Living cell imaging system and method for visually detecting cell nucleus mechanics and application

Abstract

The invention belongs to the technical field of biomolecules, and particularly relates to a living cell imaging system and method for visually detecting cell nucleus mechanics and application. The invention provides a probe composition for detecting cell nucleus mechanics. The probe composition comprises NuTS1 and NuTS2, wherein the NuTS1 and the NuTS2 are arranged in the probe composition; wherein the NuTS1 is composed of an LMNA-bd1, a TM1, a YPet fluorescent protein, a tension linker F40, an ECFP fluorescent protein and a CC-SUN1; and the NuTS2 is composed of LMNA-bd2, TM2, a YPet fluorescent protein, a tension linker F40, an ECFP fluorescent protein and a CC-SUN2. Based on the probe composition, the invention constructs a living cell imaging system of the cell nucleus mechanics, the system can perform real-time dynamic imaging of the cell nucleus mechanics in living cells, and the system has important significance for constructing a research platform of the cell nucleus mechanics and a drug screening platform related to the research platform of the cell nucleus mechanics.

Description

Technical Field

[0001] The present invention belongs to the field of biomolecule technology, and in particular relates to a living cell imaging system, method and application for visually detecting cell nuclear mechanics. Background Art

[0002] Mechanical force is an important regulator of many physiological and pathological processes, such as embryonic development, tumor metastasis, cardiovascular disease, aging, etc. Studies have shown that these physiological or pathological processes all involve the response of cells to extracellular mechanical stimuli, such as matrix stiffness, fluid shear or mechanical stretching. However, mechanical force can not only be transmitted between the extracellular matrix and the cytoskeleton, but the cytoskeleton can also transmit force inward to the cell nucleus. Therefore, the cell nucleus is also considered to be an important mechanical effector.

[0003] On the nuclear envelope (NE), the nucleoskeleton and cytoskeleton connector (LINC) complex composed of nesprin and SUN proteins transmits mechanical forces from the cytoskeleton to the nucleus, and realizes the direct transmission of mechanical forces across the inner and outer nuclear membranes. Moreover, these mechanical forces can directly or indirectly change the morphology of genetic material in the nucleus. For example, continuous mechanical stretching can regulate the distribution state of chromatin, and the stiffness of the extracellular matrix can also change the condensation of chromatin and the state of the whole genome. It can be seen that the nucleus plays a key role in the cell's perception and response to mechanical stimuli. Existing studies have shown that changes in nuclear mechanics are likely to become an important monitoring indicator in physiological or pathological processes. By deeply studying the mechanical properties of the nucleus and its biological significance, we can better understand the laws of cell life activities and provide new ideas and methods for the diagnosis and treatment of related diseases. However, at this stage, there are no good visualization measurement methods and tools to detect the dynamic changes of nuclear mechanics under the action of mechanical forces.

[0004] Some existing technologies have reported mechanical measurements at the level of intracellular protein molecules. For example, Grashoff et al. reported a method based on FRET changes to measure mechanical tension across Vinculin molecules in “Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics”; subsequently, in “Multiplexing molecular tension sensors reveals piconewton force gradient across talin-1”, a probe for measuring the mechanics within Talin molecules was developed, confirming the existence of gradient mechanical transmission within Talin molecules. At the same time, using this probe design idea, researchers developed a mechanical probe for Nesprin in “Nesprin-2G, a component of the nuclear LINC complex, is subject to myosin-dependent tension” to measure mechanical transmission from the cytoskeleton to the surface of the nucleus. However, these technologies all measure the force from the cell membrane to the surface of the nuclear membrane, and there is currently no way to measure the force directly transmitted to the nucleus. In addition, a FRET biosensor based on the nuclear skeleton protein of a nano-antibody was also reported in "Nuclear lamina strain states revealed by intermolecular force biosensor". Although this confirmed the existence of huge lateral tension in the nuclear skeleton, it could not reflect the normal force transmission of the inner and outer nuclear membranes on the surface of the nucleus. Therefore, the development of FRET-based mechanical probes is the key to visually measuring the force directly transmitted to the nucleus. Nuclear mechanics is related to physiology and pathology, but there is currently a lack of methods and tools that can truly reflect the mechanical transmission on the nuclear membrane, making it difficult to achieve dynamic monitoring of changes in nuclear mechanics during physiological and pathological processes. Summary of the invention

[0005] In order to overcome the defects of the prior art, the present invention innovatively constructs a FRET mechanical probe that can effectively detect cell nuclear mechanics, and further develops a visualized living cell imaging system and method. The system can be used for the effective detection of cell nuclear mechanics in vitro or in vivo, and realizes real-time dynamic imaging and monitoring of cell nuclear mechanics, which is of great significance for the monitoring, indication or treatment of physiological or pathological processes.

[0006] One of the objectives of the present invention is to provide a probe composition for detecting cell nuclear mechanics.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A probe composition for detecting nuclear mechanics, the probe composition comprising a NuTS1-FRET mechanical probe and a NuTS2-FRET mechanical probe; the NuTS1-FRET mechanical probe is composed of LMNA-bd1, TM1, YPet fluorescent protein, tension linker F40, ECFP fluorescent protein and CC-SUN1; the NuTS2-FRET mechanical probe is composed of LMNA-bd2, TM2, YPet fluorescent protein, tension linker F40, ECFP fluorescent protein and CC-SUN2; the amino acid sequence of the LMNA-bd1 is shown in SEQ ID NO.29, and the amino acid sequence of the LMNA-bd2 is shown in SEQ ID NO.30; the amino acid sequence of the TM1 is shown in SEQ ID NO.31, and the amino acid sequence of the TM2 is shown in SEQ ID NO.32; the amino acid sequence of the YPet fluorescent protein is shown in SEQ ID NO.33; the amino acid sequence of the tension linker F40 is shown in SEQ ID NO.34; the amino acid sequence of the ECFP fluorescent protein is shown in SEQ ID NO. The amino acid sequence of CC-SUN1 is shown in SEQ ID NO.36, and the amino acid sequence of CC-SUN2 is shown in SEQ ID NO.37.

[0009] Furthermore, the NuTS2-FRET mechanical probe and the NuTS1-FRET mechanical probe also include a GGSGGT linker sequence.

[0010] Furthermore, the nucleotide sequence of the NuTS1-FRET mechanical probe is shown in SEQ ID NO.26; the nucleotide sequence of the NuTS2-FRET mechanical probe is shown in SEQ ID NO.24.

[0011] Furthermore, the NuTS1-FRET mechanical probe uses SUN1 transmembrane protein as the target molecule; and the NuTS2-FRET mechanical probe uses SUN2 transmembrane protein as the target molecule.

[0012] Furthermore, the detection includes detecting or dynamically monitoring the force level of the nuclei of living cells in vitro, and / or detecting or dynamically monitoring the force level of the nuclei of living cells during in vivo development.

[0013] Furthermore, when the probe composition is used to detect nuclear mechanics, it is used in combination with a control probe; the control probe includes a TL1 mechanical probe with a nucleotide sequence as shown in SEQ ID NO.27 and a TL2 mechanical probe with a nucleotide sequence as shown in SEQ ID NO.25.

[0014] A second object of the present invention is to provide a live cell imaging system for nuclear mechanics.

[0015] To achieve the above object, the present invention adopts the following technical solutions:

[0016] A live cell imaging system for cell nuclear mechanics, comprising the aforementioned probe composition.

[0017] Furthermore, the living cell imaging system also contains a control probe; the control probe includes a TL1 mechanical probe and a TL2 mechanical probe; the TL1 mechanical probe is composed of LMNA-bd1, TM1, YPet fluorescent protein, tension linker F40 and ECFP fluorescent protein; the TL2 mechanical probe is composed of LMNA-bd2, TM2, YPet fluorescent protein, tension linker F40 and ECFP fluorescent protein; the amino acid sequence of the LMNA-bd1 is shown in SEQ ID NO.29, and the amino acid sequence of the LMNA-bd2 is shown in SEQ ID NO.30; the amino acid sequence of the TM1 is shown in SEQ ID NO.31, and the amino acid sequence of the TM2 is shown in SEQ ID NO.32; the amino acid sequence of the YPet fluorescent protein is shown in SEQ ID NO.33; the amino acid sequence of the tension linker F40 is shown in SEQ ID NO.34; the amino acid sequence of the ECFP fluorescent protein is shown in SEQ ID NO.35.

[0018] The control probes are CC-SUN domain deleted probes NuTS2-TL and NuTS1-TL; the NuTS2-TL and NuTS1-TL represent FRET controls under no tension, respectively.

[0019] Furthermore, the nucleotide sequence of the TL1 mechanical probe is shown as SEQ ID NO.27, and the nucleotide sequence of the TL2 mechanical probe is shown as SEQ ID NO.25.

[0020] Furthermore, the NuTS1-FRET mechanical probe uses SUN1 transmembrane protein as the target molecule; and the NuTS2-FRET mechanical probe uses SUN2 transmembrane protein as the target molecule.

[0021] Furthermore, the FRET mechanical probe and the control probe are prepared by the following method:

[0022] 1) Amplify and obtain YPet-F40-ECFP#1, YPet-F40-ECFP#2, LMNA-bd2, LMNA-bd1, TM2, TM1, CC-SUN2, and CC-SUN1 respectively;

[0023] 2) The pSIN-LADS FRET BS plasmid was digested with SpeI and EcoRI, and then the ends were treated with CIP enzyme to recover the vector sequence;

[0024] 3) The vector, LMNA-bd2, TM2, YPet-F40-ECFP#1 and CC-SUN2 were connected by the Gibson Assembly method to prepare the NuTS2 plasmid, and the vector, LMNA-bd2, TM2 and YPet-F40-ECFP#2 were connected to prepare the TL2 plasmid; the vector, LMNA-bd1, TM1, YPet-F40-ECFP#1 and CC-SUN1 were connected to prepare the NuTS1 plasmid, and the vector, LMNA-bd1, TM1 and YPet-F40-ECFP#2 were connected to prepare the TL1 plasmid; the connection was carried out at 16°C overnight, the ligation product was transfected into DH5α competent cell, the single clone was picked by plating, and the correct plasmid was identified and selected by Sanger sequencing, and finally NuTS2, TL2, NuTS1 and TL1 were obtained.

[0025] Preferably, the primers used to amplify YPet-F40-ECFP#1 include YPetF with a sequence as shown in SEQ ID NO.1 and ECFP R1 with a sequence as shown in SEQ ID NO.2; the primers used to amplify YPet-F40-ECFP#2 include YPetF with a sequence as shown in SEQ ID NO.1 and ECFP R2 with a sequence as shown in SEQ ID NO.4.

[0026] Preferably, the primers used to amplify LMNA-bd2 include LMNAF2 with a sequence as shown in SEQ ID NO.6 and LMNAR2 with a sequence as shown in SEQ ID NO.7; the primers used to amplify LMNA-bd1 include LMNAF1 with a sequence as shown in SEQ ID NO.9 and LMNAR1 with a sequence as shown in SEQ ID NO.10.

[0027] Preferably, the primers used to amplify TM2 include TM F2 with a sequence as shown in SEQ ID NO.12 and TM R2 with a sequence as shown in SEQ ID NO.13; the primers used to amplify TM1 include TM F1 with a sequence as shown in SEQ ID NO.15 and TM R1 with a sequence as shown in SEQ ID NO.16.

[0028] The primers used to amplify CC-SUN2 include CC F2 with a sequence as shown in SEQ ID NO.18 and SUN R2 with a sequence as shown in SEQ ID NO.19; the primers used to amplify CC-SUN1 include CC F1 with a sequence as shown in SEQ ID NO.21 and SUN R1 with a sequence as shown in SEQ ID NO.22.

[0029] The third object of the present invention is to provide a stable cell line obtained by transfecting the above-mentioned living cell imaging system.

[0030] A fourth object of the present invention is to provide a method for detecting the stress level of a cell nucleus using the aforementioned living cell imaging system.

[0031] To achieve the above object, the present invention adopts the following technical solutions:

[0032] Using the aforementioned live cell imaging system to detect the force level of the cell nucleus, NuTS2, TL2, NuTS1 and TL1 mechanical probes were expressed in mammalian cells in vitro, and the changes in the force level of the cell nucleus were detected by measuring the FRET / ECFP ratio.

[0033] Furthermore, the magnitude of the FRET / ECFP ratio is negatively correlated with the magnitude of the force applied to the cell nucleus.

[0034] When the cell nucleus is subjected to little or no force, the tension linker F40 is in a shorter state, the distance between the YPet fluorescent protein and the ECFP fluorescent protein is closer, the FRET signal is strong, and the FRET / ECFP ratio is higher; when the force on the cell nucleus increases, the tension linker F40 is pulled apart and becomes longer, the distance between the YPet fluorescent protein and the ECFP fluorescent protein becomes farther, the FRET signal is weakened, and the FRET / ECFP ratio is lower.

[0035] A fifth object of the present invention is to provide an application of the aforementioned probe composition and / or the aforementioned living cell imaging system in the preparation of a product for detecting and / or dynamically monitoring cell nuclear mechanics.

[0036] To achieve the above object, the present invention adopts the following technical solutions:

[0037] Use of the aforementioned probe composition and / or the aforementioned living cell imaging system in preparing products for visual detection and / or dynamic monitoring of cell nuclear mechanics.

[0038] Further, the application includes any one or more of the following 1)-4):

[0039] 1) Use of the probe composition and / or the living cell imaging system in the preparation of a product for detecting and / or dynamically monitoring changes in the nucleus mechanics of living cells when they are stimulated by substrate stiffness mechanics;

[0040] 2) Use of the probe composition and / or the living cell imaging system in the preparation of a product for detecting and / or dynamically monitoring changes in the nucleus mechanics of living cells when subjected to hypertonic and hypotonic mechanical stimulation;

[0041] 3) Use of the probe composition and / or the living cell imaging system in the preparation of a product for detecting and / or dynamically monitoring changes in the nucleus mechanics of living cells when they are subjected to local pressure mechanical stimulation;

[0042] 4) Use of the probe composition and / or the live cell imaging system in the preparation of a product for detecting and / or dynamically monitoring changes in nuclear mechanics during in vivo development.

[0043] The beneficial effects of the present invention are:

[0044] 1. The present invention innovatively constructs a real-time dynamic imaging and monitoring system for nuclear mechanics, which can be achieved through induced expression. The present invention constructs the FRET mechanical probes of SUN2 and SUN1 for the first time, which are used to achieve high-resolution dynamic tracking in living cells.

[0045] 2. The present invention innovatively constructs a cell nuclear mechanics measurement tool and its visualized FRET imaging system. The live cell dynamic imaging system can effectively detect the changes in cell nuclear mechanics of living cells when subjected to mechanical stimuli such as substrate stiffness, high and low osmotic pressure, and squeezing. It can also perform real-time dynamic imaging and monitoring of cell nuclear mechanics at the cellular and animal levels for in vivo dynamic observation with a wide detection range.

[0046] 3. The FRET mechanical probe and live cell dynamic imaging system of the present invention can be used for the effective detection of nuclear mechanics in vitro or in vivo, which is of great significance for the monitoring, indication or treatment of physiological or pathological processes, and can be used for drug screening of diseases related to nuclear mechanics.

[0047] 4. The design concept of the present invention can also be used for imaging and dynamic monitoring of other cell nuclear mechanics and for drug screening of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Schematic diagram of the detection principle of the live cell imaging system for nuclear mechanics;

[0049] Figure 2 The result diagram of the expression of FRET mechanical probes of NuTS2 and NuTS1 in mammalian cells; Figure 2 -A is a diagram showing the expression of NuTS2 plasmid in mammalian cells; Figure 2 -B is a diagram showing the expression of TL2 plasmid in mammalian cells; Figure 2-C is a diagram showing the expression of NuTS1 plasmid in mammalian cells; Figure 2 -D is the expression of TL1 plasmid in mammalian cells;

[0050] Figure 3 This is a result diagram of the specific detection of mechanical stimulation of substrate stiffness using a live cell imaging system for nuclear mechanics; Figure 3 -A is a diagram showing the stress conditions of TL2 and NuTS2 on the hard and soft substrate surfaces. Figure 3 -B is a diagram showing the stress conditions of TL1 and NuTS1 on the hard and soft substrate surfaces;

[0051] Figure 4 This is a dynamic result diagram of the specific detection of hypertonic and hypotonic mechanical stimulation using a live cell imaging system for nuclear mechanics. Figure 4 -A is a diagram showing the stress conditions of TL2 and NuTS2 in high and low osmotic environments. Figure 4 -B is a diagram showing the stress conditions of TL1 and NuTS1 in high and low osmotic environments;

[0052] Figure 5 This is a dynamic result diagram of the specific detection of local pressure mechanical stimulation using a live cell imaging system for nuclear mechanics;

[0053] Figure 6 A live cell imaging system for nuclear mechanics is used to specifically detect the dynamic results of nuclear mechanics during somatic development. DETAILED DESCRIPTION

[0054] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0055] In the embodiment of the present invention:

[0056] LMNA binding domain: connects to the nuclear skeleton protein LaminA / C molecule;

[0057] TM transmembrane domain: spans the inner nuclear membrane of the cell nucleus;

[0058] CC-SUN domain: responsible for the formation of SUN polymers and the connection of Nesprin molecules;

[0059] YPet fluorescent protein: YPet is the brightest yellow fluorescent protein developed and has good photostability;

[0060] Tension linker F40: Contains 40 amino acids of flagellar peptide (GPGGA)8, which is a commonly used linker sequence for constructing intramolecular FRET mechanical probes. It has elastic function and can realize FRET changes under stress;

[0061] ECFP: Enhanced cyan fluorescent protein.

[0062] In the embodiment of the present invention, the amino acid sequence of the LMNA binding domain (LMNA-bd1) used to construct the NuTS1 plasmid is shown in SEQ ID NO.29, and the amino acid sequence of the LMNA binding domain (LMNA-bd2) used to construct the NuTS2 plasmid is shown in SEQ ID NO.30; the amino acid sequence of the TM transmembrane domain (TM1) used to construct the NuTS1 plasmid is shown in SEQ ID NO.31, and the amino acid sequence of the TM transmembrane domain (TM2) used to construct the NuTS2 plasmid is shown in SEQ ID NO.32; the amino acid sequence of the YPet fluorescent protein is shown in SEQ ID NO.33; the amino acid sequence of the tension linker F40 is shown in SEQ ID NO.34; the amino acid sequence of the ECFP fluorescent protein is shown in SEQ ID NO.35; the amino acid sequence of the CC-SUN domain (CC-SUN1) used to construct the NuTS1 plasmid is shown in SEQ ID NO.36, and the amino acid sequence of the CC-SUN domain (CC-SUN2) used to construct the NuTS2 plasmid is shown in SEQ ID Shown in NO.37.

[0063] In the embodiment of the present invention, the LMNA binding domain, the transmembrane domain and the CC-SUN domain constitute the SUN protein domain. The SUN1 protein domain includes the LMNA binding domain (1-139aa), the transmembrane domain (416-436aa) and the CC-SUN domain (456-913aa), and the SUN1 domain sequence is referenced from NCBI: NM_024451.2. The SUN2 protein domain includes the LMNA binding domain (1-128aa), the transmembrane domain (227-247aa) and the CC-SUN domain (267-731aa), and the SUN2 domain sequence is referenced from NCBI: NM_001205345.1.

[0064] In an embodiment of the present invention, the method for live cell imaging of nuclear mechanics is as follows:

[0065] (1) The LMNA binding domain, TM transmembrane domain, YPet fluorescent protein, tension linker F40, ECFP fluorescent protein and CC-SUN domain were fused to obtain the FRET mechanical probe plasmids of NuTS2 and NuTS1;

[0066] (2) fusing the LMNA binding domain, TM transmembrane domain, YPet fluorescent protein, tension linker F40 and ECFP fluorescent protein respectively to obtain the zero-force probe plasmids of FRET mechanical probe control TL2 and TL1;

[0067] (3) The NuTS2, TL2, NuTS1, and TL1 mechanical probes were expressed in mammalian cells in vitro, and the differences in FRET / ECFP ratios were observed.

[0068] In the embodiment of the present invention, the detection principle of the live cell imaging system of cell nuclear mechanics is as follows Figure 1 As shown, the core of this patent lies in the design and application of the nuclear FRET mechanical probe. Among them, there is an artificially designed and assembled connecting sequence | (GGSGGT) between each fragment, and the sequences of the LMNA binding domain, TM transmembrane domain, YPet fluorescent protein, tension linker F40 and ECFP fluorescent protein are spliced ​​by molecular biology technology. The NuTS1-FRET mechanical probe uses the SUN1 transmembrane protein as the target molecule; the NuTS2-FRET mechanical probe uses the SUN2 transmembrane protein as the target molecule. When the FRET mechanical probe is transfected and expressed normally, the tension linker F40 is in a stressed state, the YPet fluorescent protein is far away from the ECFP fluorescent protein, and the FRET / ECFP ratio is low; while the zero-force probe used for control is in a non-stressed state, the YPet fluorescent protein is close to the ECFP fluorescent protein, the FRET signal is strong, and the FRET / ECFP ratio is high.

[0069] Example 1. Plasmid construction and evaluation of nuclear mechanics probe

[0070] (1) Design and synthesize the tension linker F40 sequence, obtain the gene fragment of tension linker F40 using custom gene synthesis service, and insert it into pUC-GW-Amp+ plasmid. Use NheI to digest the pUC-GW-Amp+ plasmid and recover the target fragment of tension linker F40. Use NheI digestion and T4 enzyme ligation to replace the EV linker in pSIN-LADS FRET BS (non-commercial plasmid) with tension linker F40.

[0071] (2) YPet F and ECFP R1 were used to amplify the YPet-F40-ECFP#1 sequence for NuTS2 / NuTS1; YPet F and ECFP R2 were used to amplify the YPet-F40-ECFP#2 sequence for TL2 / TL1. The LMNA-bd2 sequence was amplified from the cDNA of mouse C2C12 cells using LMNA F2 and LMNAR2; the LMNA-bd1 sequence was amplified using LMNAF1 and LMNAR1. The TM2 sequence was amplified from the cDNA of mouse C2C12 cells using TM F2 and TM R2; the TM1 sequence was amplified using TM F1 and TMR1. The CC-SUN2 sequence was amplified from the cDNA of mouse C2C12 cells using CC F2 and SUN R2; the CC-SUN1 sequence was amplified using CC F1 and SUN R1. The amplified sequences and primer information are shown in Table 1.

[0072] Table 1 Primer information

[0073]

[0074] (3) The pSIN-LADS FRET BS plasmid was digested with SpeI and EcoRI, and then the ends were treated with CIP enzyme to recover the vector sequence. The vector, LMNA-bd2, TM2, YPet-F40-ECFP#1 and CC-SUN2 were connected by Gibson Assembly method to prepare NuTS2 plasmid, and the vector, LMNA-bd2, TM2 and YPet-F40-ECFP#2 were connected to prepare TL2 plasmid; the vector, LMNA-bd1, TM1, YPet-F40-ECFP#1 and CC-SUN1 were connected to prepare NuTS1 plasmid, and the vector, LMNA-bd1, TM1 and YPet-F40-ECFP#2 were connected to prepare TL1 plasmid. The cells were connected overnight at 16°C, and the ligation products were transfected into DH5α competent cells. Single clones were picked out by plating, and the correct plasmids were identified and selected by Sanger sequencing. Finally, NuTS2, TL2, NuTS1 and TL1 plasmids were obtained, and their nucleotide sequences were shown in SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26 and SEQ ID NO.27, respectively. NuTS2, TL2, NuTS1 and TL1 plasmids were transiently transfected into Hela cells using the lipo3000 transfection kit. After 36 hours of transfection, the expression results were as shown in Figure 2 shown.

[0075] Example 2. Live cell imaging system for nuclear mechanics is used to specifically detect substrate stiffness mechanical stimulation

[0076] First, prepare glass-bottomed polyacrylamide hydrogels. Treat 35 mm and 18 mm pore culture dishes with 0.1 M NaOH (Sigma-Aldrich) for 40 minutes, treat with 3-aminopropyltrimethylsilane for 5 minutes, rinse with ddH2O three times, cover with 0.5% glutaraldehyde for 30 minutes, and rinse with ddH2O three times. Then, add soft and hard polyacrylamide hydrogel premixes, cover with coverslips and flatten, and peel off the coverslips in ddH2O after curing for 30 minutes. Then, immerse the gel in 0.25 mg / mL Sulfo-SANPAH (Sigma-Aldrich) crosslinker, expose to UV light for 10 minutes, and rinse with PBS three times. Finally, incubate with 20 μg / mL fibronectin at 4°C overnight and incubate with 0.1% gelatin at 37°C for 1 hour. Transfected mammalian cells were cultured at 10,000 cells / cm 2 The cells were seeded at a density of 100 μg / mL and then inoculated on soft and hard gel surfaces 12 h later. They were cultured again for 24 h and images were taken using a Dragonfly confocal microscope system (Dragonfly CR-DFLY-202-40, USA). Finally, the changes in the FRET / ECFP ratio were statistically analyzed. If NuTS showed low FRET on the hard substrate surface compared with the TL control group, it means that the cell nucleus was subjected to greater force on the hard substrate surface; conversely, the cell nucleus showed less force on the soft substrate surface. The results are shown in Figure 2. Figure 3 shown.

[0077] Example 3. Live cell imaging system for nuclear mechanics is used to specifically detect hyperosmotic and hypoosmotic mechanical stimulation

[0078] Mammalian cells were transfected with NuTS and TL plasmids for 24 hours, respectively, and then transferred to glass-bottom dishes and cultured for another 12 hours. The cells were then placed under hypertonic conditions and time-lapse images were taken in DMEM medium supplemented with 0.4M sorbitol. Then, the hypertonic medium was removed and rinsed with PBS, and the hypotonic conditions were treated with DMEM medium containing 50% water, and time-lapse images were taken again. Finally, the changes in the FRET / ECFP ratio were statistically analyzed. Compared with the TL control group, NuTS showed low FRET in a hypertonic environment, indicating that the force on the cell nucleus was reduced; if NuTS showed little change in FRET in a hypotonic environment, it means that the force on the cell nucleus may not have changed, or the force on the cell nucleus may have exceeded the measurement range of the tension linker F40. The results are shown in Figure 2. Figure 4 shown.

[0079] Example 4. Live cell imaging system for nuclear mechanics is used to specifically detect local pressure mechanical stimulation

[0080] The micropipette was prepared by a MODEL P-1000 micropipette puller. The capillary (100 mm long, 1.0 mm outer diameter, 0.8 mm inner diameter) was fixed on the instrument. Then, after 24 hours, the mammalian cells transfected with NuTS and TL plasmids were transferred into a U-shaped chamber made of PDMS and cultured for another 12 hours. Finally, the micropipette was fixed on a microscope platform connected to a Dragonfly confocal microscope system, and the needle tip was slowly lowered to the cell nucleus area and local pressure stimulation was performed. FRET images were collected at the same time. Finally, the changes in the FRET / ECFP ratio were statistically analyzed. The results are shown in Figure 2. Figure 5 As shown, compared with the TL control group, NuTS exhibited higher FRET when subjected to local pressure, indicating that NuTS was compressed.

[0081] Example 5. Live Cell Imaging System for Nuclear Mechanics Dynamically Detecting Nuclear Mechanics During Somatic Development

[0082] (1) Construction of a plasmid with a T7 promoter (SEQ ID NO. 28) and a target insert (mechanical probe sequence)

[0083] The R3C1-pSTG-ef1a-R-GECO plasmid was digested with BamHI and NotI, and then the ends were treated with CIP enzyme to recover the vector sequence. The vector and NuTS2 fragment were connected using the Gibson Assembly method to prepare the T7-NuTS2 plasmid, and the vector and TL2 were connected to prepare the T7-TL2 plasmid. The connection was carried out overnight at 16 degrees, and the connection product was transfected into DH5α competent medium. The single clone was picked by plating, and the correct plasmid was identified and selected by Sanger sequencing, and finally T7-NuTS2 and T7-TL2 plasmids were obtained.

[0084] (2) Preparation of mRNA

[0085] The plasmid with T7 promoter and target insert was transcribed in vitro using a transcription kit, and the transcription product was then collected. Using a micro-glass tube for microinjection, 1-2 nL of probe mRNA was injected into 1-2 cell zebrafish embryos (within 0.75 hours after birth). Finally, the injected zebrafish embryos were cultured at 28°C to 18-24 hpf for the next experiment. The chorion of the target zebrafish embryo was peeled off with the needle tip of a 1mL syringe. 1% low-melting point agarose was melted at 65°C, covered on the embryo when cooled to 26-30°C, and while the agarose solidified, the embryo was placed in a lateral position using a blunt-tipped micropipette. Then, FRET data was collected using the Dragonfly confocal microscope system. Finally, changes in the FRET / ECFP ratio were statistically analyzed. The results are shown in Figure 6As shown, compared with the TL control group, the FRET distribution of NuTS in the zebrafish notochord was high at the tail end and low at the front end, indicating that the cell nuclei at the tail end of the zebrafish were subjected to less force, while the cell nuclei at the front end were subjected to greater force.

[0086] The above results show that the nuclear mechanics probe imaging system of the present invention can perform real-time dynamic imaging of nuclear mechanics in living cells, and can be used to monitor changes in nuclear mechanics of living cells when they are subjected to mechanical stimuli such as substrate stiffness, hyperosmotic and hypoosmotic, and squeezing. The present invention successfully realizes dynamic monitoring of nuclear mechanics at the cellular and animal levels, and provides technical support for building a research platform for nuclear mechanics and a related drug screening platform.

Claims

1. A probe composition for detecting nuclear mechanics, characterized in that: The probe composition includes NuTS1-FRET mechanical probe and NuTS2-FRET mechanical probe; the NuTS1-FRET mechanical probe is composed of LMNA-bd1, TM1, YPet fluorescent protein, tension linker F40, ECFP fluorescent protein and CC-SUN1; the NuTS2-FRET mechanical probe is composed of LMNA-bd2, TM2, YPet fluorescent protein, tension linker F40, ECFP fluorescent protein and CC-SUN2; the amino acid sequence of the LMNA-bd1 is shown in SEQ ID NO.29, and the amino acid sequence of the LMNA-bd2 is shown in SEQ ID NO.30; the amino acid sequence of the TM1 is shown in SEQ ID NO.31, and the amino acid sequence of the TM2 is shown in SEQ ID NO.32; the amino acid sequence of the YPet fluorescent protein is shown in SEQ ID NO.33; the amino acid sequence of the tension linker F40 is shown in SEQ ID NO.34; the amino acid sequence of the ECFP fluorescent protein is shown in SEQ ID The amino acid sequence of CC-SUN1 is shown in SEQ ID NO.36, and the amino acid sequence of CC-SUN2 is shown in SEQ ID NO.

37.

2. The probe composition according to claim 1, characterized in that The nucleotide sequence of the NuTS1-FRET mechanical probe is shown as SEQ ID NO.26; the nucleotide sequence of the NuTS2-FRET mechanical probe is shown as SEQ ID NO.

24.

3. A live cell imaging system for nuclear mechanics, characterized in that The living cell imaging system comprises the probe composition according to any one of claims 1 to 2.

4. The living cell imaging system according to claim 3, characterized in that: The live cell imaging system also contains a control probe; the control probe includes a TL1 mechanical probe and a TL2 mechanical probe; the TL1 mechanical probe is composed of LMNA-bd1, TM1, YPet fluorescent protein, tension linker F40 and ECFP fluorescent protein; the TL2 mechanical probe is composed of LMNA-bd2, TM2, YPet fluorescent protein, tension linker F40 and ECFP fluorescent protein; the amino acid sequence of the LMNA-bd1 is shown in SEQ ID NO.29, and the amino acid sequence of the LMNA-bd2 is shown in SEQ ID NO.30; the amino acid sequence of the TM1 is shown in SEQ ID NO.31, and the amino acid sequence of the TM2 is shown in SEQ ID NO.32; the amino acid sequence of the YPet fluorescent protein is shown in SEQ ID NO.33; the amino acid sequence of the tension linker F40 is shown in SEQ ID NO.34; the amino acid sequence of the ECFP fluorescent protein is shown in SEQ ID NO.

35.

5. The living cell imaging system according to claim 4, characterized in that: The nucleotide sequence of the TL1 mechanical probe is shown in SEQ ID NO.27, and the nucleotide sequence of the TL2 mechanical probe is shown in SEQ ID NO.

25.

6. The living cell imaging system according to claim 3, characterized in that: The NuTS1-FRET mechanical probe uses the SUN1 transmembrane protein as the target molecule; the NuTS2-FRET mechanical probe uses the SUN2 transmembrane protein as the target molecule.

7. A stably transfected cell line obtained by transfecting the live cell imaging system according to any one of claims 3 to 6.

8. A method for detecting the force level of a cell nucleus using the living cell imaging system according to claim 7, characterized in that: In vitro, NuTS2, TL2, NuTS1 and TL1 mechanical probes were expressed in mammalian cells, and the changes in the force level of the cell nucleus were detected by measuring the FRET / ECFP ratio.

9. Use of the probe composition according to any one of claims 1 to 2 and / or the living cell imaging system according to any one of claims 3 to 6 in the preparation of a product for visual detection and / or dynamic monitoring of nuclear mechanics.

10. The use according to claim 9, characterized in that: The application includes any one or more of the following 1)-4): 1) Use of the probe composition and / or the living cell imaging system in the preparation of a product for detecting and / or dynamically monitoring changes in the nucleus mechanics of living cells when they are stimulated by substrate stiffness mechanics; 2) Use of the probe composition and / or the living cell imaging system in the preparation of a product for detecting and / or dynamically monitoring changes in the nucleus mechanics of living cells when subjected to hypertonic and hypotonic mechanical stimulation; 3) Use of the probe composition and / or the living cell imaging system in the preparation of a product for detecting and / or dynamically monitoring changes in the nucleus mechanics of living cells when they are subjected to local pressure mechanical stimulation; 4) Use of the probe composition and / or the live cell imaging system in the preparation of a product for detecting and / or dynamically monitoring changes in nuclear mechanics during in vivo development.

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