A method for detecting protein liquid phase separation and application thereof

Abstract

The application discloses a detection method for liquid phase separation of proteins and application thereof, and the method comprises the following steps: constructing a first expression vector for expressing an energy donor fluorescent molecule and a target protein, and constructing a second expression vector for expressing an energy acceptor fluorescent molecule and the target protein; transferring the first expression vector and the second expression vector into primary cultured cells; exciting the primary cultured cells into which the first expression vector and the second expression vector are transferred; collecting fluorescent microscopic imaging data by using a fluorescence microscope; calculating the change of fluorescence resonance energy transfer efficiency; and detecting the process of liquid phase separation of proteins. The application realizes, for the first time, the detection of liquid phase separation of proteins in a synapse of a living neuron on a sub-second time scale, bypasses the bottleneck of super-resolution fluorescence imaging technology, avoids the toxicity of strong laser to cells, and can be used for detecting the liquid phase separation phenomenon in a synapse with a diameter of only 200-800 nanometers.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for detecting liquid-liquid phase separation of proteins and application thereof. BACKGROUND

[0002] Studies have shown that a variety of synaptic proteins can undergo liquid-liquid phase separation in vitro, and some of them are directly related to neurodegenerative diseases, so some researchers have proposed a theory that protein phase transition out of control leads to neurodegenerative diseases. However, only direct observation and characterization of protein liquid-liquid phase separation in synapses can prove this theory and possibly open up new research directions.

[0003] Existing methods for detecting protein liquid-liquid phase separation in vitro include fluorescence microscopy and light scattering methods. Among them, the most common method is to adjust various physical and chemical conditions (such as temperature, protein concentration, salt concentration, etc.), use fluorescence microscopy to observe the formation of sub-micron to micron-sized droplet-shaped objects (i.e. condensed phase), and then use fluorescence recovery after photobleaching (FRAP) to determine the flowability of proteins in the condensed phase to determine the morphology of the material after phase transition. At the same time, the formation of condensed phase also increases the light scattering in the visible wavelength range, so this method can be used to detect protein liquid-liquid phase separation.

[0004] Synapses are the structural and functional units of connection and communication between all neurons, with a diameter of only 200-800 nanometers, so it is not possible to detect protein liquid-liquid phase separation in synapses by observing the formation of sub-micron to micron-sized condensed phase. At present, some researchers try to use various super-resolution fluorescence imaging techniques to observe nanoscale condensates formed by proteins. However, super-resolution fluorescence imaging has high hardware requirements (expensive microscopes) and application limitations (usually used for fixed cell samples). Since photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) are both based on repeated excitation, observation, bleaching of single activated fluorescent molecules and calculation to obtain localization, there is an upper limit on imaging speed, and the use of strong laser light can poison cells, so it is not conducive to detecting the process of liquid-liquid phase separation in living neurons. SUMMARY

[0005] The present application aims to detect the phase transition of proteins in a neural synapse under various neural stimuli on a sub-second time scale, and to solve the problems in the prior art, the present application provides a protein liquid phase separation detection method and application thereof. The process of protein phase transition (i.e. the formation of liquid phase separation) can cause changes in fluorescence resonance energy transfer efficiency, so that the protein phase transition can be detected by measuring and calculating the changes in energy transfer efficiency.

[0006] The first aspect of the present application provides a protein liquid phase separation detection method, comprising the following steps:

[0007] Constructing a first expression vector expressing an energy donor fluorescent molecule and a target protein, and a second expression vector expressing an energy acceptor fluorescent molecule and a target protein;

[0008] Transferring the first expression vector and the second expression vector into primary cultured cells;

[0009] Exciting the primary cultured cells into which the first expression vector and the second expression vector are transferred, and collecting fluorescence microscopy imaging data using a fluorescence microscope;

[0010] Calculating the change in fluorescence resonance energy transfer efficiency to detect the process of protein liquid phase separation.

[0011] Further, the target protein is a neural synapse protein.

[0012] The primary cultured cells are primary cultured neuron cells.

[0013] Further, the second expression vector comprises 2-3 energy acceptor fluorescent molecule nucleotide sequences in series.

[0014] Further, the primary cultured cells into which the first expression vector and the second expression vector are transferred are excited by an electric field exciter.

[0015] Preferably, the electric field has a strength of 25-50 volts per centimeter and a frequency of 20-50 hertz to stimulate neural activity.

[0016] Further, the fluorescence microscope has two or more excitation lasers or excitation filters.

[0017] Further, the fluorescence resonance energy transfer efficiency is calculated by a sensitized emission method, a ratiometric method or a fluorescence lifetime imaging (FLIM) method.

[0018] Preferably, the fluorescence resonance energy transfer efficiency is calculated by a sensitized emission method.

[0019] Further, the detection method is used for detecting the liquid phase separation process of proteins in the nerve synapses of living bodies.

[0020] The second aspect of the present application provides the application of the detection method in drug target screening.

[0021] The present application has the following advantages:

[0022] 1. The detection method of the present application is based on the principle of fluorescence resonance energy transfer. The distance between the molecules in the condensed phase is shortened after the liquid phase separation of the proteins, and the efficiency of fluorescence resonance energy transfer is improved. The energy donor fluorescent molecules and the target proteins, as well as the energy acceptor fluorescent molecules and the target proteins, are expressed in the primary cultured cells. After excitation, the fluorescence microscopic imaging data is collected by using a fluorescence microscope, and the change of the fluorescence resonance energy transfer efficiency is calculated, so as to detect the liquid phase separation process of the proteins. The detection method of the present application realizes the detection of the liquid phase separation of the proteins in the in vitro or living nerve cells (i.e. neurons), and can be used for studying the effects of protein phase transition on the nerve physiology and pathology, and for studying the phase transition process of the target proteins on different genetic or phenotypic neurons, so as to find new disease mechanisms and drug targets.

[0023] In a preferred scheme, the efficiency of fluorescence resonance energy transfer is further improved by connecting multiple energy acceptor fluorescent molecules, so as to enhance the accuracy of detecting the liquid phase separation of the proteins in the synapses of the neurons.

[0024] 2. The present application realizes the detection of the liquid phase separation of the proteins in the synapses of the neurons on the sub-second time scale for the first time. The present application detects the phase transition by using the fluorescence resonance energy transfer phenomenon caused by the shortening of the distance between the proteins after the liquid phase separation, and thus is not limited by the size of the condensed phase, and can be used for detecting the liquid phase separation phenomenon in the nerve synapses with a diameter of only 200-800 nanometers. Secondly, the measurement of fluorescence resonance energy transfer can be completed on the sub-second time scale in technology, bypassing the bottleneck of super-resolution fluorescence imaging technology, and avoiding the toxicity of strong laser to cells. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The detection principle and flowchart of the liquid phase separation of the proteins are shown in the figure. A. The target protein connected with the fluorescence energy donor; B. The target protein connected with the fluorescence energy acceptor; C. The target protein connected with multiple fluorescence energy acceptors in series; D. The primary cultured neurons expressing the proteins A and B (or A and C); E. Various types of fluorescence microscopes; F. The electric field exciter for exciting the neurons; G. Neurons with different genetic types or phenotypes.

[0026] Figure 2Experimental data to verify the principle and feasibility of this invention in the COS-7 cell line. (Left figure) General fluorescence imaging shows that the target protein forms a circular condensate phase after liquid phase separation; (Right figure) Imaging data acquired and calculated by sensitized luminescence method shows that the condensate phase has a higher fluorescence resonance energy transfer efficiency (average 0.22) compared to the outside of the condensate phase (average 0.11). Detailed Implementation

[0027] To better understand the present invention, it is now further described with reference to the following embodiments and accompanying drawings. The embodiments are for illustrative purposes only and do not limit the invention in any way. In the embodiments, all original reagents and materials are commercially available, and experimental methods not specifically specified are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0028] Example 1

[0029] This invention is based on fluorescence resonance energy transfer (FIR). The principle of fluorescence resonance energy transfer (FRET) is used to detect the enhancement of fluorescence resonance energy transfer in proteins within a condensed phase. The detection principle and flowchart for protein liquid-phase separation are shown below. Figure 1 As shown. A is the target protein attached to a fluorescent molecule as an energy donor, B is the target protein attached to a fluorescent molecule as an energy acceptor, and C is the target protein attached to multiple fluorescent molecules of energy acceptors. D is a primary cultured neuron expressing proteins A and B (or A and C). E is a confocal laser scanning microscope (or other type of fluorescence microscope) with two or more excitation lasers (or excitation filters). F is an electric field exciter capable of exciting neurons by generating an electric field. G are neurons of different genotypes or phenotypes.

[0030] The efficiency of fluorescence resonance energy transfer (FRET) depends on the distance between the energy donor (A) and the acceptor molecule (B or C). Since protein liquid-phase separation occurs through intermolecular interactions, it is predicted that the closer the protein molecules are in the condensed phase, the higher the efficiency of FRET. This method can detect protein liquid-phase separation within neurons of different genotypes or phenotypes.

[0031] In a preferred embodiment, by tandemly connecting multiple energy acceptor fluorescent molecules, the efficiency of fluorescence resonance energy transfer will be further improved, enhancing the accuracy of detecting protein liquid-phase separation within neuronal synapses.

[0032] The detection method for protein liquid phase separation includes the following steps:

[0033] (1) Constructing a first expression vector expressing an energy donor fluorescent molecule and a target protein, a second expression vector expressing an energy acceptor fluorescent molecule and a target protein, and transferring the first and second expression vectors into primary cultured neurons. In a preferred embodiment, the second expression vector comprises 2-3 energy acceptor fluorescent molecule nucleotide sequences in tandem.

[0034] (2) Fabricating an electric field excitation neuron by an electric field exciter.

[0035] (3) Collecting fluorescent microscopic imaging data using a confocal laser scanning microscope.

[0036] (4) Calculating the change in fluorescence resonance energy transfer efficiency by a sensitized luminescence method, thereby completing the entire detection of the phase transition process of the target protein in neurons.

[0037] Example 2

[0038] This example was verified in a COS-7 cell line expressing a target protein with a fluorescent molecule.

[0039] In this example, the first expression vector was a pcDNA3.1+ expression vector with a fluorescent donor mEGFP attached to the N-terminus of miniShank3, and the second expression vector was a pcDNA3.1+ expression vector with a fluorescent acceptor mCherry attached to the N-terminus of miniShank3. Lipo6000 from Biyun Tian was used TM and 1.5 μg of each of the two expression vectors was added to the COS-7 cell line, and data was collected after 48 hours of culture using a Plan-Apochromat 63x / 1.4 Oil DIC M27 objective lens from Zeiss with a pixel size of 40 nm x 40 nm. Fluorescence resonance energy transfer efficiency imaging was measured and calculated by a sensitized luminescence method: the laser wavelengths for exciting the fluorescent donor and acceptor were 488 and 561 nm, respectively, and the fluorescence collection wavelength bands were 490-560 nm (donor channel), 576-700 nm (resonance energy transfer channel), and 576-700 nm (acceptor channel). The calculation of fluorescence resonance energy transfer efficiency was based on Formula 8 in Gordon G.W. et al. (1998) Biophys J. 74(5):2702-13 and Formula 2 in Xia Z. and Liu Y. (2001) Biophys J. 81(4):2395-402.

[0040] As Figure 2As shown, the liquid-liquid phase separation phenomenon occurs when the neurosynaptic protein miniShank3 is overexpressed in COS-7 cells (left panel), and there is a significant difference in the fluorescence resonance energy transfer efficiency in the condensed phase (average value 0.22) and outside the condensed phase (average value 0.11) (right panel), thereby proving the principle of the present application, and the process of detecting the phase transition of the target protein by measuring the change in fluorescence resonance energy transfer efficiency.

[0041] Obviously, the above embodiments are merely exemplary and are not intended to limit the embodiments. Based on the above description, other different forms of changes or variations can be made by those of ordinary skill in the art. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for detecting protein liquid phase separation, characterized in that, The method comprises the following steps: constructing a first expression vector expressing an energy donor fluorescent molecule and a target protein, and a second expression vector expressing an energy acceptor fluorescent molecule and the target protein; transferring the first expression vector and the second expression vector into primary cultured cells; manufacturing an electric field excitation on the primary cultured cells into which the first expression vector and the second expression vector are transferred by an electric field exciter, and collecting fluorescent microscopic imaging data by a fluorescence microscope; calculating the change of the efficiency of fluorescence resonance energy transfer, and detecting the process of liquid phase separation of the protein; the target protein is a nerve synapse protein; the primary cultured cells are primary cultured neuron cells; the second expression vector comprises 2-3 energy acceptor fluorescent molecule nucleotide sequences in series.

2. The method of claim 1, wherein, The intensity of the electric field is 25-50 volts per centimeter, and the frequency is 20-50 hertz.

3. The method of claim 1, wherein, The fluorescence microscope has two or more excitation lasers or excitation filters.

4. The method of claim 1, wherein, The efficiency of fluorescence resonance energy transfer is calculated by a sensitized luminescence method, a ratio method or a fluorescence lifetime method.

5. The method of claim 4, wherein, The efficiency of fluorescence resonance energy transfer is calculated by a sensitized luminescence method.

6. The method of claim 1, wherein, The detecting method is used for detecting the process of liquid phase separation of a protein in a nerve synapse in vivo.

7. Use of the detecting method according to any one of claims 1-6 in drug target screening.

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