Method for realizing synthesis of specific membrane-free organelles by utilizing optogenetics technology and application

Abstract

The invention relates to a method for realizing synthesis of a specific membrane-free organelle by utilizing an optogenetics technology and application, and belongs to the technical field of biology. The method comprises the following steps: firstly, screening a recombinant protein for specifically synthesizing the membrane-free organelle, transfecting the recombinant protein into a to-be-observed cell, and accurately regulating and controlling the dynamic assembly process of the membrane-free organelle in the cell in real time by utilizing a light-operated tool. The method not only avoids the limitation of static observation of a fixed cell technology, but also overcomes the problems of large interference of a fluorescence labeling and living cell imaging technology on an intracellular environment and inaccurate dynamic observation, and provides a powerful technical support for deeply researching abnormal behaviors of neurodegenerative disease related proteins in a membraneless organelle and a disease occurrence mechanism.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method and application for synthesizing specific membraneless organelles using optogenetics. Background Technology

[0002] Research on neurodegenerative diseases has always been a hot topic and a challenge in the life sciences. These diseases, including Alzheimer's and Parkinson's, impose a heavy burden on patients and their families. With the continuous advancements in molecular and cell biology, it has become increasingly clear that the occurrence of neurodegenerative diseases is closely related to the abnormal behavior of various intracellular macromolecules. Among them, non-membrane organelles play a crucial role in the development and progression of these diseases. Non-membrane organelles are intracellular microstructures without membrane enclosures. They are formed through interactions between biomolecules such as proteins and nucleic acids, and participate in various intracellular physiological processes, such as signal transduction and gene expression regulation. However, the dynamic assembly process of non-membrane organelles and their specific mechanisms of action in neurodegenerative diseases remain poorly understood.

[0003] In traditional cell biology research, cell fixation is widely used to observe intracellular structures. Researchers typically treat cells with chemical fixatives (such as paraformaldehyde) to immobilize intracellular macromolecules in specific locations, and then observe them using techniques such as electron microscopy. This method can reveal, to some extent, the morphology and composition of membraneless organelles within cells. For example, when observing fixed cells under a transmission electron microscope, membraneless organelles can be found to exhibit structures with a certain density and morphology. Immunoelectron microscopy combined with specific antibodies can also locate and identify protein components. However, cell fixation has significant drawbacks. First, it only provides static images of cells at a fixed point in time, failing to capture the dynamic assembly and deassembly processes of membraneless organelles. The dynamic nature of membraneless organelles is one of their important physiological characteristics, and this technique cannot meet the needs of studying their dynamic behavior. Second, the use of fixatives may interfere with the structure and interactions of intracellular macromolecules, leading to discrepancies between the observed structures and components and the actual situation in living cells. Existing techniques cannot meet the need for real-time observation of the dynamic assembly process of membraneless organelles. There is a problem in continuously and accurately capturing the complete dynamic process of non-membrane organelles from formation to depolymerization. This dynamic process is crucial for understanding the role of non-membrane organelles in neurodegenerative diseases. For example, during the occurrence of neurodegenerative diseases, the assembly process of non-membrane organelles may exhibit abnormalities, such as accelerated assembly speed and difficulty in depolymerization. These dynamic changes may be closely related to the occurrence and development of the disease, but current technologies struggle to accurately monitor them. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problems of existing cell imaging technology, which has great interference with the intracellular environment, inaccurate dynamic observation, and inability to continuously and accurately capture the complete dynamic process of non-membrane organelles from formation to depolymerization.

[0005] To address the aforementioned technical problems, this invention provides a method and application for the synthesis of specific membraneless organelles using optogenetics. The recombinant protein of this invention is transfected into cells to be observed, and using light-controlled tools, the dynamic assembly process of membraneless organelles can be precisely and in real-time regulated within the cell. This not only avoids the limitations of static observation using fixed cell techniques but also overcomes the problems of significant interference with the intracellular environment and inaccurate dynamic observation caused by fluorescent labeling and live-cell imaging techniques. This provides strong technical support for in-depth research on the abnormal behavior of proteins related to neurodegenerative diseases in membraneless organelles and the mechanisms of disease development.

[0006] The working principle of this invention is as follows: This invention utilizes a dual-module optogenetic system. The first module is iLID-EGFP-FTH1, where FTH1 ferritin can self-aggregate into 12 nm diameter spherical 24-mers, forming a core. iLID can bind to SspB in the second module, IDR-mCherry-SspB, under blue light irradiation. SspB can fuse with various intrinsically disordered regions (IDRs) with phase-separation properties as an intrinsic driving force (in this invention, amino acid sequences 1-251 of the motor neuron survival protein are IDRs), thereby bringing the protein to the core. Through the interaction of iLID-IDRs, aggregates are formed. Proteins without phase-separation properties cannot form aggregates. By adding nuclear localization signals (NLS) to the iLID-EGFP-FTH1 module, the assembly process and properties of non-membrane organelles (Cajal bodies) in the protein nucleus can be specifically studied.

[0007] The first objective of this invention is to provide a recombinant protein for constructing membraneless organelles, the recombinant protein comprising a first recombinant protein and a second recombinant protein, the first recombinant protein containing a nuclear localization sequence, a photoinducible dimer downstream of the nuclear localization sequence, and ferritin heavy chain 1 downstream of the photoinducible dimer, the second recombinant protein comprising motor neuron survival protein and SspB downstream of the motor neuron survival protein, wherein the amino acid sequence of the motor neuron survival protein is shown in SEQ ID NO. 2.

[0008] Furthermore, SEQ ID NO.2:

[0009] MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSDIWDDTALIKAYDKAVASFKHALKNGDICETSGPKTTPKRKPAKKNKSQKKNTAASLQQWKVGDKCSAIWSEDGCIYPATIASIDFKRETCVVV YTGYGNREEQNLSDLLSPICEVANNIEQNAQENENESQVSTDESENSRSPGNKSDNIKPKSAPWNSFLPPPPMPGPRLGPGKPGLKFNGPPPPPPPPPPHLLSCWLPPFPSGPPIIPPPPPPICP.

[0010] Furthermore, the nuclear localization sequence is shown in SEQ ID NO.1, the NCBI number of the photoinduced dimer is 4WF0_A, and the NCBI number of the ferritin heavy chain 1 is AAI05803.

[0011] Furthermore, SEQ ID NO.1: AEPRSKRPRVTLPACPAHSGEF.

[0012] Furthermore, the amino acid sequence encoding the SspB is shown in SEQ ID NO.3.

[0013] Furthermore, SEQ ID NO.3:

[0014] SSPKRPKLLREYYDWLVDNSFTPYLVVDATYLGVNVPVEYVKDGQIVLNLSASATGNLQLTNDFIQFNARFKGVSRELYIPMGAALAIYARENGDGVMFEPEEIYDELNI.

[0015] Furthermore, the membraneless organelles include the Cajal body.

[0016] A second object of the present invention is to provide a recombinant cell containing a non-membrane organelle forming protein, wherein the recombinant cell contains the aforementioned recombinant protein.

[0017] A third objective of this invention is to provide a method for synthesizing membraneless organelles by introducing the aforementioned recombinant protein into a host cell.

[0018] The fourth objective of this invention is to provide a method for dynamically monitoring the assembly and disassembly of membraneless organelles, the method comprising the following steps:

[0019] S1. The recombinant protein is introduced into cells to be observed and cultured to obtain labeled cells to be observed, wherein the first recombinant protein is labeled with a first fluorescent protein and the second recombinant protein is labeled with a second fluorescent protein;

[0020] S2. Place the labeled cells to be observed from S1 into a fluorescence imaging device and perform fluorescence excitation. Record the fluorescence labeling signal within the cells. When fluorescence excitation is performed, the membraneless organelles assemble; when fluorescence excitation is not performed, the membraneless organelles disassemble.

[0021] Furthermore, the wavelength of the fluorescence is between 405nm and 499nm.

[0022] Furthermore, the first fluorescent protein and the second fluorescent protein are independently selected from any one of green fluorescent protein, red fluorescent protein, enhanced green fluorescent protein, and blue fluorescent protein, and are not the same as each other.

[0023] A fifth object of the present invention is to provide the use of the above-described recombinant protein or the above-described recombinant cells in the preparation of diagnostic or therapeutic products for neurodegenerative diseases.

[0024] Furthermore, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, and ALS.

[0025] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0026] (1) Real-time dynamic observation: Through light-controlled technology, the dynamic assembly process of membraneless organelles can be precisely and continuously regulated within cells. Researchers can control the formation and disassembly of membraneless organelles like operating a switch, thereby clearly observing their formation mechanism. This overcomes the limitation of fixed cell technology, which can only provide static images, and can continuously and accurately capture the complete dynamic process of membraneless organelles from formation to disassembly.

[0027] (2) Reduced interference with the intracellular environment: The light-controlled technology avoids the interference of fixatives on the structure and interactions of intracellular biomolecules, ensuring that the observed structures and components are closer to the real situation in living cells. This overcomes the problems of large interference with the intracellular environment and inaccurate dynamic observation caused by fluorescent labeling and live-cell imaging technologies.

[0028] (3) In-depth study of disease mechanisms: This method provides strong technical support for in-depth research on the abnormal behavior of proteins related to neurodegenerative diseases in non-membrane organelles and the mechanisms of disease development. By precisely controlling the dynamic assembly of non-membrane organelles, researchers can better understand the specific mechanisms of action of non-membrane organelles in neurodegenerative diseases, providing new ideas and methods for the diagnosis and treatment of diseases. Attached Figure Description

[0029] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0030] Figure 1 This invention is the SMN-Corelet NLS System working principle diagram;

[0031] Figure 2 This is a diagram showing the experimental results of the present invention for monitoring the dynamic assembly process of membraneless organelles;

[0032] Figure 3 This is an experimental result diagram showing the ability of the dual-module identification system of the present invention to detect the dynamic assembly of membraneless organelles. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0034] Example 1: SMN-Corelet NLS System preparation

[0035] 1. Gene cloning (taking pcDNA3.1-NLS-ILID-EGFP-FTH1 as an example)

[0036] 1.1 PCR

[0037] The reaction system (25 µL) is shown in Table 1.

[0038] Table 1 Reaction System

[0039]

[0040] Gently mix the mixture, centrifuge, and run the PCR program. The PCR program settings are shown in Table 2.

[0041] Table 2 PCR reaction system

[0042]

[0043] 1.2 Agarose NMR Gel Electrophoresis

[0044] Place an appropriate amount of agarose powder in a beaker, add an appropriate amount of 1×TAE buffer, microwave for 2-3 minutes to fully dissolve the agarose, let stand for 3-5 minutes, then add GelGreen (7 µL of GelGreen per 100 mL of 1×TAE), gently shake to mix, and pour smoothly into an agarose nucleic acid gel loading plate. Insert the comb and let stand for 20 minutes. After the nucleic acid gel solidifies, carefully remove the comb and place the loading plate smoothly into a horizontal electrophoresis tank, immersing the gel in the 1×TAE solution. Mix the PCR product with an equal proportion of 10×Loading Buffer, carefully load the sample into the wells, and add an appropriate volume of DNA Marker every other well. After carefully assembling the electrophoresis tank, turn on the power and perform electrophoresis at a constant voltage of 140 V. Note the electrophoresis time; generally, the electrophoresis time is set to 30 minutes.

[0045] 1.3 Glue Recycling

[0046] Gel recovery was performed using a gel recovery kit provided by Novizan Biotechnology Co., Ltd., following these steps:

[0047] (1) After electrophoresis is completed, turn off the electrophoresis apparatus, transfer the gel to the blue light irradiation device, quickly cut off the gel block containing DNA fragments, put it into a clean 1.5 mL EP tube prepared in advance, thoroughly break up the gel with the pipette tip, and weigh the gel (weigh the empty tube in advance).

[0048] (2) Add an equal volume of Buffer GDP according to the principle that 1 mg of gel is equivalent to 1 µL of Buffer GDP, and place it in a 55°C water bath for 8-10 min to allow the gel block to dissolve completely.

[0049] (3) After the gel block has completely melted, use a palm-sized centrifuge to briefly centrifuge to remove the liquid on the tube wall, transfer the reaction solution to the DNA adsorption column, and centrifuge at 12000 rpm for 1 min.

[0050] (4) Discard the filtrate, add 300 µL Buffer GDP, let stand for 1 min, and centrifuge at 12000 rpm for 1 min.

[0051] (5) Discard the filtrate and add 700 µL of Buffer GW (containing anhydrous ethanol) to the adsorption column. Centrifuge at 12000 rpm for 1 min.

[0052] (6) Repeat the previous step.

[0053] (7) Discard the filtrate and centrifuge at 12,000 rpm for 2 min.

[0054] (8) Transfer the adsorption column to a 1.5 mL clean EP tube, carefully drop 30 µL ddH2O (sterile) into the center of the adsorption column, and let stand for 1 min.

[0055] (9) Centrifuge at 12000 rpm for 1 min to obtain DNA aqueous solution.

[0056] 1.4 Enzyme digestion (taking pcDNA3.1-NLS-ILID-EGFP-FTH1 plasmid as an example)

[0057] (1) The NLS-ILID-EGFP-FTH1 DNA fragment was digested with enzymes, and the system is shown in Table 3.

[0058] Table 3 System configuration of NLS-ILID-EGFP-FTH1

[0059]

[0060] (2) The pcDNA3.1 / Myc-HisA vector (20 µL) was digested with enzymes. The plasmid system is shown in Table 4.

[0061] Table 4 System configuration of plasmids

[0062]

[0063] Place in a 37℃ bath for enzyme digestion for 2-4 hours.

[0064] 1.5 Agarose NMR Gel Electrophoresis

[0065] The operating steps are as described in 1.2.

[0066] 1.6 Glue Recycling

[0067] The operation steps are as described in 1.3. Finally, the product is recovered by eluting with 12 µL ddH2O (note the labeling of fragments and carriers).

[0068] 1.7 Connection

[0069] The ligation system consisted of Solution I (6 µL), vector digestion product (1 µL), and fragment digestion product (5 µL). The ligation system was placed in a PCR instrument and ligated at 16°C for 1–2 h.

[0070] 1.8 Transformation

[0071] (1) Place the LB culture plate containing Ampi+ resistance stored in a 4℃ refrigerator in a 37℃ biochemical constant temperature incubator in advance to dry the surface moisture. At the same time, turn on the water bath and set the temperature to 42℃ to prepare for the subsequent steps.

[0072] (2) After taking the competent state out of the -80℃ freezer, quickly place it on ice to thaw for 5-7 minutes.

[0073] (3) Light the alcohol lamp, disinfect the product, carefully aspirate the ligation product into the competent bacteria, gently blow and mix, and let stand on ice for 20 minutes.

[0074] (4) Quickly place the mixture in a 42°C water bath for 80 seconds, then quickly place it on ice for 3-5 minutes.

[0075] (5) Mark the dried LB plates, spread the bacteria evenly on the plates with a sterilized spreader, and invert them in a 37°C biochemical incubator for 13-15 hours (the spreader should be brought back to room temperature before use).

[0076] 1.9 Inoculation with bacteria

[0077] (1) Light the alcohol lamp and pour 5 mL of LB liquid culture medium containing Amp+ resistance into a 50 mL clean centrifuge tube next to the alcohol lamp.

[0078] (2) Carefully pick up the single clone of the strain growing on the LB plate with the tip of a 200 µL pipette, place it in LB liquid medium, and mix it by repeatedly pipetting.

[0079] (3) Place the bottle at an angle in a 37℃ biochemical constant temperature incubator. Do not tighten the cap. Set the rotation speed to 250 rpm and incubate for 13-15 hours.

[0080] 1.10 Preservation of bacteria

[0081] Take a clean 1.5 mL EP tube, and separately pipette 400 µL of 80% glycerol and 600 µL of cultured bacteria. Mix well, label, and store at -80℃.

[0082] 1.11 Plasmid Extraction

[0083] Plasmid extraction was performed using a small-scale extraction kit provided by Novizan Biotechnology Co., Ltd., following these steps:

[0084] (1) Add all of the RNase A solution provided by the small sample kit to Buffer P1 in advance and store it in a 4°C refrigerator for later use. Add 80 mL of anhydrous ethanol to Buffer PW2.

[0085] (2) Place the centrifuge tube containing 5 mL of bacterial solution in a benchtop centrifuge and centrifuge at 5000 rpm for 7 min. After centrifugation, pour out the supernatant.

[0086] (3) Add 250 μL of Buffer P1 to the centrifuge tube containing the bacterial precipitate, gently pipette, and transfer the well mixed bacterial solution to a clean 1.5 mL EP tube.

[0087] (4) Add 250 μL Buffer P2 and invert it 8-10 times. Be careful not to invert it too much.

[0088] (5) Add 350 μL Buffer P3, invert 8-10 times, being careful not to invert too much, and centrifuge at 12000 rpm for 15 min.

[0089] (6) Carefully aspirate the supernatant onto the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the filtrate.

[0090] (7) Add 500 μL Buffer PW1 to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0091] (8) Add 600 μL Buffer PW2 to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0092] (10) Repeat the previous step.

[0093] (11) Centrifuge at 12,000 rpm for 1 min (to completely remove the residual washing liquid in the adsorption column).

[0094] (12) Place the adsorption column in a clean 1.5 mL EP tube, carefully drop 30 µL ddH2O (sterile) into the center of the adsorption column, and let stand for 1 min.

[0095] (13) Centrifuge at 12,000 rpm for 1 min to obtain DNA aqueous solution, label it and store it at 4℃.

[0096] 1.12 Enzyme digestion identification

[0097] The enzyme digestion reaction system is shown in Table 5.

[0098] Table 5. Preparation of the enzyme digestion system

[0099]

[0100] The enzyme was digested in a 37°C water bath for 1 hour, and the cloning success was determined based on the size of the target fragment.

[0101] 1.13 Sequencing

[0102] The cloning-related sequencing was performed by Suzhou Genewiz Biotechnology Co., Ltd., and the sequencing results were compared and verified using SnapGene software. Similarly, pcDNA3.1-SMN-mCherry-SspB was prepared according to the above method.

[0103] 2.1 Cell resuscitation

[0104] (1) Adjust the water bath temperature to 37°C in advance, put on protective goggles, carefully take the frozen cells out of the liquid nitrogen tank, and quickly place them in a 37°C water bath to thaw.

[0105] (2) Transfer the completely thawed cells into a clean 5 mL EP tube, rinse the cryovial with 1 mL of preheated cell culture medium containing 10% FBS, and then add the thawed cells to the tube. Centrifuge at 1000 rpm for 3 min.

[0106] (3) Discard the supernatant, gently pipette the cells with 1 mL of cell culture medium containing 10% FBS, combine the cell suspension with 3 mL of cell culture medium and add it to a cell culture dish (middle dish) with a diameter of 60 mm. Mix well and place in a 37℃, 5% CO2 cell culture incubator.

[0107] 2.2 Cell passage (using a 60 mm diameter cell culture dish as an example)

[0108] Observe the cell state and density under a microscope. When the cell density is about 90%, it is ready for passage.

[0109] (1) Preheat cell culture medium containing 10% FBS, 0.05% trypsin, and 1×PBS.

[0110] (2) Discard the old culture medium and slowly add 1 mL of 1×PBS along the wall of the dish to wash once.

[0111] (3) Discard the PBS, slowly add 1 mL of 0.05% trypsin along the wall of the dish, and quickly transfer it to a 37°C, 5% CO2 cell culture incubator for 1 min of digestion.

[0112] (4) After digestion is complete, add 1 mL of cell culture medium containing 10% FBS to stop digestion, place 2 mL of cell suspension in a clean 5 mL EP tube, and centrifuge at 1000 rpm for 3 min.

[0113] (5) Discard the supernatant, gently pipette the cells with 1 mL of cell culture medium containing 10% FBS, take 200-250 µL of cell suspension into a medium dish containing 4 mL of fresh culture medium, mix well, label the cell type, passage number and date, and place in a 37℃, 5% CO2 cell culture incubator.

[0114] 2.3 Cell transfection (taking one well of a 24-well plate as an example)

[0115] Observe the cell state and density under a microscope. When the cell density is about 60%, the transfection experiment can be carried out.

[0116] (1) Prepare two clean 1.5 mL EP tubes, labeled A and B respectively. Add 50 µL of OMEM to tube A and tube B respectively.

[0117] (2) Add 1.0 µg pcDNA3.1-NLS-ILID-EGFP-FTH1 and 1.2 µg pcDNA3.1-SMN-mCherry-SspB to tube A, and add 2-2.2 µL of Hieff Transfection reagent to tube B. TM Liposomal Transfection Reagent.

[0118] (3) Add the liquid in tube B to tube A, mix thoroughly, and let stand for 15-20 minutes.

[0119] (4) Discard the old culture medium in the well plate, add 100 µL of fresh OMEM to the mixture in tubes A and B, mix well and slowly add it to the well plate along the well wall.

[0120] (5) After 2-4 hours, add 200-250 µL of cell culture medium containing 10% FBS.

[0121] 2.4 Live-cell imaging

[0122] Cell status and transfection efficiency are observed under a microscope. When the cell status is good and the transfection efficiency reaches the expected level, live cell imaging experiments can be performed.

[0123] (1) Prepare imaging equipment

[0124] Ensure that the live cell imaging system (such as a confocal microscope) is correctly installed and calibrated, including setting environmental parameters such as temperature and CO2 concentration to 37°C and 5% CO2 to maintain normal cell growth conditions.

[0125] Check that the microscope's objectives, filter sets, laser, and other key components are in optimal working condition to ensure image quality.

[0126] Preparation of cell culture dishes: Ensure that the bottom of the culture dish is clean and free of residual culture medium or impurities, so as not to affect the imaging effect.

[0127] (2) Imaging condition settings

[0128] Choose appropriate excitation and emission wavelengths based on the cell's fluorescent labeling. For example, for EGFP-labeled cells, the excitation wavelength is typically 488 nm and the emission wavelength is 507 nm; for mCherry-labeled cells, the excitation wavelength is 587 nm and the emission wavelength is 610 nm.

[0129] To obtain a sharp image without overexposure, you need to set appropriate exposure time and gain. Preliminary experiments are usually required to optimize these parameters.

[0130] (3) Imaging process

[0131] Place the cell culture dish on the microscope stage and adjust the focus to make the cells clearly visible.

[0132] Choose a suitable field of view, ensuring there are enough cells for observation. You can use a low-power objective (e.g., 10×) for initial positioning, then switch to a high-power objective (e.g., 40× or 60×) for detailed observation.

[0133] Initiate imaging and record the fluorescence signals of the cells. The imaging time interval can be set (e.g., every 10 seconds) to capture dynamic changes in the cells. Depending on experimental needs, imaging can last for hours or even days.

[0134] (4) Immunofluorescence labeling of important components of non-membrane organelles

[0135] At the end of the experiment, the cells were washed once with PBS, then fixed with 4% PFA, perforated with 0.1% Triton X-100, and washed with PBS in sequence. Coilin (a constituent protein of the Cajal body) primary antibody dilution buffer was added and incubated at room temperature for 2-3 hours. The cells were washed three times with PBST, and fluorescently conjugated secondary antibody dilution buffer was added, followed by incubation in the dark for 1-2 hours. The processed cell samples were washed and soaked with PBS, stored at 4°C, and observed and recorded using a laser confocal microscope.

[0136] (5) Data recording and analysis

[0137] During imaging, information such as cell fluorescence intensity and morphological changes is recorded in real time. Data acquisition and preliminary analysis can be performed using the microscope's accompanying software.

[0138] The results are as follows Figure 2 and Figure 3 As shown, SMN was overexpressed in the HEK293 cell line. TudorThe SMN-Corelets dual-carrier system did not respond quickly to 488 nm light stimulation and form intranuclear condensates. However, the SMN-Corelets dual-carrier system, upon stimulation with a 488 nm laser, rapidly forms condensates in the cell nucleus. This process can be observed and recorded in real-time under laser confocal microscopy. Subsequent studies using Coilin staining, a marker of condensate formation, confirmed that the condensates formed by light stimulation using the SMN-Corelets dual-carrier system are indeed condensates. The successful establishment of this model provides a tool for our subsequent photocontrolled regulation of biologically functional intracellular membraneless organelles.

[0139] Comparative Example 1: SMN Tudor - Construction of the Corelets Dual-Carrier System

[0140] The SMN portion in the plasmid pcDNA3.1-SMN-mCherry-SspB was replaced with SMN-Tudor to obtain the plasmid pcDNA3.1-SMN-Tudor-mCherry-SspB. The remaining steps were the same as in Example 1 to obtain SMN. Tudor -Corelets dual-carrier system. The amino acid sequence of the Tudor domain of the motor neuron survival protein is shown in SEQ ID NO.4 (GDKCSAIWSEDGCIYPATIASIDFKRETCVVVYTGYGNREEQNLSDLLS). Figure 2 As shown, SMN was overexpressed in the HEK293 cell line. Tudor The Corelets dual-carrier system does not respond quickly to 488 nm light stimulation and form intranuclear condensates.

[0141] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A recombinant protein for constructing membraneless organelles, characterized in that, The recombinant protein includes a first recombinant protein and a second recombinant protein. The first recombinant protein contains a nuclear localization sequence, a photoinducible dimer downstream of the nuclear localization sequence, and ferritin heavy chain 1 downstream of the photoinducible dimer. The second recombinant protein includes motor neuron survival protein and SspB downstream of the motor neuron survival protein. The amino acid sequence of the motor neuron survival protein is shown in SEQ ID NO.

2.

2. The recombinant protein according to claim 1, characterized in that, The nuclear localization sequence is shown in SEQ ID NO.1, and the amino acid sequence encoding the SspB is shown in SEQ ID NO.

3.

3. The recombinant protein according to claim 1, characterized in that, The NCBI number of the photoinduced dimer is 4WF0_A, and the NCBI number of the ferritin heavy chain 1 is AAI05803.

4. The recombinant protein according to claim 1, characterized in that, The membraneless organelles include the Cajal body.

5. A recombinant cell containing proteins that form non-membrane organelles, characterized in that, The recombinant cells contain the recombinant protein according to any one of claims 1-4.

6. A method for synthesizing membraneless organelles, characterized in that, The recombinant protein according to any one of claims 1-4 is introduced into a host cell and then excited with fluorescence.

7. A method for dynamically monitoring the assembly and disassembly of membraneless organelles, characterized in that, The method includes the following steps: S1. The recombinant protein according to any one of claims 1-4 is introduced into cells to be observed and cultured to obtain labeled cells to be observed, wherein the first recombinant protein is labeled with a first fluorescent protein and the second recombinant protein is labeled with a second fluorescent protein; S2. Place the labeled cells from S1 into a fluorescence imaging device and perform fluorescence excitation to record the fluorescent labeling signal within the cells.

8. The method according to claim 7, characterized in that, The wavelength of the fluorescence is between 405 nm and 499 nm.

9. The method according to claim 7, characterized in that, The first fluorescent protein and the second fluorescent protein are independently selected from any one of green fluorescent protein, red fluorescent protein, enhanced green fluorescent protein, and blue fluorescent protein, and are not the same as each other.

10. The use of the recombinant protein according to any one of claims 1-4 or the recombinant cell according to claim 5 in the preparation of diagnostic or therapeutic products for neurodegenerative diseases.

Images