Polypeptide for marking cytoskeleton and application of polypeptide as cytoskeleton biomarker
By developing the CSK-Tracker polypeptide sequence, actin and microtubules can be labeled simultaneously in the cytoskeleton, solving the problem of difficulty in simultaneous labeling in the prior art, and achieving no impact on the cell division process, providing a new biomarker selection.
Patent Information
- Application Number
- CN202510159865.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-13
- Publication Date
- 2025-05-09
AI Technical Summary
The prior art is difficult to effectively label actin and microtubules in the cytoskeleton at the same time, and it has a significant impact on biological processes such as cell division.
A polypeptide sequence, called CSK-Tracker, was developed, which can label actin and microtubule simultaneously and introduce it into cells through gene editing techniques to achieve labeling without affecting cell division.
Simultaneous labeling of actin and microtubules is achieved without affecting the cell division process, providing a new biomarker selection, filling the gap in the absence of live microtubules in the prior art.
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Figure CN119954912A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of cytoskeleton biomarkers, and in particular to a polypeptide for marking cytoskeleton and its application as a cytoskeleton biomarker. Background Art
[0002] The cytoskeleton generally refers to the network structure of fibers composed of proteins in the cytoplasm of cells. It is a dynamic structure, part of which is constantly destroyed, renewed or newly built, and some cells can move by destroying and rebuilding the skeleton. Cytoskeleton is found in cells of all biological domains of life (archaea, bacteria, eukaryotes) (especially in all eukaryotic cells, including human, animal and plant cells, and even bacteriophages).
[0003] The cytoskeleton not only plays an important role in maintaining cell morphology, bearing external forces, and maintaining the orderliness of the internal structure of cells, but also participates in many important life activities, such as: in cell division, the cytoskeleton pulls chromosome separation; in the transport of cell substances, various vesicles and organelles can be transported along the cytoskeleton; in muscle cells, the cytoskeleton and its binding proteins form a power system; in the migration of white blood cells (leukocytes), the movement of sperm, the extension of axons and dendrites of nerve cells, etc., they are all related to the cytoskeleton. In addition, in plant cells, the cytoskeleton guides the synthesis of cell walls. Therefore, in-depth research on the cytoskeleton has important significance for the development of the entire field of biology, especially biophysics.
[0004] Due to the large scale of the cytoskeleton, ordinary fluorescence microscopes in general laboratories can be used to observe the process of cell movement and cell division, and then study the biophysical changes of the whole process. The premise of observing the cytoskeleton is to fluorescently label the cytoskeleton, so as to observe the changes of the cytoskeleton more clearly and dynamically. The main components of the cytoskeleton are microfilaments (actin filaments) and microtubules. There are currently three main methods for dynamic labeling of the cytoskeleton of living cells, namely: 1. Nano-antibody labeling method. Nano-antibodies for actin have been developed, but nano-antibodies for microtubules have not yet been applied; 2. Phalloidin conjugate labeling method. This dye can dynamically stain actin, but dynamic imaging dyes for microtubules have not yet been developed, and the developed paclitaxel analogs destroy the cell division process; 3. Gene editing method. This method uses gene editing (knock-in) to knock fluorescent proteins, such as GFP, RFP and miRFP, into the genome after the ATG (start codon) of actin or microtubules to achieve fluorescent labeling, but this method will consume a lot of time and energy, especially editing two genes at the same time is extremely difficult. Summary of the invention
[0005] In view of the current limitations of biomarkers for dynamic observation of cytoskeleton, the present invention provides a polypeptide for labeling cytoskeleton and its application as a cytoskeleton biomarker.
[0006] A polypeptide for marking a cytoskeleton, whose amino acid sequence is shown in SEQ ID NO. 2 or SEQ ID NO. 4. The polypeptide is used as a cytoskeleton biomarker.
[0007] A polypeptide that simultaneously labels actin and microtubules, and its amino acid sequence is shown in SEQ ID NO.2. The polypeptide is obtained by translating the DNA with the base sequence shown in SEQ ID NO.1. The polypeptide is used as a biomarker that simultaneously labels actin and microtubules. A polypeptide sequence (named cytoskeleton tracker, CSK-Tracker) that can simultaneously label actin and microtubules, and has no significant effect on cell division and cell morphology, that is, cells can still divide and proliferate normally and maintain their original characteristics. This new cytoskeleton biomarker provides great convenience for the study of the cytoskeleton and provides a new biomarker option for cytoskeleton research.
[0008] A polypeptide for independently marking microtubules, whose amino acid sequence is shown in SEQ ID NO. 4. The polypeptide is obtained by translating the DNA with the base sequence shown in SEQ ID NO. 3. The polypeptide is used as a biomarker for independently marking microtubules.
[0009] Compared with the prior art, the present invention has the following advantages:
[0010] 1. The present invention is the first to use a single biomarker to simultaneously label actin and microtubules, while existing methods can only use different protein dyes or biomarkers to label them separately;
[0011] 2. The labeling of cytoskeletal proteins by the method of the present invention has no significant effect on biological processes such as cell division, while previously microtubules could not be labeled in living cells using biomarkers. For example, paclitaxel analogs, a marker of microtubules, can significantly inhibit the cell division process;
[0012] 3. The method of the present invention can conveniently and quickly label actin and microtubules without affecting processes such as cell division, and the stably transfected cell lines constructed by the method can be stored in liquid nitrogen for long-term use;
[0013] 4. If only the dynamic process of microtubules needs to be observed, the method of the present invention also provides MT-Tracker, a derivative of CSK-Tracker, to individually mark cytoskeleton microtubules, filling the gap in the field of lack of microtubule living cell markers. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 The CSK-Tracker of the present invention can successfully label the actin and microtubules constituting the cytoskeleton.
[0015] Figure 2 Destroying the cell cortex does not affect the spindle structure composed only of microtubules.
[0016] Figure 3 MT-Tracker only labels the spindle composed of microtubules and no longer labels actin.
[0017] Figure 4 This is the plasmid map of pcDNA3.1(+)-GFP-CSK-Tracker.
[0018] Figure 5 This is the map of pcDNA3.1(+)-GFP-MT-Tracker plasmid. DETAILED DESCRIPTION
[0019] CSK-Tracker (whose amino acid sequence is shown in SEQ ID NO.2, obtained by translating the DNA with the base sequence shown in SEQ ID NO.1) is relatively easy to use. It only needs to transfect the expression plasmid carrying the polypeptide sequence into the target cells for labeling (if the target cells need to be used in large quantities for a long time, stable transfection strains can be screened). Figure 1 As shown in Figure 2, CSK-Tracker can accurately locate to the cell cortex during the division phase by binding to actin enriched in the cortex. Figure 2 (E) is the localization of actin itself in dividing cells (the filamentous protrusions around the cells are the unique localization characteristics of actin filaments). After adding the actin inhibitor (ML-7), actin dissociates from the cortical layer, and the localization of CSK-Tracker in the cortical layer also begins to disappear ( Figure 2 D), thus CSK-Tracker can accurately label actin in living cells.
[0020] CSK-Tracker can also accurately label microtubules, another cytoskeletal protein. Figure 1 The spindles assembled by tubulin in the mitotic cells are clearly visible, and the addition of actin inhibitors does not affect the localization of CSK-Tracker on the spindles ( Figure 2 D), indicating that CSK-Tracker can accurately label microtubules independently of actin.
[0021] In addition, we have discovered a new application of CSK-Tracker by mutation. After the F35 of CSK-Tracker was mutated to A35, a new biomarker MT-Tracker (MicrotubuleTracker, MT-Tracker, whose amino acid sequence is shown in SEQ ID NO.4) that only marks intracellular microtubules was obtained. Figure 3 As shown, MT-Tracker localizes only to the spindle composed of microtubules and no longer labels actin.
[0022] Conclusion: CSK-Tracker can label actin and microtubules simultaneously without affecting the cell division process. Moreover, the labeling of the two cytoskeletal proteins is independent of each other. Therefore, it can be used as a new biomarker of the cytoskeleton.
[0023] Specific usage of CSK-Tracker and MT-Tracker:
[0024] 1. Construct a gene expression vector according to the following CSK-Tracker DNA sequence: Select a gene synthesis company to perform gene synthesis according to the following sequence, select XbaI as the restriction site, and the final constructed expression vector sequence and plasmid map are attached (pcDNA3.1(+)-GFP-CSK-Tracker, such as Figure 4 As shown in ); For MT-Tracker, the construction method is the same as CSK-Tracker, the complete expression vector sequence and plasmid map are attached (pcDNA3.1(+)-GFP-MT-Tracker, as shown in Figure 5 shown).
[0025] 2. Preparation of HEK293 cells: Cultured HEK293 cells (other types of mammalian cells such as MCF-10A and U2OS can also be used) are plated into 6-well plates, with about 3 x 10 cells per well. 5 The cells were cultured in DMEM medium containing 10% FBS for 24-36 hours (the specific time was determined according to the cell status) to make the final cell density between 70% and 90%.
[0026] 3. Cell transfection experiment: a. Mix the liposome transfection reagent and the expression plasmid (2-3μg) in a 1:1 ratio and let it stand for 15 minutes (the operation methods of different brands of transfection reagents are slightly different, please refer to the instructions); b. Aspirate the original culture medium in the 6-well plate, then add fresh complete culture medium and culture in a 37℃ cell culture incubator for 6-12 hours (less than 6 hours will reduce the transfection efficiency, and more than 12 hours will cause the transfection reagent to be toxic to the cells); c. After 12 hours, replace with fresh complete culture medium and continue to culture for 24 hours.
[0027] 4. Positive cell screening: a. After culturing for 24 hours, discard the original culture medium and add fresh complete culture medium containing G418 antibiotic (working concentration is 100 μg / ml) for positive cell screening; b. Replace the complete culture medium containing G418 antibiotic every 2 days to maintain the antibiotic within the working concentration range; c. After about 10-14 days, almost all HEK293 cells that are not stably transfected will die, and the remaining cells are the successfully transfected cells; d. Use the limiting dilution method to plate the stably transfected cells in a 96-well plate and continue to culture for 2 weeks, then select the monoclonal clone with green fluorescence under fluorescence microscopy for expansion culture; e. Freeze 3-5 tubes of cells for future use, and plate the remaining cells in a 24-well plate (5x 10 4 cells / well) for subsequent observation experiments.
[0028] 5. Observation of labeled cells: After 24 hours of culture, the stably transfected cells in the 24-well plate can be observed using a fluorescence microscope. The localization of CSK-Tracker on actin and microtubules can be clearly observed using a 60x or 100x lens. At this point, the CSK-Tracker labeled cells prepared by the above steps can be used for downstream experiments related to cell movement and cell division.
Claims
1. A polypeptide for labeling a cytoskeleton, characterized in that: Its amino acid sequence is shown in SEQ ID NO.2 or SEQ ID NO.
4.
2. Use of the polypeptide according to claim 1 as a cytoskeleton biomarker.
3. A polypeptide for simultaneously labeling actin and microtubules, characterized in that: Its amino acid sequence is shown in SEQ ID NO.
2.
4. The polypeptide according to claim 3, characterized in that Obtained by translating the DNA with the base sequence shown in SEQ ID NO.
1.
5. Use of the polypeptide according to claim 3 or 4 as a biomarker for simultaneously labeling actin and microtubules.
6. A polypeptide for individually labeling microtubules, characterized in that: Its amino acid sequence is shown in SEQ ID NO.
4.
7. The polypeptide according to claim 6, characterized in that Obtained by translating the DNA with the base sequence shown in SEQ ID NO.
3.
8. Use of the polypeptide according to claim 6 or 7 as a biomarker for independently marking microtubules.