Intermolecular structural domain tension probe and application

Abstract

The invention provides construction and application of an intermolecular structural domain tension probe, the probe comprises a first structure, a second structure and a fluorescent protein pair eCFP and eYFP, the first structure and the second structure are respectively connected to two ends of the fluorescent protein pair, and the eCFP and the eYFP are connected through a short peptide; the first structure is a structural domain or protein capable of being combined with a cytoskeleton connecting protein, and the second structure is a structural domain or protein capable of being combined with a cytoskeleton; the cytoskeleton connecting protein is selected from talin, SYNE4, SYNE3, SYNE2, Desmoplakin and the like; the cytoskeleton is a microfilament, a microtubule or an intermediate filament. The intermolecular structural domain tension probe can be used for detecting intracellular mechanical signals, the limitation of a traditional tension probe is overcome, the adverse effect of function enhancement caused by gene cloning is avoided, a new opinion is provided for a mechanical transduction molecular mechanism, and the application has the advantages that the application range is wide, and the application prospect is wide. And a more accurate experimental tool is provided for the research of cell mechanics biology, and the effect of a mechanical signal in cell behavior regulation and control is disclosed.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and biotechnology, and relates to intermolecular domain tension probes for monitoring intracellular mechanical signals and effects. Background Technology

[0002] Protein-protein interactions (PPIs) play a crucial role in the regulation of cellular function. Research on PPIs not only reveals the biological functions of proteins but also provides a theoretical basis for new drug development, disease diagnosis, and treatment. In recent years, PPI research has moved beyond static protein interactions, increasingly focusing on their dynamic characteristics, particularly their role in the intracellular mechanical environment. PPIs regulate cellular morphology, polarization, adhesion, migration, and other biological processes through their interaction with mechanical signals. Intracellular and extracellular signal transduction networks are highly dependent on the formation and regulation of PPIs. Many key physiological and pathological processes involve complex PPI mechanisms. However, the dynamic nature and diversity of PPIs make studying their mechanisms exceptionally complex. The intracellular mechanical environment, in particular, plays a vital role in the occurrence of PPIs. The interplay of mechanical signals with chemical and electrical signals allows cells to adjust their biological behavior in response to various external stimuli, thereby influencing organ development, tissue repair, and pathological changes. Therefore, elucidating the role of PPIs in mechanical signals and their effects is crucial for understanding the mechanisms by which cells respond to the external physical environment.

[0003] While traditional experimental techniques such as co-immunoprecipitation (Co-IP), mass spectrometry, and two-hybrid assays have provided valuable technical support for the discovery of intracellular protein-protein interactions (PPIs), these methods are often limited by experimental conditions and cannot achieve real-time monitoring of dynamic PPIs in living cells. Traditional methods for studying intracellular mechanical signals, such as the soft-hard matrix method, traction microscopy, and atomic force microscopy, cannot detect real-time mechanical changes within living cells and lack sufficient resolution. To address the challenge of detecting mechanical signals between PPIs in living cells, fluorescence resonance energy transfer (FRET)-based biosensors have gradually become a key research focus. The core advantage of FRET probes lies in their ability to monitor protein-protein binding and its dynamic changes in living cells in real time, providing a novel perspective on the dynamic changes of intracellular PPIs. However, traditional distance-dependent whole-genome FRET probes still have certain limitations in studying protein-protein interactions, especially under the influence of protein orientation and dynamic rearrangement, which may lead to signal misinterpretation and biased results.

[0004] Talin is a typical cytoskeleton-associated protein, and its role in cellular mechanotransduction has attracted much attention. As a bridge between transmembrane integrins and the cellular microfilament cytoskeleton, Talin is not only responsible for signal transduction but also plays a crucial role in cell morphology, migration, and mechanotransduction. Traditional Talin-M tension probes insert a highly fluorescent donor eCFP and a receptor eYFP linked by a 7-amino acid peptide between the Talin-Head and Talin-Rod. When the target protein is mechanically stretched, the probe increases the fluorescent protein-to-protein (FRET) angle with increasing tension; the magnitude of the tension is inversely proportional to its FRET efficiency. Compared to traditional distance-dependent FRET probes, Talin-M probes, as representatives of angle-dependent FRET probes, have made some progress in mechanotransduction studies. However, overexpression of Talin-M probes may cause unwanted changes in cellular function, such as overactivation of integrin signaling, alteration of the original structure and function of Talin protein, and impact on cellular behaviors such as cell adhesion, migration, invasion, and polarization. These issues limit the use of whole-genome Talin-M probes under certain conditions.

[0005] To address this issue, this application proposes an intermolecular structural domain tension probe for monitoring intracellular mechanical signals. Summary of the Invention

[0006] To overcome the problems of existing technologies, this invention provides an intermolecular structural domain tension probe for monitoring intracellular mechanical signals. By reducing the overexpression effect, it can monitor the intracellular mechanical signal transduction process in living cells in real time, rather than simply enhancing or altering protein function, thus avoiding excessive changes in cell function.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] (I) This invention provides an intermolecular domain tension probe, the probe comprising: a first structure, a second structure, and a fluorescent protein pair eCFP and eYFP, wherein the first structure and the second structure are respectively connected to the two ends of the fluorescent protein pair, and the eCFP and eYFP are connected by a short peptide; the first structure is a domain or protein capable of binding to a cytoskeleton linker protein, and the second structure is a domain or protein capable of binding to the cytoskeleton; the cytoskeleton linker protein is talin, SYNE4, SYNE3, SYNE2, or Desmoplakin; the cytoskeleton is a microfilament, microtubule, or intermediate filament.

[0009] Furthermore, the eCFP and eYFP are linked by a short peptide to form an α-helix, and the amino acid sequence of the short peptide is GGGPGGG.

[0010] Furthermore, the cytoskeleton linker protein is talin, and the cytoskeleton is the microfilament molecule β-actin; that is, the probe is a Talin-microfilament intermolecular domain tension probe. The first structure is the EF domain of α-actinin, and the second structure is the SH3 domain of srGAP2, the PDZ domain of ZO1, the LIM domain of Zyxin, or the FERM domain of Ezrin. Four Talin-microfilament intermolecular domain tension probes are used: EF-cpstFRET-SH3, EF-cpstFRET-PDZ, EF-cpstFRET-LIM, and EF-cpstFRET-FERM, preferably EF-cpstFRET-LIM.

[0011] Furthermore, the amino acid sites in β-actin that bind to the srGAP2-SH3 domain are one or more of the following: 133aa, 139aa, 143aa, 148aa, 166–170aa, 288–289aa, and 345–375aa (tail); the amino acid sites in β-actin that bind to the Ezrin-FERM domain are: 30–67aa (head), 83–99aa, 125–127aa, 202–208aa, and 240–245aa. One or more of the following amino acid sites in β-actin are: 110aa, 113aa, 116aa, 133aa, 148–149aa, 166–175aa, 284–292aa, and 346–375aa (tail); one or more of the following amino acid sites in β-actin are: 37–68aa (head), 194–208aa, and 243–244aa.

[0012] Furthermore, the β-actin site sequences (345–375aa), (30–67aa), (346–375aa), and (37–68aa) are shown in SEQ ID NO:1–4, respectively.

[0013] Furthermore, the cytoskeleton linker protein is SYNE4, and the cytoskeleton is composed of microtubule molecules α-tubulin or β-tubulin; that is, the probe is a SYNE4-microtubule intermolecular domain tension probe. The first structure is the 3NF1 or 5OJ8 domain of KLC1 (Kinesinlight chain 1), and the second structure is the CAP-Gly1 or CAP-Gly2 domain of Clip-170 (CAP-Gly domain-containing linker protein 170). Specifically, this is represented as follows:

[0014] SYNE4~①:::3NF1(KLC1)~cpstFRET~CAP~Gly1(Clip170):::③~α, βtubulin;

[0015] SYNE4~②:::5OJ8(KLC1)~cpstFRET~CAP~Gly1(Clip170):::③~α, βtubulin;

[0016] SYNE4~②:::5OJ8(KLC1)~cpstFRET~CAP~Gly2(Clip170):::④~α, βtubulin.

[0017] Furthermore, the amino acid sites in SYNE4 that bind to the 3NF1 (KLC1) domain are one or more of the following: 181aa, 185aa, 187–188aa, 191–192aa, 195aa, 198–199aa, 202–203aa, 206aa, 248–249aa, 251aa, 344–376aa, 388aa, 391aa, and 393aa; SYNE The amino acid sites that bind to the 5OJ8(KLC1) domain in the four groups are: 187–188 aa, 191–192 aa, 195 aa, 198–199 aa, 202–203 aa, 206 aa, 209–210 aa, 213 aa, 243–245 aa, 247–249 aa, 251–252 aa, 255 aa, 283–286 aa, 347–374 aa, 386– One or more of 391aa and 393aa; the sites where αtubulin binds to the CAP-Gly1 (Clip170) domain are one or more of: 11aa, 15aa, 71aa, 73aa, 98aa, 100–102aa, 105aa, 176–182aa, 210aa, 214aa, 220–224aa, and 403–408aa; αtubulin binding The sites of the CAP-Gly2 (Clip170) domain are one or more of the following: 158-159aa, 162-166aa, 192aa, 195-199aa, 253aa, 253-266aa, 313-314aa, 346-350aa, 414aa, 417aa, 423-424aa, 434-435, 437-439aa, and 442-443aa.

[0018] Furthermore, the SYNE4 site sequence (344–376aa), SYNE4 site sequence (347–374aa), αtubulin site sequence (176–182aa), and αtubulin site sequence (253–266aa) are shown in SEQ ID NO:5–8, respectively.

[0019] Furthermore, the cytoskeleton linker protein is SYNE2, and the cytoskeleton is the microfilament molecule β-actin; that is, the probe is a SYNE2-microfilament intermolecular domain tension probe. The first structure is the GBD domain of FHOD1 (FH1 / FH2 domain-containing protein 1), and the second structure is the ABS domain of FHOD1 (FH1 / FH2 domain-containing protein 1) or the ABD domain of α-actinin. Specifically, as follows:

[0020] SYNE2~①:::GBD(FHOD1)~cpstFRET~ABS(FHOD1):::②~β~actin;

[0021] SYNE2~①:::GBD(FHOD1)~cpstFRET~ABD(actinin):::③~β~actin.

[0022] Furthermore, the sites where SYNE2 binds to the GBD(FHOD1) domain are 1340–1678 aa. The sites where β-actin binds to the ABS(FHOD1) domain are one or more of the following: 22–25 aa, 28 aa, 59–62 aa, 143–148 aa, 167–169 aa, 207–208 aa, 210–211 aa, 215 aa, 238–243 aa, 332–355 aa (tail), and 375 aa. The sites where β-actin binds to the ABD (actinin) domain are one or more of the following: 2–6 aa, 21–32 aa, 44–61 aa (head), 83–101 aa, 126 aa, 130 aa, 215 aa, 218 aa, 235–236 aa, 238 aa, 254 aa, 305 aa, 307–308 aa, 311 aa, 333–337 aa, and 341 aa.

[0023] Furthermore, the SYNE2 site sequence (1340–1678aa), the β-actin site sequence (332–355aa), and the β-actin site sequence (44–61aa) are shown in SEQ ID NO:9–11, respectively.

[0024] Furthermore, the cytoskeleton linker protein is SYNE3, and the cytoskeleton is the intermediate filament molecule Vimentin or Keratin14, meaning the probe is a SYNE3-intermediate filament intermolecular tension probe; the first structure is the ABD domain of Plectin, the ABD domain of MACF1 (Microtubule-actin cross-linking factor 1), or the ABD domain of DYST (Dystonin); the second structure is the filament-head of Desmin or the binding region of TRADD (Tumornecrosis factor receptor type 1-associated DEATH domain protein). Specifically, this is represented as follows:

[0025] SYNE3~①:::ABD(Plectin)~cpstFRET~Filament-head(Desmin):::②~Vimentin;

[0026] SYNE3~③:::ABD(MACF1)~cpstFRET~Filament-head(Desmin):::②~Vimentin;

[0027] SYNE3~④:::ABD(DYST)~cpstFRET~Filament-head(Desmin:::②~Vimentin;

[0028] SYNE3~⑤:::ABD(Plectin)~cpstFRET~region(TRADD):::⑥~Keratin14.

[0029] Furthermore, the SYNE3 site sequence (220–325aa) binds to the ABD (FHOD1, MACF1, or DYST) domain at spectrin1, as shown in SEQ ID NO:12.

[0030] The sites where Vimentin binds to the Filament-head (Desmin) domain are one or more of the following: 104–132 aa, 190–209 aa, 366–397 aa (rod region), and 438–461 aa. The Vimentin site sequence (366–397 aa) is shown in SEQ ID NO:13, and the IF Rod domain [103–411 aa] is shown in SEQ ID NO:14.

[0031] The sites where Keratin14 binds to the region (TRADD) domain are one or more of the following: 243aa, 246-247aa, 250aa, 257aa, 261aa, 274-276aa, and 281-285aa. The Keratin14 site sequence (243-285aa) is shown in SEQ ID NO: 15.

[0032] Furthermore, the cytoskeleton linker protein is Desmoplakin, and the cytoskeleton is the intermediate filament molecule Vimentin; that is, the probe is a Desmoplakin-intermediate filament intermolecular tension probe. The first structure is the head of Plakophilin-2 or the head of Plakophilin-1, and the second structure is the Filament-head of Desmin. Specifically, it is represented as: Desmoplakin-①:::PKP2-head(Plakophilin-2)-cpstFRET-Filament-head(Desmin):::③-Vimentin; Desmoplakin-②:::PKP1-head(Plakophilin-1)-cpstFRET-Filament-head(Desmin):::③-Vimentin.

[0033] Furthermore, the sites where Desmoplakin binds to the PKP2-head domain are one or more of the following: 174–212aa, 250–257aa, 280–295aa, 316–366aa, 468–472aa, 488–492aa, 504–506aa, 934–948aa, 997–1022aa, 2461–2501aa, 2550–2563aa, and 2682–2719aa.

[0034] Furthermore, the Desmoplakin site sequence (316–366aa (head-side)) is shown in SEQ ID NO:16, and Region 1 (Interaction with PKP1, JUP, PKP2) [1–548aa] is shown in SEQ ID NO:17.

[0035] Furthermore, the Desmoplakin binding sites to the PKP1-head domain are one or more of the following: 209–212 aa, 277–295 aa, 323–366 aa, 468–492 aa, 789–795 aa, 952–983 aa, 1014–1018 aa, 1298–1308 aa, 1834–1841 aa, 2278–2293 aa, 2357–2366 aa, 2410–2416 aa, 2464–2474 aa, 2640–2646 aa, 2772–2791 aa, and 2803–2852 aa. The Desmoplakin site sequence (323–366 aa) is shown in SEQ ID NO:18.

[0036] Furthermore, the sites where Vimentin binds to the Desmin-Filament-head domain are one or more of the following: 104–132 aa, 190–209 aa, 366–397 aa (rod region), and 438–461 aa. The Vimentin site sequence (366–397 aa) is shown in SEQ ID NO:13, and the IF Rod domain [103–411 aa] is shown in SEQ ID NO:14.

[0037] Furthermore, the design method for intermolecular domain tension probes is as follows: Protein-protein interaction analysis is performed using the STRING database. Based on PPI strength and affinity, protein 1, which binds with high affinity to cytoskeleton linkers, and protein 2, which binds with high affinity to the cytoskeleton, are identified. AlphaFold2 is used to predict the structural binding of cytoskeleton linkers and protein 1, and the structural binding of cytoskeleton linkers and protein 2. Then, PDBePISA is used to visualize the AlphaFold interaction prediction binding energies and binding interfaces. The solvation energy effects of the binding and the P-value of the interaction are statistically analyzed to determine whether protein 1 / protein 2 can interact with cytoskeleton linkers / cytoskeleton. When the solvation energy effect is below -5 kcal / mol, it indicates stable and effective binding with high affinity between the bound proteins. When the P-value of the solvation energy effect is less than 0.5, the interaction interface is specific.

[0038] Furthermore, when using AlphaFold2 for prediction, the process is as follows: (1) Enter the sequences of the two domain proteins in the query_sequence field, separated by a colon ":", to create the docking sequence; (2) Customize the name of the protein docking prediction job to be performed in the job_name field; (3) Set the MSA options of AlphaFold2: MMseqs2(UniRef_Environmental), pair_mode(unpaired_paired), model_type(auto), num_recylces(3); (4) Execute the Run all program to run the program, and wait for all AlphaFold2 programs to finish executing; (5) Download the predicted protein interaction structure in PDB format. After successfully obtaining the protein domain interaction prediction PDB file, use PDBePISA (https: / / www.ebi.ac.uk / msd-srv / prot_int / ) to analyze the protein-protein docking results. This program can help evaluate the interaction interface between the two protein domains and the solvation energy effect (Δ). i G, kcal / mol), Δ iGP-value, interacting amino acid residues, and hydrogen bonds or salt bridges between two protein chains.

[0039] (ii) The present invention also provides a coding gene for encoding the above-mentioned intermolecular domain tension probe.

[0040] (iii) The present invention also provides a recombinant plasmid, comprising a recombinant vector and a gene encoding an intermolecular structural domain tension probe cloned into the recombinant vector.

[0041] (iv) The present invention also provides a transformant, the transformant comprising an expression vector and the above-mentioned recombinant plasmid expressed in the expression vector.

[0042] (V) This invention also provides the application of the above-described intermolecular domain tension probes in monitoring intracellular mechanical signals, wherein,

[0043] Talin-microfilament intermolecular domain tension probes are used to monitor mechanical signals between Talin-microfilament backbones (the tensile tension of microfilaments on talin proteins).

[0044] The SYNE4-microtubule intermolecular structural domain tension probe is used to monitor the mechanical signals between the cell nucleus and microtubules (the tensile tension of microtubules on the cell nucleus).

[0045] The SYNE2-microfilament intermolecular structural domain tension probe is used to monitor the mechanical signals between the cell nucleus and the microfilament (the tensile tension of the microfilament on the cell nucleus).

[0046] The SYNE3-intermediate filament intermolecular structural domain tension probe is used to monitor the mechanical signals between the nucleus and intermediate filaments (the tensile tension of the intermediate filaments on the nucleus).

[0047] Desmoplakin-intermediate filament intermolecular structural domain tension probes are used to monitor mechanical signals between the plasma membrane and intermediate filaments (the tensile tension of the intermediate filaments on the plasma membrane).

[0048] The beneficial effects of this invention are:

[0049] (1) This invention successfully constructed four tension probes (EF-SH3, EF-PDZ, EF-LIM, and EF-FERM) based on the interaction interface of Talin-microfilament intermolecular domains, and verified their effectiveness in detecting the mechanical signals of the Integrin-Talin-microfilament scaffold through multidimensional verification. These Talin-microfilament intermolecular tension probes overcome the limitations of traditional Talin-M probes, exhibiting higher specificity, more flexible design, and less impact on protein activity and cell function;

[0050] (2) The intermolecular structural domain tension probe constructed in this invention can effectively detect tension signals without over-activating Integrin. That is, the probe can reduce the effects of overexpression-induced cell function enhancement or cell damage while ensuring the function of effective transduction force signals, and does not affect the natural activity of Integrin while detecting mechanical signals.

[0051] (3) This invention optimizes the molecular weight of the probe, resulting in a smaller molecular weight. This improves the flexibility and penetration of the tension probe, reduces interference from other intracellular mechanical signals, enhances the flexibility and transfection rate of real-time measurement in living cells, reduces protein expression-induced cytotoxicity, and facilitates real-time measurement within cells. Compared to the full-length Talin-M tension probe (molecular weight >250kDa), the Talin-microfilament intermolecular domain tension probe contains only the protein core domains (EF, SH3, PDZ, LIM, FERM), significantly reducing the molecular weight (~75kDa) and alleviating the cell transfection burden. Simultaneously, it avoids Talin gain-of-function interference, such as overactivation and enhanced migration of downstream integrin signals like FAK / Src. Furthermore, this optimization provides higher sensitivity and accuracy for the application of intermolecular domain tension probes in cell mechanics research, particularly demonstrating significant advantages in real-time monitoring of intracellular mechanical signals.

[0052] (4) The intermolecular domain tension probe constructed in this invention did not exhibit self-association or aggregation in domain binding prediction and Native-PAGE, demonstrating the reliability of intermolecular domain binding. Our results not only validate the stability of the probe in the cellular environment but also show that it can effectively avoid unnecessary self-aggregation or misbinding, thereby ensuring the accuracy of experimental results. This intermolecular probe can bind to the target protein in situ and naturally without significantly altering the protein's native state or causing overexpression. This approach allows the probe to provide interaction information that more closely approximates physiological conditions.

[0053] (5) The novel Talin-microfilament intermolecular structural domain tension probe developed in this invention, by combining AlphaFold2 structural prediction and FRET biosensing technology, successfully achieves real-time monitoring of intracellular PPI and mechanical signal transduction. This invention not only provides new insights into the molecular mechanisms of mechanical transduction but also offers a more precise experimental tool for research in cell mechanobiology. In the future, the application of this probe will help reveal the role of mechanical signals in the regulation of cell behavior and provide important references for research on the treatment of various diseases such as cancer, fibrosis, and cardiovascular diseases. Attached Figure Description

[0054] Figure 1The diagram shows the construction pattern of Talin-microfilament intermolecular domain tension probes; where A is a schematic diagram of the mTurquoise2-7aas-YFP2(cpstFRET) probe; B is a schematic diagram of the construction of four Talin-microfilament intermolecular domain tension probes and EF-free negative probes; C is a schematic diagram of the Talin-microfilament intermolecular domain tension probe interaction model.

[0055] Figure 2 This study demonstrates the effectiveness and localization analysis of Talin-microfilament intermolecular domain tension probes. A represents representative CFP and CFP / FERT images of each tension probe after 24 hours of MnCl2 treatment and stimulation of U87 cells under transfection conditions with Talin-microfilament intermolecular domain tension probes and EF-free negative probes. B represents representative CFP and CFP / FERT images of each tension probe after 24 hours of MnCl2 treatment and stimulation of PC12 cells under low-dose transfection conditions (150 ng Talin-microfilament intermolecular domain tension probes and EF-free negative probes). C–D represent images of the intermolecular domain tension probe after 24 hours of low-dose transfection conditions (150 ng Talin-microfilament intermolecular domain tension probes). Representative immunofluorescence images of U87 and PC12 cells localized by 594 phalloidin (red), DAPI (blue), and CFP fluorescent probe (green) after transfection 24 h.

[0056] Figure 3 The image shows the tension changes of Talin-microfilament intermolecular domain probes under high transfection conditions; (A, C, E, G) represent representative CFP and CFP / FERT images of four intermolecular domain tension probes in U87 cells after 15 min of stimulation with hypotonic (0 mOsm / kg) and Carbachol (100 μM) treatment at 1000 ng of intermolecular domain tension probes under high transfection conditions; (B, D, F, H) represent the normalized CFP / FERT ratios detected by the four intermolecular domain tension probes after 15 min of stimulation with different treatments under high transfection conditions.

[0057] Figure 4The results show that the EF-LIM intermolecular domain probe can effectively detect tension changes under low transfection conditions. A represents the CFP and CFP / FERT images of the EF-LIM intermolecular domain tension probe in U87 cells stimulated for 15 min after low transfection with 150 ng of the intermolecular domain tension probe and treatment with hypotonic (0 mOsm / kg) and Carbachol (100 μM). B represents the normalized CFP / FERT ratio detected by the EF-LIM intermolecular domain tension probe under low transfection conditions and stimulation for 15 min with different treatments.

[0058] Figure 5 This indicates that Talin-microfilament intermolecular structural domain probes can effectively detect Mn under high transfection conditions. 2+ Induced Integrin-Talin mechanomechanical signals; where (A,C,E,G) are representative CFP and CFP / FERT images of four intermolecular domain tension probes after 15 min of stimulation with MnCl2, ATN-161 or both under high transfection conditions of 1000 ng intermolecular domain tension probes; (B,D,F,H) are normalized CFP / FERT ratios detected by four intermolecular domain tension probes after 15 min of stimulation with different treatments under high transfection conditions.

[0059] Figure 6 This indicates that the EF-LIM intermolecular structural domain probe can effectively detect Mn under low transfection conditions. 2+ Induced Integrin-Talin mechanomechanical signals; where A is a representative CFP and CFP / FERT image of EF-LIM intermolecular domain tension probe in U87 cells stimulated for 15 min under low transfection conditions of 150 ng intermolecular domain tension probe, after treatment with MnCl2, ATN-161 or both for 15 min; B is the normalized CFP / FERT ratio detected by EF-LIM intermolecular domain tension probe under low transfection conditions and different treatment stimulation for 15 min.

[0060] Figure 7This indicates that Talin-microfilament intermolecular domain probes avoid excessive activation of Integrin and enhanced cell function; A shows the immunofluorescence signal of pFAK, the downstream activation signal of Integrin, in U87 cells transfected with Talin-microfilament intermolecular domain tension probes, Talin-M probes, and blank plasmid templates, after 24 hours of MnCl2 stimulation. Red fluorescence represents TRITC-labeled pFAK, green fluorescence represents CFP-labeled tension probes, and blue fluorescence represents DAPI-labeled cell nuclei; B shows the analysis of pFAK fluorescence signals in U87 cells expressing different tension probes and blank plasmids; C shows the immunofluorescence signal of pSrc, the downstream activation signal of Integrin, in each group of U87 cells detected using the same method as in A; D shows the immunofluorescence signal of pSrc, the downstream activation signal of Integrin, in U87 cells expressing different tension probes and blank plasmids. Analysis of pSrc fluorescence signal in cells; E is a representative image of the invasion ability of U87 cells transfected with blank plasmid and different tension probes as detected by Transwell assay; F is a representative image of the migration ability of U87 cells transfected with blank plasmid and different tension probes over 24 hours as detected by scratch wound healing assay; G is the number of invading cells per unit area in the Transwell assay; H is the migration area of ​​cells expressing empty plasmid and different probes over 24 hours in the scratch wound healing assay; I represents the migration ability of U87 cells transfected with different probes over 21 hours as shown by long-term live-cell imaging; J represents the total migration distance of cells over 21 hours in the long-term live-cell imaging assay; K represents the migration rate of cells every 20 minutes over 21 hours in the long-term live-cell imaging assay.

[0061] Figure 8 This indicates that Talin-microfilament intermolecular domain probes can effectively detect the mechanomechanical signals of NGF-induced short-term neural polarization under high transfection conditions; (A,C,E,G) are representative CFP and CFP / FERT images of EF-SH3, EF-PDZ, EF-LIM, and EF-FERM intermolecular domain tension probes in U87 cells stimulated for 15 min with NGF (500 ng / mL) and Aggrecan (500 μg / mL) or both, under high transfection conditions (1000 ng), and after stimulation with both. (B,D,F,H) are the normalized CFP / FERT ratios detected by EF-SH3, EF-PDZ, EF-LIM, and EF-FERM intermolecular domain tension probes after 15 min of stimulation with different treatments under high transfection conditions.

[0062] Figure 9The results show that the EF-LIM intermolecular domain probe can effectively detect NGF-induced neuromechanical signals under low transfection conditions; A is a representative CFP and CFP / FERT image of the EF-LIM intermolecular domain tension probe in U87 cells stimulated for 15 min after low transfection with NGF (50 ng / mL) and Aggrecan (50 μg / mL) or both; B is the normalized CFP / FERT ratio detected by the EF-LIM intermolecular domain tension probe under low transfection conditions and different treatments for 15 min.

[0063] Figure 10 The image shows the effect of Talin-microfilament intermolecular domain tension probes on NGF-induced neural polarization. Image A shows representative immunofluorescence images of neural polarization in each group 24 hours after transfection of PC12 cells with four intermolecular domain tension probes and Talin-M tension probe or blank plasmid vector, followed by NGF-induced neural polarization. In this image, TRITC-labeled neurocrosslinking protein 43 (GAP43) (red) marks neurites, CFP (green) marks probe localization, and DAPI (blue) marks cell nuclei. Image B shows the length of neurites in PC12 cells expressing each tension probe or blank plasmid vector during the neural polarization experiment.

[0064] Figure 11 This indicates that Talin-microfilament intermolecular domain tension probes effectively detect mechanical signals from the Integrin-Talin complex to the microfilament backbone; (A,C,E,G) are representative CFP and CFP / FERT images of four intermolecular domain tension probes after 15 min of stimulation with MnCl2 and CytoB or both, under high transfection conditions of 1000 ng of intermolecular domain tension probes; (B,D,F,H) are the normalized CFP / FERT ratios detected by the four intermolecular domain tension probes after 15 min of stimulation with different treatments under high transfection conditions.

[0065] Figure 12 The results show that the EF-LIM intermolecular domain probe can effectively detect mechanical signals from the Integrin-Talin complex to the microfilament backbone under low transfection conditions; A is a representative CFP and CFP / FERT image of the EF-LIM intermolecular domain tension probe after stimulating U87 cells with MnCl2 and CytoB or both for 15 min under low transfection conditions of 150 ng of the intermolecular domain tension probe; B is the normalized CFP / FERT ratio detected by the EF-LIM intermolecular domain tension probe after stimulating U87 cells with different treatments for 15 min under low transfection conditions.

[0066] Figure 13 The tension signals detected by Talin-microfilament intermolecular domain tension probes under high transfection conditions are not affected by microtubule forces; (A,C,E,G) are representative CFP and CFP / FERT images of four intermolecular domain tension probes after 15 min of stimulation with MnCl2 and Nocodazole or both, under high transfection conditions of 1000 ng intermolecular domain tension probes; (B,D,F,H) are the normalized CFP / FERT ratios detected by the four intermolecular domain tension probes after 15 min of stimulation with different treatments under high transfection conditions.

[0067] Figure 14 This indicates that the tension signal detected by the EF-LIM intermolecular domain probe under low transfection conditions is not affected by microtubule forces; A shows representative CFP and CFP / FERT images of the EF-LIM intermolecular domain tension probe in U87 cells stimulated for 15 min with MnCl2 and Nocodazole or both under low transfection conditions of 150 ng; B shows the normalized CFP / FERT ratio detected by the EF-LIM intermolecular domain tension probe under low transfection conditions and different treatments for 15 min.

[0068] Figure 15 The images show the tension changes of SYNE4-microtubule and SYNE2-microfilament intermolecular domain probes under high transfection conditions. (A, C, E, G) represent representative CFP and CFP / FERT images of the three intermolecular domain tension probes in 293T cells stimulated for 15 min under high transfection conditions of 1000 ng, after treatment with hypotonic (0 mOsm / kg) and hypertonic (540 mOsm / kg). (B, D, F, H) represent the normalized CFP / FERT ratios detected by the four intermolecular domain tension probes after 15 min of stimulation under different treatments under high transfection conditions. Detailed Implementation

[0069] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0070] (I) Design of intermolecular domain tension probes

[0071] To construct Talin-microfilament intermolecular domain probes, protein-protein interaction analysis was performed using the STRING database (https: / / cn.string-db.org / ). Based on PPI strength and affinity, proteins with high affinity binding to Talin1 were identified, including α-actinin1, α-actinin4, Talin2, Paxillin, and Zyxin, as well as proteins with high affinity binding to β-actin, including Ezrin, TJP1 / ZO1, α-actinin1, Zyxin, srGAP2, Talin1, and Talin2. The EF domain of α-actinin can directly bind to the ABD (Actin-Binding Domain) at the end of Talin-Rod, the SH3 domain of srGAP2 and the PDZ domain of ZO1 can indirectly bind to the cytofilament cytoskeleton, while the LIM domain of Zyxin and the FERM domain of Ezrin directly interact with the cytofilament cytoskeleton. In the structure and function of Talin, Talin-Head is mainly responsible for binding to the β subunit of Integrin, while Talin-Rod is mainly responsible for binding to microfilaments and microfilament-related proteins. In this way, Talin can bidirectionally transmit mechanical signals of Integrin and microfilaments.

[0072] Similarly, protein-protein interaction analysis was performed using the STRING database (https: / / cn.string-db.org / ) to construct probes for the intermolecular domains of SYNE4-microtubules / SYNE2-microfilaments / SYNE3-IF and Desmoplakin-intermediate filaments. Based on PPI strength and affinity, proteins with high affinity for SYNE4 were identified, including Kinesin-1 heavy chain, Kinesin light chain 1, Plectin, Kinesin light chain 3, and KASH5; proteins with high affinity for tubulin1A, including CAP-Gly domain-containing linker protein 1 and EB1; proteins with high affinity for SYNE2, including FH1 / FH2 domain-containing protein 1 and α-actinin1 which binds to β-actin; Microtubule-actin cross-linking factor 1, Dystonin, and Plectin which bind to SYNE3; and Desmin which binds to Vimentin; and Plakophilin-2 and Plakophilin-1 which are linked to Desmoplakin.

[0073] Based on the above analysis, the present invention designs the following types of probes:

[0074] Four Talin-microfilament intermolecular domain probes were used. The N-terminus of each probe is the EF domain of α-actinin, which can directly bind to the ABD domain at the Talin-Rod terminal. The C-terminus of each probe contains a β-actin-binding domain, including the SH3 domain of srGAP2, the PDZ domain of ZO1, the LIM domain of Zyxin, and the FERM domain of Ezrin, which are used for interaction with β-actin.

[0075] Three types of SYNE4-microtubule intermolecular probes are used. The N-terminus of the probe is the 3NF1 or 5OJ8 domain of Kinesin lightchain 1, which can bind to SYNE4; the C-terminus is the CAP~Gly1 or CAP~Gly2 domain of CAP-Gly domain-containing linker protein 1, which can bind to α-tubulin.

[0076] Two types of SYNE2-microfilament intermolecular probes are used. The N-terminus of the probes is the GBD domain of FH1 / FH2 domain-containing protein 1, which can bind to SYNE2, and the C-terminus is the ABS or ABD domain, which can bind to β-actin. They are derived from FH1 / FH2 domain-containing protein 1 and α-actinin1, respectively.

[0077] Four SYNE3-intermediate filament intermolecular probes were developed. The N-terminus of the probes contained an ABD domain that could bind to SYNE3, derived from Microtubule-actin cross-linking factor 1, Dystonin, and Plectin, respectively. The C-terminus contained the Filament-head domain of Desmin that could bind to Vimentin, and the domain of Tumornecrosis factor receptor type 1-associated DEATH domain protein that could bind to Keratin14.

[0078] Two types of Desmoplakin-intermediate filament intermolecular probes are used. The N-terminus of the probes contains the head domain of either Plakophilin-2 or Plakophilin-1, which can bind to Desmoplakin. The C-terminus of both probes contains the head domain of Desmin, which can bind to Vimentin.

[0079] Subsequently, this invention uses AlphaFold2 to predict the structural binding of each domain, and the prediction results are visualized using PDBePISA to visualize the AlphaFold interaction prediction binding energy and binding interface.

[0080] The results show that the binding free energy (Δ) between the α-actinin-EF domain and the Talin-ABD domain is... i G) is -11.6 kcal / mol, and the Δ binding of srGAP2-SH3, ZO1-PDZ, Zyxin-LIM, and Ezrin-FERM domains to β-actin... i The binding energies of G are -9.1 kcal / mol, -9.8 kcal / mol, -14.3 kcal / mol, and -15.8 kcal / mol, respectively. The binding energies of 3NF1 and 5OJ8 domains with SYNE4 are -16.4 kcal / mol and -24.9 kcal / mol, respectively; the binding energies of CAP~Gly1 and CAP~Gly2 with α-tubulin are -7.5 kcal / mol and -7.4 kcal / mol, respectively; the binding energy of the GBD domain with SYNE2 is -7.2 kcal / mol; and the binding energies of ABS and ABD domains with β-actin are -32.6 kcal / mol and -5.3 kcal / mol, respectively. ABD-p and ABD-m domains... The binding energies of the ABD-d domain to SYNE3 are -6 kcal / mol, -13 kcal / mol, and -5.8 kcal / mol, respectively; the binding energy of the Filament-head domain to Vimentin is -16.1 kcal / mol; and the binding energy of the region-t domain to Keratin14 is -8.0 kcal / mol. The binding energies of the PKP2-head and PKP1-head domains to Desmoplakin are -13 kcal / mol and -32.3 kcal / mol, respectively. The ΔE ratio of their binding interactions... iAll G values ​​were below -5 kcal / mol. The results showed that the interactions between the α-actinin-EF domain and the Talin-ABD domain, as well as the binding of β-actin to the srGAP2-SH3, ZO1-PDZ, Zyxin-LIM, or Ezrin-FERM domains; the interactions between the 3NF1 and 5OJ8 domains and SYNE4, and the binding of α-tubulin to the CAP-Gly1 and CAP-Gly2 domains; the interactions between the GBD domain and SYNE2, and the binding of β-actin to the ABS and ABD domains; the interactions between the ABD-p, ABD-m, and ABD-d domains and SYNE3, as well as the binding of Vimentin to the Filament-head domain, Keratin14 to the region-t domain, and the binding of the PKP2-head and PKP1-head domains to Desmoplakin were all stable and effective, with high affinity between the binding proteins.

[0081] Simultaneously, the Δ interaction between the α-actinin-EF domain and the Talin-ABD domain... i The GP-value is 0.343. Similarly, the ΔGAP2-SH3, ZO1-PDZ, Zyxin-LIM, or Ezrin-FERM domains binding to β-actin also exhibit similar characteristics. i The GP-values ​​were 0.334, 0.230, 0.063, and 0.141, respectively; the Δ values ​​of the 3NF1 domain and SYNE4 were... i The GP-value is 0.173, and the Δ value of the 5OJ8 structural domain is similar to that of SYNE4. i The GP-value is 0.212, and the Δ value of CAP ~ Gly1 is similar to that of α-tubulin. i The GP-value is 0.326 kcal / mol, and the Δ value of CAP~Gly2 and α-tubulin is... i The GP-value is 0.335; the Δ value between the GBD domain and SYNE2 is... i The GP-value is 0.479, and the Δ value between the ABS domain and β-actin is... i The GP-value is 0.438, and the Δ value between the ABD domain and β-actin is... i The GP-value is 0.421; the ABD-p domain has a Δ value similar to SYNE3. i The GP-value is 0.385, and the ABD-m structural domain has a Δ value similar to SYNE3. i The GP-value is 0.350, and the ABD-d structural domain has a Δ value similar to SYNE3. iThe GP-value is 0.416, and the Δ value between the Filament-head structural domain and Vimentin is... i The GP-value is 0.438, and the region-t structural domain has a Δ value similar to Keratin14. i The GP-value is 0.327; the Δ value of the PKP2-head structural domain is similar to that of Desmoplakin. i The GP-value is 0.496, and the Δ value of the PKP1-head structural domain is similar to that of Desmoplakin. i The GP-value is 0.338; all are below 0.5. When Δ i A GP-value less than 0.5 is considered to indicate that the interaction interface is specific. Therefore, this result shows that these interfaces exhibit hydrophobicity significantly higher than the average value of their respective structures, and that the interaction interfaces of the two protein domains have the potential for specific interactions.

[0082] The results in summary indicate that each domain meets the construction requirements for intermolecular probes of Talin-microfilaments, SYNE4-microtubules, SYNE2-microfilaments, SYNE3-intermediate filaments, and Desmoplakin-intermediate filaments. Taking the Talin-microfilament intermolecular domain tension probe as an example, the α-actinin-EF domain can serve as the N-terminal binding protein of the Talin-microfilament intermolecular domain tension probe, responsible for binding Talin-Rod; while the srGAP2-SH3, ZO1-PDZ, Zyxin-LIM, or Ezrin-FERM domains can serve as C-terminal binding proteins of probes binding to the cellular microfilament cytoskeleton in diverse forms, among which the Zyxin-LIM domain exhibits stronger protein affinity and binding specificity.

[0083] (II) Construction of intermolecular domain tension probes

[0084] 1. Extraction and purification of total RNA:

[0085] (1) Centrifuge to collect 1×10 7 Up to 5×10 7(1) Add 500 μL of Trizol lysis buffer to the cell pellet, vortex to mix, and let stand at room temperature for 5 min; (2) Add 200 μL of chloroform and shake vigorously for 15 s, and let stand for 2 min to form a three-phase layer; (3) Centrifuge at 12000 g for 15 min at 4 ℃, and carefully aspirate about 500 μL of the upper aqueous phase; (4) Add an equal volume of isopropanol and mix gently, and let stand at room temperature for 10 min to induce nucleic acid precipitation; (5) Centrifuge at 12000 g for 10 min at 4 ℃, remove the liquid phase and retain the precipitate; (6) Resuspend the precipitate in 1 mL of pre-cooled 75% ethanol, centrifuge at 7500 g for 5 min at 4 ℃, and discard the washing solution; (7) Repeat the ethanol washing operation once; (8) Dry at room temperature for 5 min, and add 30 μL of LEPC-treated water to dissolve the RNA precipitate; (9) Use a micro spectrophotometer to measure the A260 / A280 absorbance value to evaluate the nucleic acid concentration and purity.

[0086] 2. cDNA Synthesis

[0087] After total RNA extraction, reverse transcription is performed to obtain total cDNA for amplification of the target fragment. The reverse transcription reaction system is shown in Table 1-1. After adding the system, a brief centrifugation is performed, followed by incubation at 37°C for 15 minutes, termination at 85°C, and final extension protection at 4°C. After the reaction, the obtained cDNA solution is stored at -20°C for later use.

[0088] Table 1-1: cDNA Reverse Transcription Amplification System

[0089] Components volume 5×PrimeScript Buffer 2μL Total RNA (0.5 μg / μL) 2μL <![CDATA[RNase Free dH2O]]> To 10μL

[0090] 3. Acquisition of plasmid vector fragments

[0091] The plasmid vector pEGFP-C1 (pCMV vector variant) was obtained by double digestion using existing plasmid systems within the research group. Preliminary experiments demonstrated that double digestion of the pEGFP-C1 fragment with probes such as srGAP2-cpstFRET and Vimentin-cpstFRET yielded the highest efficiency. The double digestion reaction system is shown in Table 1-2. After adding the reaction mixture, a brief centrifugation was performed. Then, the mixture was first digested with the cryoenzyme ApaI at a lower temperature of 25°C for 3–3.5 h; followed by digestion with the cryoenzyme NheI at a higher temperature of 37°C for 3–3.5 h. Finally, the reaction was terminated by heating at 80°C for 20 min. The digested fragments were stored at -20°C for later use.

[0092] Table 1-2: Plasmid vector double enzyme digestion reaction system

[0093] Components volume Vimentin-cpstFRET plasmid (1 μg / μL) 1μL 10×rCutsmart buffer 5μL NheI restriction endonuclease 1μL ApaI restriction endonuclease 1μL <![CDATA[ddH2O]]> 42μL Final volume 50μL

[0094] 4. Acquisition of the target fragment

[0095] Primers with homologous arms for homologous recombination of the target fragment were designed using NEBuilder (https: / / nebuilder.neb.com / #! / ). The primer designs are shown in Table 1-3.

[0096] Table 1-3: Primers for homologous recombination of the target fragment

[0097]

[0098]

[0099] Gradient PCR was used to amplify the target fragments. The following fragments were amplified from existing plasmids: srGAP2-SH3, ZO1-PDZ, Zyxin-LIM, Ezrin-FERM, cpstFRET, Ezrin-cpstFRET, Vimentin-cpstFRET, and Clip170-cpstFRET, yielding fragments srGAP2-SH3, ZO1-PDZ, Zyxin-LIM, Ezrin-FERM, cpstFRET, 2E3I, and 2E3H. α-actinin-EF, 3NF1, 5OJ8, GBD, ABS, ABD, ABD-m, ABD-p, ABD-d, DES-H, PKP2-H, and PKP1-H were amplified from cDNA. The PCR reaction systems are shown in Tables 1-4.

[0100] Table 1-4: PCR amplification system for target fragments

[0101] Components volume <![CDATA[ddH2O]]> 33μL 10×Buffer 5μL 2mM dNTPs 5μL <![CDATA[25mM MgSO4]]> 3μL upstream primer 1μL Downstream primer 1μL DNA template 1μL KOD enzyme 1μL Total volume 50μL

[0102] After adding the reagents, perform a brief centrifugation and then proceed with PCR amplification according to the reaction procedure in Table 1-5:

[0103] Table 1-5: PCR reaction procedure for the target fragment

[0104] Step Name Procedure Steps Predenature 94℃, 2min Denature 98℃, 10sec Annealing 60℃ (Tm), 30sec Extension 68℃, 30sec / kb Cycle 30-35 cycles

[0105] The gradient annealing temperature can be set according to the changes in the primer Tm value, generally fluctuating within ±5℃. After amplification, store at -20℃ for later use.

[0106] 5. Agarose gel electrophoresis verification and gel cutting and recovery of the target fragment.

[0107] 5.1 Preparation of agarose gel: To prepare 1% agarose gel, dissolve 2.0g of agarose powder in 200mL of 1×TAE buffer, microwave until completely dissolved, and after cooling to 60℃, add nucleic acid dye at a final concentration of 1× and mix gently.

[0108] 5.2 Agarose gel horizontal electrophoresis: (1) Inject the gel mold to form sample wells, and place it in the horizontal electrophoresis system after solidification. Equilibrate the gel surface by immersing it in buffer for 10 min; (2) Mix the enzyme digestion product and PCR amplification product with 6× loading buffer at a ratio of 5:1, and accurately add the samples to the corresponding wells; (3) Electrophore at a constant voltage of 120V for 40 min, and confirm the migration position of the target fragments through the ultraviolet imaging system.

[0109] 5.3 Recovery of target fragment: (1) Cut specific bands according to molecular weight standard, transfer them to sterile centrifuge tubes, weigh and record (mass-volume conversion: 1mg≈1μL); (2) Add 3 times the volume of gel dissolution buffer, incubate at 55℃ for 10min, and vortex to accelerate dissolution during the process; (3) After the solution is clear, add 3M sodium acetate to adjust the pH according to the color development, and add an equal volume of isopropanol for fragments <1kb.

[0110] 5.4 Purification of the target fragment: (1) Transfer the mixture to a silica membrane adsorption column, let it stand at room temperature for 2 min, then centrifuge at 12000g for 1 min and discard the flow-through liquid; (2) Add 700μL of washing buffer and centrifuge under the same conditions to remove impurities; (3) Centrifuge empty for 2 min to completely remove residual ethanol, then open the cap and dry at room temperature for 5 min; (4) Elute with 50μL of preheated nuclease-free water (60℃), let it stand for 3 min, then centrifuge to collect the purified product and freeze at -20℃ for later use.

[0111] 6. One-step cloning and homologous recombination: The target fragment probe was recombined into the pEGFP-C1 template plasmid vector using one-step directed cloning and homologous recombination. The specific reaction system is shown in Table 1-6. After adding the system, the sample was briefly centrifuged, and then reacted at a constant temperature of 50℃ for 30 min. After the reaction was completed, the sample was stored at -20℃ for later use.

[0112] Table 1-6: Homologous Recombination System

[0113] reagents 3-segment connection Molar mass ratio of carrier to insert fragment 1：2 2×ClonExpress Mix 5μL Recombinase 1μL <![CDATA[ddH2O]]> To 10μL

[0114] Various types of Talin-microfilament intermolecular structural domain tension probes were constructed using the above homologous recombination method.

[0115] By attaching EF domains and β-actin binding domains (SH3, PDZ, LIM, FERM) to both ends of the angle-dependent eCFP and eYFP fluorescent pairs, namely cpstFRET, four effective intermolecular domain probes and a negative probe EF-cpstFRET (EF-free) were constructed: EF-cpstFRET-SH3, EF-cpstFRET-PDZ, EF-cpstFRET-LIM, and EF-cpstFRET-FERM. These are hereinafter referred to as EF-SH3, EF-PDZ, EF-LIM, EF-FERM, and EF-free.

[0116] By attaching the 5OJ8 / 3NF1 domain and the α-tubulin binding domain (CAP~Gly1 / CAP~Gly2) to both ends of cpstFRET, three effective intermolecular domain probes were constructed: 3NF1-cpstFRET-CAP~Gly1, 5OJ8-cpstFRET-CAP~Gly1, and 5OJ8-cpstFRET-CAP~Gly2, hereinafter referred to as 3NF1-cpstFRET-2E3H, 5OJ8-2E3H, and 5OJ8-2E3I.

[0117] By attaching GBD domains and β-actin binding domains (ABS / ABD) to both ends of cpstFRET, two effective intermolecular domain probes, GBD-cpstFRET-ABS and GBD-cpstFRET-ABS, are constructed, hereinafter referred to as GBD-ABS and GBD-ABD.

[0118] By attaching ABD-p, ABD-m, ABD-d domains and a Vimentin-binding domain (Filament-head) or a Keratin14-binding domain (TRADD) to both ends of cpstFRET, four effective intermolecular domain probes were constructed: ABD-p-cpstFRET-Filament-head, ABD-m-cpstFRET-Filament-head, ABD-d-cpstFRET-Filament-head, and ABD-p-cpstFRET-TRADD, hereinafter referred to as ABD-p-Filament-head, ABD-m-Filament-head, ABD-d-Filament-head, and ABD-p-TRADD.

[0119] By attaching the head domains of Plakophilin-2 or Plakophilin-1 and the head domain of Desmin, which can bind to Vimentin, to both ends of cpstFRET, two effective intermolecular domain probes, PKP2-head-cpstFRET-DES-H and PKP1-head-cpstFRET-DES-H, were constructed, hereinafter referred to as PKP2-head-DES-H and PKP1-head-DES-H.

[0120] Figure 1 This diagram illustrates the construction of Talin-microfilament intermolecular domain tension probes. A shows the mTurquoise2-7aas-YFP2 (cpstFRET) probe, where mTurquoise2 (cyan fluorescent protein, CFP) acts as the donor and sYFP2 (yellow fluorescent protein, YFP) as the acceptor. Without external tension, the two fluorophores align in parallel. Applying external force changes the angle between the probe fluorescent pairs, causing FRET efficiency to decrease with increasing angle of the cpstFRET module. B shows the construction of four Talin-microfilament intermolecular domain tension probes and an EF-free negative probe; C shows the interaction model of the Talin-microfilament intermolecular domain tension probe. The N-terminal EF domain binds to the activated Talin-Rod tail, while the C-terminal X domains (SH3, PDZ, LIM, FERM) interact with the cytofilament cytoskeleton.

[0121] (III) Effectiveness and localization analysis of Talin-microfilament intermolecular domain tension probes

[0122] First, four constructed Talin-microfilament intermolecular domain probes and EF-free negative probes were transfected into U87 cells and PC12 cells using a low-transfection method with 150 ng, respectively. Mn was then used as the transfection medium. 2+ (200 μM) Continuous stimulation of cells with Integrin for 24 h resulted in activation. The results showed that U87 cells expressing EF-free negative probes did not exhibit any change or increase in intracellular tension even under Integrin activation, and the tension detected by the probes was homogenized intracellularly. In contrast, U87 cells transfected with low levels of all four probes showed significant changes in intracellular tension, and stronger force signals were observed at the cell membrane epitopes. Figure 2 A). Meanwhile, under low transfection conditions, EF-free negative probes and four Talin-microfilament intermolecular domain probes all showed significant localization on the cell membrane. Figure 2AF). The same experimental phenomena were observed in mouse PC12 type neurons. The EF-free negative probe and the four probes were mainly localized to the cell membrane, and the four constructed probes had the ability to detect changes in cell tension compared to the EF-free negative probe. Figure 2 B). This indicates that the four probes are normally expressed in cells and have the ability to effectively detect the tension transferred from the extracellular matrix to the cytofilament cytoskeleton after Integrin activation. Simultaneously, the Talin-microfilament intermolecular domain probe, after intracellular expression, can target Integrin-Talin adhesion sites on the cell membrane.

[0123] To further confirm the relationship between the four Talin-microfilament intermolecular domain probes and the cellular microfilament cytoskeleton and Talin protein, fluorescence co-localization was used to study the spatiotemporal connections among the three. First, the four Talin-microfilament intermolecular domain probes were expressed in U87 and PC12 cells using a low-transfection method 24 hours in advance. Then, intracellular F-actin was labeled with CL594-labeled phalloidin, or Talin was labeled with TRITC-labeled Talin antibody. Intracellular EF-SH3, EF-PDZ, EF-LIM, and EF-FERM intermolecular domain tension probes were located using a 488 nm laser, while F-actin or Talin was located using a 554 nm laser. Three regions of interest (ROIs) located on the cytoplasmic membrane were selected from each cell, and the Pearson correlation coefficient between the two fluorescence sources was calculated. The results showed that the average Pearson coefficients between the fluorescence of EF-SH3, EF-PDZ, EF-LIM, and EF-FERM probes and the fluorescence of the U87 microfilament scaffold in three different ROI regions were 0.86, 0.877, 0.877, and 0.79, respectively. The average Pearson coefficients between the Talin fluorescence in U87 cells and the CFP fluorescence of the EF-SH3, EF-PDZ, EF-LIM, and EF-FERM probes in three different ROI regions were 0.78, 0.77, 0.78, and 0.703, respectively. Figure 2 E) also confirmed that the microfilament cytoskeleton and Talin protein of U87 cells colocalize with EF-SH3, EF-PDZ, EF-LIM and EF-FERM probes on the cell membrane.

[0124] Similar to the results in U87 cells, corresponding experimental results were also observed in mouse PC12 cells: the average Pearson coefficients of the three different ROI regions between the fluorescence of EF-SH3, EF-PDZ, EF-LIM, and EF-FERM probes and the fluorescence of the microfilament scaffold were 0.847, 0.873, 0.897, and 0.83, respectively. Figure 2D); The average Pearson coefficients of Talin with EF-SH3, EF-PDZ, EF-LIM, and EF-FERM probes in three different ROI regions were 0.647, 0.697, 0.807, and 0.79, respectively. Figure 2 F). This indicates that the four Talin-microfilament intermolecular structural domain tension probes also co-localize with the cellular microfilament cytoskeleton and Talin protein on the cell membrane in PC12 cells.

[0125] (IV) Study on the aggregation, self-linking and effectiveness of Talin-microfilament intermolecular domain tension probes

[0126] I. Self-linking detection of Talin-microfilament intermolecular structural domain tension probes

[0127] To further ensure that the Talin-microfilament intermolecular domain probes do not exhibit self-inhibition or aggregate into dimers or polymers, we first used AlphaFold2 to predict protein-protein interactions between the N-terminal and C-terminal domains of the probes, using the same method described above. This involved detecting whether the α-actinin-EF domain could interact with domains such as srGAP2-SH3, ZO1-PDZ, Zyxin-LIM, and Ezrin-FERM. AlphaFold2 predictions indicated that the ΔE ratio of α-actinin-EF binding to srGAP2-SH3, ZO1-PDZ, Zyxin-LIM, and Ezrin-FERM was [not specified in the original text]. i The G values ​​were 2.3 kcal / mol, -1.0 kcal / mol, -0.6 kcal / mol, and -3.9 kcal / mol, respectively, all greater than -5 kcal / mol. This indicates that the binding force and interaction affinity between α-actinin-EF and the four domains are not effective, and self-linking or the formation of dimers or polymers cannot occur between the same probes. In contrast, the ΔG values ​​for the binding of α-actinin-EF to srGAP2-SH3, ZO1-PDZ, Zyxin-LIM, and Ezrin-FERM are significantly higher than those for srGAP2-SH3, ZO1-PDZ, Zyxin-LIM, and Ezrin-FERM. i The GP-values ​​were 0.668, 0.579, 0.530, and 0.501, respectively, all higher than 0.5. This indicates that these domains do not possess specific binding capabilities in their interactions. AlphaFold2's predictions confirm that the four Talin-microfilament intermolecular domain tension probes do not possess the affinity or specificity to self-associate or generate dimerization or polymerization.

[0128] In addition, this invention also verified through non-denaturing electrophoresis (Native-PAGE) that the four Talin-microfilament intermolecular domain tension probes expressed in cells do not self-link or undergo dimerization or polymerization. First, all four intermolecular domain tension probes were highly transfected into U87 cells. Proteins were extracted from the cells without SDS denaturation treatment, followed by non-denaturing electrophoresis. The results showed that the EF-SH3, EF-PDZ, EF-LIM, and EF-FERM intermolecular domain tension probes each exhibited exactly one target protein band at their respective molecular weights, without protein aggregation or fragmentation. This result confirms that when the four intermolecular domain tension probes are expressed in cells, they exist only as single molecules and do not undergo dimerization or polymerization.

[0129] II. Effectiveness of Talin-microfilament intermolecular domain tension probe for tension signal detection

[0130] This invention transfects four Talin-microfilament intermolecular tension probes into U87 cells using both low-transfection and high-transfection methods. The cells are stimulated with hypotonic solution (0 mOsm / kg) and Carbachol (100 μM) to generate intracellular traction force, and the fluorescence-tension changes of the cells are recorded within 15 min to calculate the CFP / FERT ratio.

[0131] In the high transfection assay, at the 15-minute treatment timeline, the CFP / FERT ratio in cells expressing EF-SH3, EF-PDZ, EF-LIM, and EF-FERM intermolecular domain tension probes was significantly increased in the hypotonic solution stimulation group compared to the control group. Figure 3 AH). Similar fluorescence-tension enhancement was also observed in the Carbachol-treated group. Figure 3 AH). It was confirmed that four Talin-microfilament intermolecular structural domain probes can effectively detect tension changes in the Talin-microfilament structure.

[0132] Subsequently, the intermolecular tension probe was transfected into U87 cells using a low-transfection method, and the cells were then stimulated for 15 min with hypotonic solution (0 mOsm / kg) and Carbachol (100 μM). The results showed that the EF-LIM intermolecular domain tension probe, while maintaining its membrane localization under low-transfection conditions, exhibited significantly enhanced mechanical signaling upon stimulation with both hypotonic solution and Carbachol. Figure 4 (A and B).

[0133] (V) Study of Talin-microfilament intermolecular domain tension probes and mechanical signals and functions of Integrin

[0134] I. Talin-microfilament intermolecular structural domain tension probes effectively detect Integrin-Talin mechanical signals

[0135] To investigate whether four intermolecular domain tension probes can detect changes in force signals between Integrin-Talin microfilaments in real time, EF-SH3, EF-PDZ, EF-LIM, and EF-FERM intermolecular domain tension probes were first transfected into U87 cells for 24 h, and then treated with Mn, a highly effective Integrin activator. 2+ Talin-microfilament intermolecular tension probes were used to stimulate or inhibit cellular integrin activation with 1 mM, the integrin-specific inhibitor ATN-161 (1 μM), or a mixture of both. The CFP / FERT ratio of four intermolecular probes was measured within 15 min. The results showed that under high transfection conditions, none of the four Talin-microfilament intermolecular tension probes exhibited precise plasma membrane localization. Figure 5 A, C, E, G). When cytokines are affected by Mn 2+ When stimulated, the CFP / FERT ratio detected by the intermolecular probe was significantly enhanced. Figure 5 AH). The addition of ATN-161 can reverse Mn. 2+ The induced increase in the CFP / FERT ratio ( Figure 5 AH). Furthermore, when the integrin inhibitor ATN-161 was used alone, the CFP / FERT ratio detected by the tetramolecular probe showed no significant change. Figure 5 (AH). There were no significant differences among the EF-SH3, EF-PDZ, EF-LIM, and EF-FERM intermolecular probes. These results indicate that all four Talin-microfilament intermolecular domain tension probes effectively detect the mechanical signals transmitted to Talin generated by Integrin activation, meaning that Talin-microfilament intermolecular domain tension probes can effectively detect mechanical changes from the extracellular matrix to the intracellular matrix in real time.

[0136] Four intermolecular domain tension probes were transfected into cells using a 150 ng low-dose transfection method. Similarly, Mn... 2+ Mn (1 mM), ATN-161 (1 μM), or a mixture of both were used to stimulate integrin changes in U87 cells. Under low transfection conditions, only the EF-LIM probe effectively detected Mn in real time while maintaining effective membrane localization. 2+ Activated integrin mechanical signals; similar to high transfection results, the addition of ATN-16 significantly reduced the probe-detected CFP / FERT ratio, indicating that integrin activation was inhibited. Figure 6 (A and B). ATN-161 stimulation alone did not produce significant changes in tone.

[0137] II. Talin-microfilament intermolecular domain tension probes do not affect integrin and changes in cell function.

[0138] To investigate whether the intermolecular domain tension probes EF-SH3, EF-PDZ, EF-LIM, and EF-FERM can address the issues of excessive molecular weight, enhanced Talin function, and impaired cell motility associated with traditional Talin-M probes, we first transfected and expressed these four intermolecular domain tension probes into U87 cells, and then used Mn... 2+ After stimulating integrin activation, the signaling of its downstream phosphorylated focal adhesion kinase (p-FAK) and phosphorylated proto-oncogene tyrosine-protein kinase Src (p-Src) was detected. FAK and Src, as non-receptor tyrosine kinases, synergistically play a role in integrin signaling: responsible for activating downstream signaling pathways and promoting cell survival, migration, and proliferation.

[0139] Immunofluorescence results showed that using Mn 2+ After activation of Integrin, compared with the untransfected and empty vector transfected control groups (Con-plasmid), the expression of the four intermolecular domain tension probes had almost no effect on p-FAK signaling; while the intracellular expression of the Talin-M probe showed high fluorescence intensity of p-FAK, indicating that Integrin was overactivated. Figure 7 A and B). Similarly, we can also observe a similar trend in p-Src ( Figure 7 (C and D). The experimental results confirm that the Talin-microfilament intermolecular structural domain tension probe does not affect the activity of Integrin and can still effectively detect force transmission; while the transduction of the Talin-M probe affects the activity of Integrin, leading to the hyperphosphorylation of its downstream FAK and Src.

[0140] The enhanced activity of integrin inevitably leads to changes in Talin signal transduction function and alterations in cell adhesion, invasion, and migration. To investigate the effects of EF-SH3, EF-PDZ, EF-LIM, and EF-FERM intermolecular domain tension probes on cell function compared to the Talin-M probe, we conducted the following experiments and assays.

[0141] To investigate the effects of four intermolecular domain tension probes and Talin-M probes on cell invasion ability, we performed Transwell assays on U87 cells expressing these five types of tension probes. The results showed that the number of cells detected in the lower chamber of the Transwell assay in the Talin-M probe transfection group was significantly higher than that in the four intermolecular domain tension probe groups, while there were no significant differences among the four intermolecular domain tension probe transfection groups. Figure 7 E and G). It was confirmed that the transduction of the Talin-M probe did indeed enhance the cell's invasive ability, while the intermolecular domain tension probes of EF-SH3, EF-PDZ, EF-LIM and EF-FERM did not affect the cell's normal invasive ability.

[0142] To cross-validate the reduced effect of the four intermolecular domain tension probes on cell migration compared to the Talin-M probe, we also conducted scratch wound healing assays and long-term live-cell imaging assays. In cells transfected with the four Talin-microfilament intermolecular domain tension probes and Talin-M, the cell migration range and area in the Talin-M probe transfection group were significantly higher than those in the four probe groups. Figure 7 F and H). There were no significant differences among the four probe transfection groups. Similar to the scratch assay, cells transfected with the Talin-M probe migrated over a wider range and moved to more distal locations compared to cells transfected with the four intermolecular domain tension probes. Figure 7 I). In terms of total migration distance, the cells transfected with the four intermolecular domain tension probes also migrated a significantly shorter total distance within 21 hours than the Talin-M probe group. Figure 7 J). The motility per minute of the four probe transfection groups was also significantly lower than that of the Talin-M group (J). Figure 7 K). There were no significant differences among the four probe transfection groups. These results confirm that, compared to the Talin-M probe, the intermolecular domain tension probes EF-SH3, EF-PDZ, EF-LIM, and EF-FERM do not affect cell migration and motility to a minimum.

[0143] (VI) Study of Talin-microfilament intermolecular domain tension probes and their mechanical signals and functions in nerve growth

[0144] I. Talin-microfilament intermolecular structural domain tension probes effectively detect Talin mechanical signals induced by nerve growth.

[0145] To investigate the ability of Talin-microfilament intermolecular tension probes to monitor tension changes during neural polarization in real time, this invention induced neurogenesis or neural inhibition in U87 cells highly expressing four intermolecular tension probes for 15 min using higher concentrations of NGF (500 ng / mL) and Aggrecan (500 μg / mL), thus constructing a short-acting model. The results showed that NGF (500 ng / mL) stimulation promoted an increase in tension detected by the four Talin-microfilament intermolecular tension probes, while co-treatment with Aggrecan (500 μg / mL) and NGF eliminated or reduced this increase in tension. Figure 8 (AH). Similar to the long-term treatment group with high probe expression, there were no significant differences among the four low-probe transfection groups. In summary, the Talin-microfilament intermolecular domain tension probe is highly sensitive to changes in Integrin-Talin mechanotransduction, providing a powerful tool for real-time tension monitoring.

[0146] Subsequently, to investigate the correlation between the localization of intermolecular domain tension probes and NGF-induced tension changes, we transfected four intermolecular domain tension probes into cells using a low-transfection method with 150 ng of the probes, and then stimulated the cells for 15 min with NGF (500 ng / mL) and Aggrecan (500 μg / mL). The results showed that only the EF-LIM intermolecular domain tension probe, while maintaining its plasma membrane localization under low-transfection conditions, also produced a significant enhancement of mechanical signal in the NGF-induced neural polarization model. Figure 9 (A and B).

[0147] II. Talin-microfilament intermolecular domain tension probes do not affect changes in neural polarization function.

[0148] To investigate whether Talin-microfilament intermolecular domain tension probes affect neural polarization compared to Talin-M, we transfected PC12 cells with EF-SH3, EF-PDZ, EF-LIM, and EF-FERM intermolecular domain tension probes, as well as Talin-M tension probe. A neural polarization model was then constructed using NGF (50 ng / mL) induction to observe and compare the effects of different tension probes on neural polarization. The results showed that, under transfection conditions, there was no significant difference in neurite length between the four intermolecular domain tension probes and the blank control group (Control) and the vector control group (Con-plasmid), indicating that the tension of the four Talin-microfilament intermolecular domains does not affect neural polarization function. Figure 10 A and B). In cells expressing the Talin-M tension probe, neurites showed significantly greater growth compared to other groups, confirming the beneficial effect of the Talin-M probe on neural polarization function. Figure 10 (A and B). Meanwhile, under normal transfection conditions, there was no difference in neurite length among the intermolecular structural domain tension probe groups of EF-SH3, EF-PDZ, EF-LIM, and EF-FERM.

[0149] In summary, the four Talin-microfilament intermolecular structural tension probes can effectively detect the mechanical signals during intracellular neural polarization, and the EF-LIM probe exhibits stronger adaptability and core competitiveness. Neural polarization experiments confirmed that none of the four Talin-microfilament intermolecular structural tension probes affected neural polarization function; however, Talin-M showed a significant enhancement of neural polarization function. This indicates that the Talin-microfilament intermolecular structural tension probes demonstrate optimization and advancement in their impact on cellular function compared to the Talin-M probe.

[0150] (VII) Correlation Study of Talin-Microfilament Intermolecular Tension Probes with Cytoskeleton

[0151] I. Effective Detection of Talin Microfilament Skeletal Mechanical Signals Using Intermolecular Tension Probes

[0152] To investigate whether four Talin-microfilament intermolecular domain probes can effectively detect intracellular signal changes similar to cytoskeleton remodeling, we used cytochalasin B to depolymerize the intracellular microfilament skeleton in U87 cells highly transfected with Talin-microfilament intermolecular domain tension probes, thereby testing the functional effectiveness of the intermolecular domain probes.

[0153] The results are the same as before; using Mn alone... 2+ When Integrin was activated by (1 mM) stimulation, the CFP / FRET signal ratio detected by the four Talin-microfilament intermolecular structural domain tension probes showed a significant enhancement on the time axis; however, when CytoB (10 μM) was used, the cytofilament cytoskeleton was excited to disaggregate, even with the addition of Mn 2+ Stimulation of Integrin signaling activation resulted in no significant enhancement of FRET signals detected by the probe. Figure 11 AH). Even when using CytoB depolymerization alone, without Mn 2+ Upon activation, the tension signals detected by the EF-SH3 and EF-FERM probes even showed a decreasing trend. Figure 11A, B, G, H). This indicates that in the state of microfilament framework depolymerization, the integrin-microfilament framework tension signal transmission is hindered, and the depolymerization of the microfilament framework also leads to changes in integrin activity. Under microfilament framework depolymerization, the FRET signal values ​​of microfilament framework-Integrin detected by the four Talin-microfilament intermolecular structural domain tension probes no longer show significant changes, indicating that all four Talin-microfilament intermolecular structural domain tension probes can effectively detect the mechanical signal transmission between the microfilament framework and Talin. This also shows that under the condition of microfilament framework depolymerization, the activity of integrin usually decreases.

[0154] U87 cells expressing low levels of Talin-microfilament intermolecular domain tension probes were stimulated with Mn (1 mM) and CytoB (10 μM) for 15 min to investigate the correlation between the localization of the intermolecular domain tension probes and the changes in mechanical signals after microfilament depolymerization. The results showed that only the EF-LIM intermolecular domain tension probe, under conditions of low transfection maintaining plasma membrane localization, effectively detected Mn. 2+ The stimulation enhances the mechanical signal and the mechanical signal transduction is impaired after the microfilament skeleton depolymerization. Figure 12 (A and B).

[0155] In summary, these results demonstrate that disruption of the cellular microfilament cytoskeleton disrupts the mechanotransmission pathway between the Integrin-Talin microfilament cytoskeleton. They also confirm that four Talin-microfilament intermolecular tension probes can effectively monitor the mechanotransduction process between the Talin-microfilament cytoskeleton. Compared to the three intermolecular probes EF-SH3, EF-PDZ, and EF-FERM, the EF-LIM intermolecular probe exhibits both membrane localization specificity and FRET signal detection capability at low transfection levels.

[0156] II. Talin-microfilament intermolecular domain tension probes are unaffected by microtubule forces.

[0157] To investigate the relationship between the tension signals detected by four Talin-microfilament intermolecular domain probes and the cellular microtubule skeleton, the changes in CFP / FRET signal values ​​of U87 cells transfected with 1000 ng of intermolecular domain probes were first detected by depolymerizing the cellular microtubule skeleton using Nocodazole (Noc, 20 μM), and then the tension changes were analyzed.

[0158] When Nocodazole was used alone to depolymerize microtubules, the changes in FRET signal values ​​detected by the four Talin-microfilament intermolecular structural domain tension probes were almost identical to those in the Control group, indicating that the depolymerization of microtubules had no effect on Integrin activity and the Integrin-Talin mechanotransduction pathway, or the effect was negligible. Figure 13 AH); and under the depolymerization effect, the Integrin activator Mn is used in combination. 2+ Upon stimulation, the FRET tension signals detected by the four intermolecular structural domain probes were significantly enhanced, but lower than those detected by Mn alone. 2 +Stimulation Activation Group ( Figure 14 The presence of microtubules (AH) suggests that microtubules may be involved in signal transduction within the Integrin-Talin-microfilament framework, and that microtubule depolymerization weakens the transduction capacity of this system. However, in practice, microtubule depolymerization does not affect the bidirectional transduction function of the four intermolecular domain probes. They can still effectively detect changes in extracellular mechanical signals mediated by Integrin, indicating that the four intermolecular probes are unaffected by microtubule forces.

[0159] Subsequently, this invention used a low-dose transfection method to transfect four Talin-microfilament intermolecular structural domain tension probes into cells, and then used Nocodazole (20 μM) to depolymerize the microtubule backbone and Mn. 2+ (1mM) stimulation activates Integrin, allowing for a more precise investigation of the effect of microtubule forces on the Talin-microfilament signal transduction pathway. Similar to the results of microfilament depolymerization experiments, the EF-LIM probe demonstrated effective membrane localization while detecting the mechanical signals of Integrin activation. Figure 14 (A and B). This confirms that the EF-LIM probe has stronger detection capabilities and binding specificity compared to the EF-SH3, EF-PDZ, and EF-FERM probes.

[0160] Figure 15 The image shows the tension changes detected by the SYNE4-microtubule and SYNE2-microfilament intermolecular structural domain probes under high transfection conditions, indicating that the SYNE4-microtubule and SYNE2-microfilament intermolecular structural domain probes can effectively detect the mechanical changes from the extracellular matrix to the cell in real time.

[0161] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principle of the present invention should be considered within the scope of protection of the present invention.

Claims

1. An intermolecular domain tension probe, characterized in that, The probe includes: a first structure, a second structure, and fluorescent protein pairs eCFP and eYFP, wherein the first structure and the second structure are respectively connected to the two ends of the fluorescent protein pair, and the eCFP and eYFP are connected by a short peptide. The first structure is a domain or protein capable of binding to cytoskeleton linker proteins, and the second structure is a domain or protein capable of binding to the cytoskeleton. The cytoskeleton linker protein is talin, SYNE4, SYNE3, SYNE2, or Desmoplakin; The cytoskeleton is composed of microfilaments, microtubules, or intermediate filaments.

2. The intermolecular structural domain tension probe according to claim 1, characterized in that, The cytoskeleton linker protein is talin, and the cytoskeleton is the microfilament molecule β-actin. Therefore, the probe is a Talin-microfilament intermolecular tension probe. The first structure is the EF domain of α-actinin, and the second structure is the SH3 domain of srGAP2, the PDZ domain of ZO1, the LIM domain of Zyxin, or the FERM domain of Ezrin.

3. The intermolecular structural domain tension probe according to claim 1, characterized in that, The cytoskeleton linker protein is SYNE4, and the cytoskeleton is composed of microtubule molecules α-tubulin or β-tubulin. That is, the probe is a SYNE4-microtubule intermolecular domain tension probe. The first structure is the 3NF1 or 5OJ8 structure domain of KLC1, and the second structure is the CAP~Gly1 or CAP~Gly2 structure domain of Clip-170.

4. The intermolecular domain tension probe according to claim 1, characterized in that, The cytoskeleton linker protein is SYNE2, and the cytoskeleton is the microfilament molecule β-actin. Therefore, the probe is a SYNE2-microfilament intermolecular domain tension probe. The first structure is the GBD domain of FHOD1, and the second structure is the ABS domain of FHOD1 or the ABD domain of α-actinin.

5. The intermolecular structural domain tension probe according to claim 1, characterized in that, The cytoskeleton linker protein is SYNE3, and the cytoskeleton is the intermediate filament molecule Vimentin or Keratin14. That is, the probe is a SYNE3-intermediate filament intermolecular domain tension probe. The first structure is the ABD structure domain of Plectin, the ABD structure domain of MACF1, or the ABD structure domain of DYST. The second structure is the combined region of Desmin's Filament-head or TRADD.

6. The intermolecular structural domain tension probe according to claim 1, characterized in that, The cytoskeleton linker protein is Desmoplakin, and the cytoskeleton is the intermediate filament molecule Vimentin. Therefore, the probe is a Desmoplakin-intermediate filament intermolecular domain tension probe. The first structure is the head of Plakophilin-2 or the head of Plakophilin-1, and the second structure is the Filament-head of Desmin.

7. A gene encoding a gene, characterized in that: The encoding gene is used to encode the intermolecular domain tension probe according to any one of claims 1 to 6.

8. A recombinant plasmid, characterized in that: The invention includes a recombinant vector and the gene encoding the intermolecular domain tension probe as described in any one of claims 1 to 6, which is cloned into the recombinant vector.

9. A transformant, characterized in that: It includes the expression vector and the recombinant plasmid of claim 8 expressed in the expression vector.

10. The application of the intermolecular structural domain tension probe according to any one of claims 1 to 6 in monitoring intracellular mechanical signals.

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