YE5S (yeast extract supplemented with 0.225 g / L uracil, lysine, histidine, adenine and leucine) and imaged in EMM5S (Edinburgh minimal medium supplemented with 0.225 g / L uracil, lysine, histidine, adenine and leucine). Yeast cells were grown overnight at 32 ° C and shaken at 200 rpm to reach OD 595nm Exponential period between 0.3 and 0.5.

[0279] Microscopic examination

[0280] Cells were imaged on a 25% gelatin pad at room temperature on a Nikon TiE inverted microscope (Nikon, Tokyo, Japan) with a CSU-W1 confocal scanning unit (Yokogawa Electric Corporation, Tokyo, Japan), under a CFI Plan Apo 100X / 1.45NA phase objective (Nikon, Tokyo, Japan). Images were acquired with an iXon Ultra888EMCCD camera (Andor, Belfast, UK). The mEGFP marker strain was excited with a 488nm argon ion laser and filtered through a Spectra X with a single bandpass filter 510 / 25. The mScarlet-I marker strain was excited with a 561nm argon ion laser and filtered through a Spectra X with a single bandpass filter 575 / 25. The fluorescence signal from the entire cell was collected with 21 optical sections spaced 0.5 μm apart, and maximum projection was performed to create a 2D image. Laser excitation and image acquisition settings were identical for the same fluorophore (mEGFP or mScarlet-I) for all imaged strains. Images were displayed and analyzed using the Fiji distribution of ImageJ (NIH, USA).

[0281] LatA processing

[0282] Before LatA (Thermo Fisher, MA, USA) is processed, cells are cleaned with EMM5S and loaded into CellASIC microfluid chamber (Y04C-02-5PK, Millipore-Sigma, Saint Louis, USA). Culture medium exchange is controlled by CellASIC ONIX2 microfluid system (Millipore-Sigma, Saint Louis, USA) that flow pressure is 4psi. LatA is diluted to a final concentration of 100 μM in EMM5S. Fluorescence signal from whole cells is collected with 21 optical sections with 0.5 μm thickness respectively, and stacks are projected with average intensity. Cells are imaged in every 5 minutes, and final image is corrected for photobleaching (exponential fitting) before analysis.

[0283] Protein Aggregate Analysis

[0284] For quantification of End4p protein aggregates, an ImageJ plugin was created that identifies protein aggregates based on fluorescence intensity (900-65535), size (4-500 pixels) and circularity (0.2-1.0) and excludes End4p plaques. The plugin showed good agreement with manual selection. Examples of automatically identified protein aggregates are shown in Figure 6 Shown in.

[0285] Example 2: Creation of a force-sensitive coiled-coil library

[0286] Here, an existing set of coiled-coils was expanded to create a complete library of coiled-coil force sensors that each defold at a different threshold force (spanning the range of ~3-20 pN) ( Fig.15 )(Ren, Y. et al., 2021, bioRxiv, 2021.06.29.450294). This range of forces covers most known biomolecules that generate forces in vivo. Coiled coils whose average opening forces differ by less than 1 pN from each other were developed. The library started with several starting coiled coil sequences (GCN4, peptide Oakley, peptide Velcro, dynein stem, VBP). Reasonable mutations within residues at the interface between two α-helices and changes in the length of the coiled coil were used, because extensive studies of coiled coils have shown that the number of hydrophobic residues at this interface largely controls the affinity between α-helices. For a given coiled coil base, the force threshold is correlated with affinity, a trend that has been confirmed in previously calibrated force sensors (Ren, Y. et al., 2021, bioRxiv, 2021.06.29.450294).

[0287] Following the protocol used for the previously calibrated force sensor ( Fig.15 Error! Reference source not found. ), the new coiled coil was purified from Escherichia coli and calibrated by pulling with optical tweezers in vitro (Ren, Y. et al., 2021, bioRxiv, 2021.06.29.450294). For each base design, a positive control sensor and a negative control sensor were systematically created based on preliminary data, which were either a) always unfolded (by removing hydrophobic residues of the hydrophobic core or shortening the linker between α-helices) or b) always folded (by inducing the formation of disulfide bonds between cysteines introduced in the hydrophobic core).

[0288] The unfolding force of the force sensor is not different in vitro and in vivo. First, the pulling rate (i.e., the speed at which force is applied to the protein) can significantly change the opening force threshold of the coiled coil, however, the pulling rate in cells is in the same range as the pulling rate used in optical tweezers experiments (10 nm / s), and theory and experiments show that the unfolding force increases by ∼20% when the pulling rate changes by more than 1-2 orders of magnitude (Xi, Z. et al., 2012, PNAS, 109:5711-5716; Goktas, M. et al., Chem Sci, 9:4610-4621).

[0289] It is expected that inserting force sensors into the proteins of interest will not change their unfolding forces. FRET-based force sensors have been shown to work similarly in vivo and in vitro (Cost, A.-L. et al., 2015, Cel Mol Bioeng, 8:96-105; Grashoff, C. et al., 2010, Nature, 466:263-6). To ensure that this is the case for the current force sensors, optical tweezers were used to determine the unfolding force thresholds of selected purified End4p constructs containing different sensors at different locations. The unfolding forces of these constructs were the same as those of the sensors alone.

[0290] Several different coiled-coil bases with overlapping force threshold ranges have been designed and they will be used to cross-validate the strategy. Disulfide bonds are not usually formed in the reducing environment of the cell, however, if the cysteines are kept in close proximity (e.g., in coiled coils), disulfide bonds are usually formed (Huang, H. et al., 2000, Chemistry & Biology, 7: 355-364; He, C. et al., 2002, Chemistry & Biology, 9: 1297-1303). In addition, the force sensor with the highest force threshold still provides relatively good 'folding' control for most proteins of interest. In order to expand the sensor range to higher forces, protein domains with more complex folding and higher unfolding force thresholds are used (e.g., previously characterized artificial SNARE complex constructs) (Zorman, S. et al., 2014, Elife, 3: e03348; Gao, Y. et al., 2012, Science, 337: 1340-3).

[0291] The data show that the standard deviation of the mean force of the unfolding force threshold of the coiled coil is ~20% (Ren, Y. et al., 2021, bioRxiv, 2021.06.29.450294). At present, it remains unknown how these extensions can be rationally controlled by the amino acid sequence. Similarly, how to control the refolding force remains unknown. However, these properties do not have a big impact on in vivo measurements. The library contains enough coiled coils that have similar average opening forces, but have different standard deviations and different refolding forces. By systematically introducing these sensors between different domains of End4p, the effects of the sensor's opening force standard deviation and refolding force on the ability to discern the force load of the protein are elucidated. In addition, the data identify general rules that affect the distribution of coiled coil opening and refolding forces.

[0292] Not all positions within a protein are suitable for insertion of a force sensor. It is important not to insert force sensors within known domains, but rather to insert them preferably in linker regions between domains, avoiding residues predicted to undergo post-translational modifications or known motifs. Controls were included to ensure that insertion of the force sensor did not affect protein function by using an always folded version of the sensor and confirming proper function of the protein and the subcellular processes in which it participates. Since domain boundaries are not always unambiguously known, the insertion site was moved a few residues if the first insertion attempt was unsuccessful. This was confirmed by inserting an "always open" positive control sensor.

[0293] Typically, natural interactions between the domains of a POI do not interfere with force measurements. A false negative situation is conceivable, in which a coiled-coil force sensor is inserted between two domains of a POI and does not open even if these domains are actually subjected to forces above the coiled-coil force threshold. If these domains interact strongly with each other and the force separating them (e.g., 7 pN) is greater than the coiled-coil force threshold (e.g., 3 pN), the domains may be subjected to forces greater than the threshold of the force sensor (e.g., 6 pN), while the sensor remains closed. In order to limit this problem as much as possible, the structure of the protein (when available) or a prediction thereof is used to determine the position at which the force sensor is inserted, which does not destroy the domains of the protein and is not between two domains that interact strongly. However, if it is necessary to insert a force sensor at these positions (e.g., where it is strongly suspected that the domains are subjected to force), it is important not to interpret a negative result as the absence of force. As a control, a force sensor with a different threshold is used and the force is measured at different positions on the protein and on the binding partner.

[0294] Example 3: Intrinsic fluorescence readings of force sensors

[0295] In some data, unfolding of the force sensor within End4p was detected due to aggregation of these open constructs. However, this readout is not always ideal because aggregates of the POI construct may not be easily distinguished from normal POI localization. This readout also lacks spatial and temporal resolution. In addition, force sensors with lower thresholds (<7pN) are not prone to aggregation, most likely because the affinity between α-helices is too low to be conducive to intermolecular binding as a core for aggregation, or the POI into which the coiled-coil sensor has been inserted lacks domains or residues that are prone to phase separation. Therefore, the readout of the force sensor is expanded by introducing a) split-GFP or b) conformation-sensitive fluorescent proteins within the loop of the α-helix connecting the sensor. These direct fluorescence readouts expand the potential applications of the inventor's force sensors by making them easier to use and adding spatial and temporal resolution.

[0296] The "split-GFP strategy" is based on previously engineered GFP so that it is split into two fragments, a short 16-amino acid peptide (GFP11) and the rest of the protein (GFP1-10) that do not fluoresce alone, but can be detected if the two constructs bind to each other ( Fig.16A)(Cabantous, S. et al., 2005, Nat Biotechnol, 23102-107). Therefore, the GFP11 peptide was introduced into the loop of the coiled-coil force sensor. When the force applied to the sensor is below its threshold force, the coiled-coil remains closed and GFP11 cannot approach GFP1-10, so no fluorescence can be detected. If the force is greater than the threshold, the sensor opens and exposes GFP11, which binds to GFP1-10. This strategy is particularly suitable for antiparallel coiled coils ( Fig.16 A).

[0297] As a proof of concept, 3pN GFP 11 The sensor cc-3pN-GFP11 was inserted before or after the THATCH domain into End4p ( Fig.16 A) When the coiled-coil is placed after the THATCH domain, expression of the insert construct and GFP is observed 1-10 The cells did not produce fluorescence, but when cc-3pN-GFP11 was inserted before THATCH, fluorescence was observed ( Fig.16 C).

[0298] The split-GFP system was further investigated in Ent1p, an additional protein involved in force redistribution during clathrin-mediated endocytosis. Three different force sensors (cc-3pN-GFP11, cc-5pN-GFP11, and cc-7pN-GFP11) were inserted into Ent1p. In yeast expressing the cc-5pN-GFP11 and cc-7pN-GFP1 constructs, expression of the constructs in fission yeast cells did not produce fluorescence, indicating that the peak force applied to Ent1p was less than 5pN. However, cells expressing the cc-3pN-GFP11 construct produced visible fluorescence, indicating that the force applied to Ent1p was between 3pN and 5pN ( Fig.17 A to Fig.17 C).

[0299] The second strategy (hereinafter referred to as the "cpFP strategy") is based on circular permutation fluorescent proteins (cpFPs), whose photophysical properties depend on their conformation. cpFP conformational changes modulate their absorption and emission spectra, which can be directly monitored using a fluorescence microscope by measuring the intensity of the emitted fluorescence near its peak wavelength (e.g., 510 nm for cpEGFP) or the ratio of the fluorescence emitted when excited at two absorption peak wavelengths (e.g., 400 nm and 490 nm for cpEGFP). Fig.16 B) cpFP has two ligands on both sides that can bind specific ligands (e.g., Ca 2+, GTP, glutamate, etc.), cpFP has been widely used to develop a variety of genetically encoded biosensors to detect and quantify ions or metabolites in vivo (Germond, A. et al., 2016, Biophys Rev, 8:121-138; Kostyuk, A. et al., 2017, International Journal of Molecular Sciences, 20:4200; Nasu, Y. et al., 2021, Nature Chemical Biology, 17:509-518; Kroning, K. E. et al., 2021, Angewandte Chemie, 133:13470-13477; Sun, F. et al., 2018, Cell, 174:481-496.e19; Patriarchi, T. et al., 2018, Science, 360:eaat4422). Upon binding, the ligand binding domain changes conformation, which then propagates to the cpFP and alters its photophysical properties.

[0300] Using a similar idea, cpFP was introduced into the joint region of the coiled-coil force sensor ( Fig.16 ). When the force is greater than the threshold force, the unfolding of the coiled coil causes a conformational change in the cpFP, which modulates its photophysical properties. This strategy is exemplified in two designs based on: a GCN4-based parallel force sensor and an Oakley antiparallel force sensor. The prototype fluorescent force sensor is inserted before the THATCH actin binding domain of End4p, where, under standard conditions, they are subjected to force and thus unfold. End4p is also labeled with mScarlet-I at its C-terminus to normalize the measurements to differences in expression levels and local concentrations. The same cells were treated with 100 μM red sea sponge A for 40 minutes to terminate actin assembly, thereby terminating the force on End4p, and the force sensor remained folded. This assay allows the determination of the fluorescent properties of sensors that are subjected to high forces or are not subjected to force and can be easily scaled for optimization of sensors. As needed, open coiled coils subjected to high forces are simulated by using variants of these sensors in which all hydrophobic residues at the interface between α-helices are mutated to hydrophilic residues.

[0301] The circular arrangement of green fluorescent protein (cpGFP) of the GCaMP6s calcium sensor is inserted into the linker region between the two α-helices of the force sensor (Kostyuk, AI et al., 2017, International Journal of Molecular Sciences, 20: 4200). This cpGFP has a response amplitude up to 100 times higher than that of previously developed calcium sensors. Since the force sensor has a very simple fold and is based on a previously crystallized peptide, structure prediction software (e.g., Alphafold, RoseTTa, and RaptorX) allows for the rational design of a linker between the α-helix and cpGFP that best keeps the coiled coil and cpGFP in a closed conformation (Jumper, J. et al., 2012, Nature, 596:583-589; Simons, KT et al., 1997, Journal of Molecular Biology, 268:209-225; Baek, M. et al., 2021, Science, 373:871-876; Wang, S. et al., 2017, PLOS Computational Biology, 13:e1005324). Several initial designs were tested to select the best candidate. For each construct, the intensity and ΔF / F were measured using the in vivo assay mentioned in the previous paragraph. min ratio, where ΔF is the difference in fluorescence intensity between the open version of the fluorescent force sensor and the closed version of the fluorescent force sensor and F min For open version fluorescence. The ideal initial design is to make ΔF / F min Between 0.25 and 1 for further optimization (Nasu, Y. et al., 2021, Nature Chemical Biology, 17:509-518).

[0302] From the same screen, constructs with the greatest difference in fluorescence when excited at 400 nm and 490 nm (hereafter referred to as 400 / 490 ratio) were identified ( Fig.16 B). Such ratio sensors have the advantage of not requiring additional fluorescent proteins to standardize concentrations. Typically, ratio sensors are less bright and therefore more difficult to develop and use (Barnett, LM et al., 2017, PLOS ONE, 12: e0170934).

[0303] These constructs were improved by rational selection of residues closest to cpGFP. First, the amino acids in the coiled-coil linker region directly adjacent to the "post" residues of cpGFP at positions 145 and 148 (as numbered in the GFP sequence) were screened (Nasu, Y. et al., 2021, Nature Chemical Biology, 17: 509-518; Kroning, KE et al., 2021, Angewandte Chemie, 133: 13470-13477; Sun, F. et al., 2018, Cell, 174: 481-496.e19; Patriarchi, T. et al., 2018, Science, 360: eaat4422). The amino acids before "post 144" were replaced by each of the other 19 amino acids, and the amino acids with the highest ΔF / F were selected. min (or the optimal 400 / 490 ratio), and repeat the strategy for the amino acids after "after 148". If necessary, repeat the strategy for the next adjacent residue to further optimize the fluorescent sensor.

[0304] A third strategy for studying the forces acting on a protein of interest revolves around the binding of a labeled fluorescent protein to a binding peptide in a linker domain. When sufficient force is applied, the binding peptide is exposed to its surroundings, allowing the labeled fluorescent protein to bind and generate an area of ​​enriched fluorescence ( Fig.17 D). When the force is removed from the protein, the coiled coil reforms and the fluorescence signal decreases. As a proof of concept, a 3pN coiled coil-IAAL-K3 peptide construct was inserted into Ent1p and the fluorescence intensity was monitored over time in yeast cells. mEGFP-IAAL-E3 was recruited to endocytic sites only when the force on Ent1p exceeded 3pN ( Fig.17 E).

[0305] Another strategy to exploit bioluminescence revolves around the use of split luciferase (NanoLuc). The HiBiT linker is inserted between the two α-helices, preventing them from forming the enzymatically active NanoLuc. When the construct is expressed in the presence of LgBiT and sufficient force is applied to the coiled coil, the two domains reassemble into the enzymatically active NanoLuc and catalyze the conversion of furimazine to furimamide, producing detectable luminescence ( Fig.17 F). Insertion of the 3pN coiled-coil HiBiT construct into several Ent1p variants further demonstrated that the minimum force applied to the protein is 3pN ( Fig.17 G).

[0306] Once one GCN4-based force sensor and one Oakley-based force sensor were optimized, it was easy to convert the other force sensors in the library into fluorescent force sensors with little further optimization required, since they are all coiled-coils and have nearly identical structures. Since many force sensors differ from these two coiled-coils by only a few residues, the fluorescent versions are actually optimal if the same cpGFP and linker are used. For the other coiled-coil-based force sensors (Dynein-I, VBP, and Velcro), the same insert linker was used as a starting point, and similar optimization strategies were repeated as needed.

[0307] Previous use of FRET-based force sensors to measure forces on proteins provided candidates for validation of force sensors. To test the full range of forces covered by the force sensor, proteins involved in focal adhesion of mammalian cells were evaluated (forces in the range of 1-10 pN) (Freikamp, ​​A. et al., 2016, Trends in Cell Biology, 26: 838-847; Ringer, P. et al., 2017, Nature Methods, 14: 1090-1096).

[0308] Focal adhesions (FAs) are adhesion complexes of mammalian cells that connect the extracellular matrix (ECM) to the cell's actomyosin cytoskeleton and participate in the transmission of forces between cells and ECM. Fig.18 ). FRET-based force sensors have been used to measure forces on the FA proteins talin and vinculin (in the range of 7-10 pN and 1-6 pN, respectively, when FAs are engaged) (Grashoff, C. et al., 2010, Nature, 466:263-266; Austen, K. et al., 2015, Nature Cell Biology, 17:1597-1606). FAs are therefore an ideal system for: a) validating the calibration of the inventors' force sensor, b) comparing the ease of implementation of the force measurement strategy with strategies that rely on FRET-based sensors, and c) demonstrating that the strategy can be applied to mammalian cells. Plasmids expressing vinculin and talin constructs were modified to replace the FRET sensor with a coiled-coil force sensor with a threshold in the appropriate range ( Fig.18 B), with or without fluorescent reporters. The reduction of forces on talin and vinculin upon disruption of actomyosin contractility using actin depolymerizing drugs (eg, erythromycin A) or myosin inhibitors (eg, blebbistatin) will be confirmed.

[0309] Example 4: Identification of proteins that regulate forces in subcellular processes

[0310] Force sensor panels allow the discovery of proteins that regulate the forces generated in subcellular processes. Once a POI is known to be subject to force, by genetically, chemically, or mechanically perturbing other proteins of the subcellular process, it can be determined whether the force on the POI increases or decreases by monitoring the open or closed state of the force sensors it contains. Fig.19 ).

[0311] This method can be applied to any subcellular process in any organism. Here, to validate the method, the technique was applied to study forces during clathrin-mediated endocytosis (CME) in fission yeast. This system has many advantages: a) almost all CME proteins have been identified, so the screening space is well defined and easily tractable, b) several proteins have been proposed to be involved in force generation or transmission and are good pilots for this method; however, c) only one CME protein has been unequivocally demonstrated to bear force, and e) for all but one CME protein, quantitative measurements or relative contributions are unknown (Lacy, MM et al., 2018, FEBS Letters, 592: 3586-3605; Ren, Y. et al., 2021, bioRxiv, 2021.06.29.450294; Goode, BL et al., 2015, Genetics, 199: 315-58; Abella, M. et al., 2021, Developmental Cell, 56: 2419-2426.e4). This method is also extended to other organisms and other subcellular processes.

[0312] Cells expressing an End4p construct containing a force sensor with a threshold of 11 pN, which aggregates under normal conditions, were treated with the actin polymerization inhibitor limnisin A ( Figure 5 I- Figure 5 K)(Ren, Y. et al., 2021, bioRxiv, 2021.06.29.450294). This treatment restored the aggregation of the End4p construct, indicating that when actin assembly is inhibited, the force on End4p is reduced to less than 11 pN (Ren, Y. et al., 2021, bioRxiv, 2021.06.29.450294). This result confirms the idea that actin assembly is involved in generating the force transmitted by End4p to deform the plasma membrane during CME.

[0313] In another experiment, it was observed that an End4p construct containing a force sensor with a 20 pN threshold near its actin-binding domain, which does not aggregate in the wild type, aggregated after the actin-binding domain of epsin Ent1p was deleted (Ren, Y. et al., 2021, bioRxiv, 2021.06.29.450294). This result demonstrates that Ent1p plays a role in reducing the force transmitted from the actin meshwork to End4p, and confirms the idea that wild-type Ent1p also bears actin forces and shares the load with End4p (Garcia-Alai, MM et al., Nature Communications, 9:328; Skruzny, M. et al., 2012, PNAS, 109:42; Skruzny, M. et al., 2015, Developmental Cell, 33:150-162; Lizarrondo, J. et al., 2021, Nat Commun, 12:2889).

[0314] It is now widely accepted that actin dynamics are important for efficient endocytosis in yeast and mammals, especially when cells are mechanically challenged (high membrane tension or turgor pressure). However, how forces are generated and transmitted during CME remains controversial. The mainstream hypothesis in the field is that actin polymerization around clathrin plaques pushes the tangled actin meshwork inward, resulting in the membrane being pulled into the pits via adaptor proteins. However, the estimated force generated by polymerization is only 1 to 2 orders of magnitude, which is too small (Lacy, MM et al., 2018, FEBS Letters, 592: 3586-3605; a, R. et al., 2021, Biophysical Journal, 120: 1625-1640; Dmitrieff, S. et al., 2015, PLoS ComputBiol, 11: e1004538; Dmitrieff, S. et al., 2016, J Cell Biol, 212: 763-766; Berro, J. et al., 2014, MBoC, 25: 3515-3527; Berro, J. et al., 2010, MBoC, 21: 2905-2915). Therefore, it is necessary to identify other proteins that contribute to force generation. Furthermore, the mechanism of force transmission is more complex than previously thought, as data demonstrate that forces are collected and redistributed by multiple proteins of the endocytic machinery before they are applied to the membrane.

[0315] To identify proteins that regulate the forces applied to the plasma membrane, we expressed End4p constructs containing either an 8 pN or 10 pN force sensor immediately following its ANTH lipid-binding domain (referred to as ANTH-cc8pN -End4p and ANTH-cc 10pN -End4p) cells to determine the effects of complete deletion, domain deletion, or point mutation on CME proteins. 8pN -End4p aggregates in wild-type cells, and if no aggregates are observed when protein X is mutated, protein X impedes force generation or force transmission to the membrane. 10pN - End4p does not aggregate in wild-type cells, if aggregates are observed when protein Y is mutated, then protein Y Enhancement Force generation or transmission ( Fig.19 ).

[0316] A mutant library was constructed using scarless and markerless CRISPR-Cas9 (Fernandez et al., 2016, Yeast, 33(10):549-557). Construction of this strain requires a single simple transformation because cells selected for plasmids expressing Cas9 and guide RNA are likely to eventually contain the expected edits (confirmed by sequencing). Since fission yeast lose plasmids when selection pressure is eliminated, the same protocol and plasmid selection are reused to introduce one of the force sensors in each mutant of the library in one transformation. This step is easily multiplexed because it is the same transformation for all mutants. The initial library focuses on mutating proteins containing actin binding domains or motifs (eliminating actin binding by domain deletion or point mutation). The second focus is on proteins suspected of binding to End4p by domain deletion or point mutation (and therefore good candidates for allocating force from and to End4p). This approach is then extended to other CME proteins to discover new proteins that have not yet been shown to be involved in force generation or transmission during CME.

[0317] Fimbrin is a protein that cross-links actin filaments. It is the second most abundant protein after actin at each site of CME. It is believed that it is subjected to high stress during CME and directly participates in the transmission of force (Berro, J. et al., 2014, MBoC, 25: 3515-3527; Picco, A. et al., 2018, MBoC, 29: 1346-1358; Planade, J. et al., 2019, PLOS Biology, 17: e3000500; Ma, R. et al., 2019, Cytoskeleton, 76: 346-354; Ma, R. et al., 2018, PLOS Computational Biology, 14: e1006150). Therefore, it is an excellent candidate as a reporter of stress generated in the actin meshwork.

[0318] First, a force sensor library was used to determine the tension between the domains of fascin. Fibroin has an EF-hand (EH) domain and two actin-binding domains, ABD1 and ABD2. Previous data suggest that all three domains are involved in actin binding (not just ABD1 and ABD2 as commonly believed). The force sensor library was used with a split-GFP reporter in the ring to measure the force on fascin, because the force on fascin is likely to be less than 8pN.

[0319] The generated mutant library was then used to introduce force sensors with thresholds slightly below and slightly above the measured force, thereby determining in which mutant the stress on the filamentous protein was increased or decreased. A set of proteins was identified that partially overlapped with the proteins identified above. Proteins present only in the above dataset correspond to proteins that transmit force from the actin meshwork to the membrane, and proteins present in both datasets correspond to proteins directly involved in force generation or regulators upstream of them. Proteins identified only in this dataset correspond to proteins involved in actin stress that is not transmitted to the membrane by End4p, and therefore point to other pathways of force transmission to the membrane or other mechanisms that do not conform to the "push-pull" paradigm of force generation during CME.

[0320] Example 5: Mapping the forces exerted on proteins during subcellular processes

[0321] After the weakest force sensor (~3 pN) is inserted into the POI, if a change in the reporter state is detected (aggregation, appearance of GFP signal, or change in the photophysical properties of the reporter), Figure 1-5 , Fig. 9 and Fig.16 ), then determine if the POI is under force. Using a force sensor that is always folded or has a maximum force threshold allows verification that: a) insertion of a folded coiled-coil at the chosen site is harmless, and b) the change in the reporter state is indeed due to force ( Fig.15 and Fig. 20 By systematically using this pair of force sensors on a set of POIs, proteins that are subject to forces during their normal cellular function are identified. In addition, the location of the force exerted on the POI is determined by systematically introducing these two force sensors between consecutive domains of the POI ( Fig. 20 ), and by testing force sensors with increasing force thresholds to measure the magnitude of force with high accuracy ( Fig.15 & Fig. 20 ).

[0322] Methods for determining which proteins in subcellular processes are subject to forces and determining the location and magnitude of those forces are universal, but CME was chosen here because enough is known to allow predictive examples that are likely to work, and there is still much unknown about the forces in the process for which this discovery is influential.

[0323] Many proteins identified in the first two data sets are force-bearing, especially proteins that are identified as regulators of force on End4p rather than on Fim1p. Therefore, this strategy focuses on these proteins and extends to other proteins suspected of being stressed because they contain known actin or membrane-bound domains or are known or presumed interactors of these proteins. For each candidate, a test sensor (e.g., 3pN threshold, using split-GFP reporter) and a control sensor (e.g., non-openable or 20pN threshold) are introduced between two continuous domains. Domains are identified from database annotations (e.g., Uniprot or Pombase) from disclosed (PDB) or predicted (AlphaFold) structures. Control sensors allow determination of whether the insertion site is harmless to the localization of the protein, which is similar to the wild type, and does not disrupt endocytosis, as measured by FM4-64 dye internalization. Insertion sites within the predicted unstructured region are selected and regions containing known interaction motifs or residues that are prone to post-translational modification are avoided. Initially, sensors were not inserted into long intrinsically disordered regions because they potentially mediate phase separation, but forces within these regions were probed in subsequent studies when relevant. Sites adjacent to actin or membrane binding domains were investigated first. When the protein does experience forces, this strategy and inserted sensors between consecutive domains were used to localize forces on the protein.

[0324] Once force is confirmed to be exerted at a certain location on the protein, a collection of force sensors with split-GFP reporters are used to accurately measure the forces at these locations. Forces on the identified proteins are systematically measured to map the forces within the CME mechanism. Proteins with actin or membrane binding domains are prioritized, followed by known or putative partners of these proteins. The same strategy as above is used to narrow down the role of protein domains in force transmission and identify or confirm mechanical relationships between CME partners. Finally, force sensors with cpGFP reporters are used to improve measurements of selected proteins to determine at which stage CME forces of different magnitudes are generated.

[0325] Example 6: Application in multicellular organisms

[0326] The use of force sensors in single-cell organisms such as yeast provides insight into certain cellular mechanisms, but does not shed significant light on how such forces affect development in humans or other multicellular organisms. Therefore, the developed force sensor was used to conduct experiments in a species of nematode worm, Caenorhabditis elegans. As a transparent multicellular organism, C. elegans is an excellent model for optical force sensors. C. elegans also has the advantage of having one of the most in-depth developmental pathways.

[0327] The C. elegans UNC-70 protein, encoded by the unc-70 gene, was chosen as the first example because of its homology to human spectrin β. To investigate the forces acting on UNC-70, a variety of strains with various insertions between 1166R and 1167D were obtained (Table 1). The strains were grown to the L4 larval stage, at which time they were mounted on 10% agarose patches and paralyzed in a drop of 10 mM levamisole diluted in M9 medium for imaging ( Fig.21 Of particular interest in UNC-70 expression in C. elegans is the association with the membrane-associated periodic cytoskeleton (MPS / spectrin lattice, Fig.21 B) Related posterolateral microtubule cells (PLM neurons, Fig.21 A), which is involved in the sensation of force and tactile response on the body. GFP was observed in both wild-type and unc-115(ko) backgrounds. 11 Expression of the construct resulted in minimal GFP fluorescence. In contrast, animals of the wild-type background expressing the 3pN sensor construct exhibited significant GFP fluorescence, while those expressing the 10pN construct produced almost no fluorescence, indicating that UNC-70 is subjected to forces between 3pN and 10pN in the MPS. It is interesting to observe that unc-115(ko) animals expressing the 3pN sensor construct do not exhibit an increase in fluorescence. Since the UNC-115 protein is required for actin binding and periodic MPS stability, in the absence of the UNC-115 protein, no force is acting on UNC-70, resulting in a lack of fluorescence.

[0328] Table 1: Caenorhabditis elegans force sensor strains

[0329]

[0330] Microscopic examination

[0331] Images were acquired with a DMi8 inverted microscope (Leica) equipped with a VT-iSIM system (Biovision) and an ORCA-Flash 4.0 camera (Hamamatsu). The microscope was controlled by MetaMorph advanced confocal acquisition software. Images were acquired at 488nm laser line with an HC PL APO 40x / 1.30NAOIL CS2 objective. The original images were processed and analyzed in Fiji / ImageJv2.3.0 / 1.53f51 (Schneider, CA et al., 2012, Nat Methods, 9, 671-675; Schindelin, J. et al., 2012, Nat Methods, 9, 676-682). Images were acquired in a single layer and then stacked as maximum projections. In order to capture the intensity signal along the entire length of the PLM, which may not be available in a single field of view, multiple images along the length of the neurite were taken and stitched into a single image using a linear blending fusion method using the pairwise stitching plugin (Preibisch, S. et al., 2009, Bioinformatics, 25, 1463-1465).

[0332] Fluorescence quantification

[0333] To determine the average fluorescence along the PLM neurite, a 5 pxl thick line was drawn along the center of the neurite, and a signal intensity profile was generated using the plot profile function, and the signal intensities were averaged. To subtract background fluorescence, the same signal intensity profile was obtained by moving the drawn line from the center of the neurite to the non-neuronal tissue that directly contacts the neurite by a few pxl. Signal intensity was calculated in arbitrary units as I 平均 =I 平均 (Neurite)-I 平均 (background).

[0334] Statistical evaluation

[0335] Statistical evaluation was performed using GraphPad Prism (version 7). The data set was first tested for normality distribution by using D'Agostino and Pearson tests to determine whether to use nonparametric or parametric statistical tests.

[0336] Example 7: Sequence of force sensor protein

[0337] The sequences of exemplary force-sensing peptides are provided below along with their unfolding force thresholds (if available). Linkers were added before and after each coiled coil to allow for changes in coiled coil orientation after insertion into the target protein. All linkers (before, between, and after the α-helix) are underlined. The α-helix is ​​shown in bold.

[0338] 1. α-helical sequence:

[0339]

[0340]

[0341]

[0342] 2. Linker sequence:

[0343] SEQ ID NO:41- GGGGGGATNFSLLKLAGDVELNAGAGGGGG

[0344] SEQ ID NO:42- GGGGGGGGGGGATNFSLLKLAGDVELNAGAGGGGGGGGGG

[0345] SEQ ID NO:43- GGGSGGGSGGGSGGGSGGGSGATNFSLLKLAGDVELNAGAGGGSGGGSGGGSGGG SGGGS

[0346] SEQ ID NO:44- GGSSGG

[0347] SEQ ID NO:45- GGGGG

[0348] 3. Functional connector:

[0349] SEQ ID NO:46-GFP11: reassembled with GFP1-10 to have fluorescence.

[0350] GGSRDHMVLHEYVNAAGITSGG

[0351] SEQ ID NO:47-FIB1: Increased protein aggregate size through intrinsically disordered sequence.

[0352] MAYTPGSRGGRGGSRGGRGGFNGGRGGFGGGRGGARGGGRGGARGGRGGRGGARGGRGGSSGGRGGAKG G

[0353] SEQ ID NO:48—Short linker (for parallel coiled-coils): increases protein aggregate size by forcing coiled-coils to open.

[0354] GGGGGSSGGGGG

[0355] SEQ ID NO:49—Rigid linker (for antiparallel coiled-coils): increases protein aggregate size by forcing coiled-coils to open.

[0356] GGRQLLAEKRELEEKKRREEEKKREEEERERERAQRGG

[0357] SEQ ID NO:50-IAAK-K3: for reversible binding with IAAK-E3

[0358] GGKIAALKEKIAALKEKIAALKEGG

[0359] SEQ ID NO:51-IAAK-E3: for reversible binding with IAAK-K3

[0360] EIAALEKEIAALEKEIAALEK

[0361] SEQ ID NO:52—HiBiT: reassembles with LgBit to emit light.

[0362] GGVSGWRLFKKISGG

[0363] SEQ ID NO:53—SsrA: for reversible binding to SspB.

[0364] AANDENY

[0365] SEQ ID NO:54—TRAP Peptide 1: for reversible binding to different TRAP proteins (eg, TRAP2 and TRAP4) with different affinities.

[0366] MEEVF

[0367] SEQ ID NO:55—TRAP Peptide 2: for reversible binding to different TRAP proteins (eg, TRAP2 and TRAP4) with different affinities.

[0368] MEEVW

[0369] SEQ ID NO:56—TEV linker: for recognition by TEV protease for cleavage.

[0370] ENLYFQS

[0371] 4. GCN4 series (parallel coiled coil; modified from natural coiled coil):

[0372] SEQ ID NO:57-GCN4-pIL-I16N-5E3R-2A-Mut-GCN4-pIL-I16N-5R3E(pJB289)

[0373] Unfolding force threshold: 11pN

[0374]

[0375] Unfolding force threshold: 8pN

[0376]

[0377] Unfolding force threshold: 6 / 9 / 14pN

[0378]

[0379] SEQ ID NO:62-GCN4-p1-5E3R-2A-Mut-GCN4-p1-5R3E (pJB296) unfolding force threshold: 6.5 pN

[0380]

[0381]

[0382] SEQ ID NO:65-GCN4-pIL-5E3R-2A_Mut-GCN4-pIL-5R3E(pJB329)

[0383] Unfolding force threshold: 13.9±0.4pN

[0384]

[0385] 5. pER series (parallel coiled coil; modified from natural coiled coil):

[0386] SEQ ID NO:66-pER-E-GGGS-pER-R(pJB325)

[0387] Unfolding force threshold: 20pN

[0388]

[0389] SEQ ID NO:67-pER-E-GGGS-pER-R-LA(pJB330)

[0390] Unfolding force threshold: 17.5±0.7pN

[0391]

[0392] Velcro series (parallel curled spiral; artificial):

[0393] SEQ ID NO:68-Velcro-p1(pJB321)

[0394] Unfolding force threshold: 7.5±0.3pN

[0395]

[0396] 6. Oakley series (antiparallel coiled coil; artificial): SEQ ID NO: 71-Oakley-p1 (pJB297)

[0397] Unfolding force threshold: 7.3±0.8pN

[0398]

[0399] Unfolding force threshold: 9.95±0.31pN

[0400]

[0401] Unfolding force threshold: 7.5±0.2pN

[0402]

[0403] 7. Dynein stem series (antiparallel coiled coil; modified from native coiled coil): SEQ ID NO: 78-Dynein stem-p1 (pJB158)

[0404] Unfolding force threshold: 3pN

[0405]

[0406] 8. Caenorhabditis elegans sensor (A* indicates silent mutation):

[0407]

[0408]

[0409] SEQ ID NO:80-10pN GFP sensor

[0410] GGGG AQLEKELQALEKKLAQLEWELQALEKENAQLEKELQA GGSRDHMVLHEYVNAAGITSGG QALKKKLAQNKKKLQALKKELAQLKWKLQALKKKLAQ GGGG

[0411] SEQ ID NO:81-GFP 11 Insert UNC-70

[0412] AACTTCTCAATCAACACGCTGCCATCCGT ggtggatcacgagatcacatggttctgcacgagtacgttaacgccgcgggaatcacc tcgggcgga GAAGAA*ATTGACGGATACGCTGAGGATTACAAGAAGATGCGTGCAATGGGAGATCGTGTCAC

[0413] SEQ ID NO:82-3pN GFP sensor inserted into unc-70

[0414]

[0415] SEQ ID NO:83-10 pN GFP sensor inserted into unc-70

[0416]

[0417] 9. Binding partner of the sensor

[0418] SEQ ID NO:84-GFP1-10: Assembles with GFP11 to form fluorescent GFP.

[0419] SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYK TRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK

[0420] SEQ ID NO:85 - LgBiT: Assembles with HiBiT or SmBiT to form catalytically active NanoLuc.

[0421] MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINS

[0422] SEQ ID NO:86—TRAP 1: reversibly binds to TRAP peptides (eg, MEEVF and MEEVW).

[0423] MSYYHHHHHHDYDIPTTENLYFQGSKQALKEKELGNDAYKKKDFDTALKHYDKAKELDPTNMYILNQAAVYFEKGDYNKCRELCEKAIEVGRENREDYRLIAIAYARIGNSYFKEEKYKDAIHFYNKSLAEHRTPKVLKKCQQAEKILKEQ

[0424] SEQ ID NO:87 - TRAP 2: Reversibly binds to TRAP peptides (e.g., MEEVF and MEEVW). MSYYHHHHHHDYDIPTTENLYFQGSKQALKEKELGNDAYKKKDFDTALKHYDKAKELDPTNMYIMNQAAVYFEKGDYNKCRELCEKAIEVGRENREDYRMIAYAYARIGNSYFKEEKYKDAIHFYNKSLAEHRTPKVLKKCQQAEKILKEQ

[0425] SEQ ID NO:88 - TRAP 3: Reversibly binds to TRAP peptides (e.g., MEEVF and MEEVW). MSYYHHHHHHDYDIPTTENLYFQGSKQALKEKELGNDAYKKKDFDTALKHYDKAKELDPTNMYIMNQAAVYFEKGDYNKCRELCEKAIEVGRENREDYRMIAYAYADIGDSYFKEEKYKDAIHFYNKSLAEHRTPKVLKKCQQAEKILKEQ

[0426] SEQ ID NO:89 - TRAP4: Reversibly binds to TRAP peptides (e.g., MEEVF and MEEVW).MKQALKEKELGNDAYKKKDFDTALKHYDKAKELDPTNMTYIINQAAVYFEKGDYNKCRELCEKAIEVGRENREDYRWIAIAYARIGNSYFKEEKYKDAIHFYNKSLAEHRTPKVLKKCQQAEKILKEQ

[0427] SEQ ID NO:90—SspB: reversibly binds to SsrA.

[0428] SQLTPRRPYLLRAFYEWLLDNQLTPHLVVDVTLPGVQVPMEYARDGQIVLNIAPRAVGNLELANDEVRFNARFGGIPRQVSVPLAAVLAIYARENGAGTMFEPEAAYD

[0429] The disclosure of each patent, patent application and publication cited herein is hereby incorporated by reference in its entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the present invention may be designed by other skilled in the art without departing from the true spirit and scope of the present invention. The appended claims are intended to be interpreted as including all such embodiments and equivalent variations.

Claims

1. A force sensing peptide comprising two α-helical domains connected by a linker sequence, wherein: In the absence of external force, the two α-helical domains form a coiled-coil domain, referred to as a closed conformation, and further, wherein, in the presence of a force above a threshold level for uncoiling of the coiled-coil domain, the coiled-coil domain undergoes a conformational change to an open conformation.

2. The force-sensing peptide of claim 1, wherein the force-sensing peptide is operably linked to a target molecule of interest, such that when a force is applied to the target molecule of interest, the force-sensing peptide undergoes the conformational change.

3. The force-sensing peptide according to claim 1 or 2, wherein the linker sequence comprises at least one functional domain or portion for generating a signal when the force-sensing peptide is in an open conformation.

4. The force sensing peptide of claim 3, wherein at least one functional domain or portion is selected from the group consisting of: a fluorescent protein or a fragment thereof, a cyclic fluorescent protein, a protein cleavage domain, an RNA binding molecule, a DNA binding domain, an epitope for recognition by a binding molecule, an ion binding domain, a lipid binding domain, a peptide comprising residues for post-translational modification, a peptide comprising unnatural amino acids, a peptide that is a toxin to cells, a peptide with antibacterial activity, a peptide with antiviral activity, a peptide with antifungal activity, a peptide with enzymatic activity, a peptide that modifies the enzymatic activity of other proteins, a peptide comprising a localization signal for a cellular compartment, a peptide comprising a secretion signal, and a peptide that binds to another peptide or protein construct.

5. The force-sensing peptide of any one of claims 1-4, wherein the coiled-coil domain has an unwinding threshold level of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or greater than 25 piconewtons (pN).

6. The force-sensing peptide according to any one of claims 1-5, wherein the peptide comprises at least one α-helical domain selected from the group consisting of SEQ ID NO: 1-SEQ ID NO:

40.

7. The force-sensing peptide according to any one of claims 1 to 6, wherein the peptide comprises at least one set of α-helical domains selected from the group consisting of: a) SEQ ID NO: 1 and SEQ ID NO: 21; b) SEQ ID NO:2 and SEQ ID NO:21; c) SEQ ID NO: 2 and SEQ ID NO: 22; d) SEQ ID NO:3 and SEQ ID NO:23; e) SEQ ID NO:4 and SEQ ID NO:23; f) SEQ ID NO:4 and SEQ ID NO:24; g) SEQ ID NO:5 and SEQ ID NO:25; h) SEQ ID NO:6 and SEQ ID NO:26; i) SEQ ID NO:7 and SEQ ID NO:27; j) SEQ ID NO: 8 and SEQ ID NO: 28; k) SEQ ID NO:9 and SEQ ID NO:29; 1) SEQ ID NO: 10 and SEQ ID NO: 30; m) SEQ ID NO:11 and SEQ ID NO:31; n) SEQ ID NO:12 and SEQ ID NO:32; o) SEQ ID NO: 13 and SEQ ID NO: 33; p) SEQ ID NO: 14 and SEQ ID NO: 34; q) SEQ ID NO:15 and SEQ ID NO:35; r) SEQ ID NO:16 and SEQ ID NO:36; s) SEQ ID NO:17 and SEQ ID NO:37; t) SEQ ID NO: 18 and SEQ ID NO: 38; u) SEQ ID NO: 19 and SEQ ID NO: 39; and v) SEQ ID NO: 20 and SEQ ID NO: 40, wherein the set of α-helical domains are connected by a linker sequence.

8. The force-sensing peptide according to any one of claims 1-7, wherein the peptide comprises at least one linker sequence selected from the group consisting of SEQ ID NO:41-SEQ ID NO:

56.

9. The force-sensing peptide of any one of claims 1-8, wherein the peptide comprises a sequence selected from the group consisting of SEQ ID NO: 57-SEQ ID NO:

80.

10. The force sensing peptide of any one of claims 1-9, wherein the target molecule of interest is selected from the group consisting of a detectable moiety, a fluorescent protein, a purification tag, a targeting domain, a cellular localization signal, a DNA molecule, an RNA molecule, cAMP, cGMP, eicosapentaenoic acid, eicosatetraenoic acid, phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, an aminomethyl polystyrene linker, a chloromethyl polystyrene linker, a PEG linker, a solid phase protein synthesis resin, a C-terminal fragment of End4p, a C-terminal fragment of actin, a C-terminal fragment of clathrin, a C-terminal fragment of focal adhesion protein, a C-terminal fragment of talin, a C-terminal fragment of integrin, glycogen, cellulose, a therapeutic agent, an antibiotic, an antiviral agent, an antifungal agent, an antihelmintic agent, an anti-inflammatory molecule, and a chemotherapeutic agent.

11. A system for determining the level of force exerted on a target of interest, the system comprising at least two force-sensing peptides according to any one of claims 1 to 10, wherein the at least two force-sensing peptides have different unwinding threshold levels.

12. A nucleic acid molecule encoding the force-sensing peptide according to any one of claims 1 to 10.

13. A genetically modified host cell comprising the nucleic acid molecule according to claim 12.

14. A method for detecting a force applied to a target molecule of interest, the method comprising contacting a target molecule of interest operably linked to a force-sensing peptide according to any one of claims 1 to 10 with a sufficient level of force to induce a conformational change in the force-sensing peptide, and detecting the presence of an open conformation of the force-sensing peptide.

15. The method of claim 14, wherein the force-sensing peptide comprises a linker sequence comprising at least one functional domain or portion for generating a signal when the force-sensing peptide is in an open conformation.

16. The method of claim 15, wherein at least one functional domain or portion is selected from the group consisting of: a fragment of a fluorescent protein, a bioluminescent protein, a cyclic fluorescent protein, a protein cleavage domain, an RNA binding molecule, a DNA binding domain, an epitope for recognition by a binding molecule, an ion binding domain, a lipid binding domain, a peptide comprising residues for post-translational modification, a peptide comprising unnatural amino acids, a peptide that is a toxin to cells, a peptide with antibacterial activity, a peptide with antiviral activity, a peptide with antifungal activity, a peptide with enzymatic activity, a peptide that modifies the enzymatic activity of other proteins, a peptide comprising a localization signal for a cellular compartment, and a peptide comprising a secretion signal.

17. The method of claim 15 or 16, wherein the detection method comprises detecting a fluorescent signal generated in the presence of the open conformation of the force-sensing peptide.

18. The method of claim 15 or 16, wherein the detection method comprises detecting a bioluminescent signal generated in the presence of the open conformation of the force-sensing peptide.

19. The method of claim 15 or 16, wherein the detection method comprises detecting differential levels of protein or mRNA molecules when the force sensing peptide is in the open conformation.

20. The method of claim 15 or 16, wherein the detection method comprises detecting a change in the localization of the target molecule due to cleavage of a peptide cleavage domain exposed when the force sensing peptide is in the open conformation.

21. The method of any one of claims 14-20, wherein the peptide comprises at least one alpha-helical domain selected from the group consisting of SEQ ID NO: 1-SEQ ID NO:

40.

22. The method of any one of claims 14-21, wherein the peptide comprises at least one set of alpha-helical domains selected from the group consisting of: a) SEQ ID NO: 1 and SEQ ID NO: 21; b) SEQ ID NO:2 and SEQ ID NO:21; c) SEQ ID NO: 2 and SEQ ID NO: 22; d) SEQ ID NO:3 and SEQ ID NO:23; e) SEQ ID NO:4 and SEQ ID NO:23; f) SEQ ID NO:4 and SEQ ID NO:24; g) SEQ ID NO:5 and SEQ ID NO:25; h) SEQ ID NO:6 and SEQ ID NO:26; i) SEQ ID NO:7 and SEQ ID NO:27; j) SEQ ID NO: 8 and SEQ ID NO: 28; k) SEQ ID NO:9 and SEQ ID NO:29; 1) SEQ ID NO: 10 and SEQ ID NO: 30; m) SEQ ID NO:11 and SEQ ID NO:31; n) SEQ ID NO:12 and SEQ ID NO:32; o) SEQ ID NO: 13 and SEQ ID NO: 33; p) SEQ ID NO: 14 and SEQ ID NO: 34; q) SEQ ID NO:15 and SEQ ID NO:35; r) SEQ ID NO:16 and SEQ ID NO:36; s) SEQ ID NO:17 and SEQ ID NO:37; t) SEQ ID NO: 18 and SEQ ID NO: 38; u) SEQ ID NO: 19 and SEQ ID NO: 39; and v) SEQ ID NO: 20 and SEQ ID NO: 40, wherein the set of α-helical domains are connected by a linker sequence.

23. The method of any one of claims 14-22, wherein the peptide comprises at least one linker sequence selected from the group consisting of SEQ ID NO:41-SEQ ID NO:

56.

24. The method according to any one of claims 14-23, wherein the peptide comprises at least one sequence selected from the group consisting of SEQ ID NO: 57-SEQ ID NO: 80.

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