A method for real-time determination of motor protein mechanical parameters by capturing transport particles in membrane nanotubes using optical tweezers

Abstract

The present invention provides a method for real-time determination of the mechanical parameters of motor proteins by capturing transport particles in membrane nanotubes by optical tweezers. The present invention uses quantum dots, dyes and fluorescent proteins as fluorescent markers to mark the particles in membrane nanotubes, microfilaments, microtubules and motor proteins respectively. Through single-particle tracing technology, the transport process of particles and related motor proteins in membrane nanotubes is imaged in real time, and optical tweezers are used to capture the target particles being transported and measure their force spectrum information, so as to achieve real-time determination of the mechanical parameters of the motor proteins that drive the movement of particles in membrane nanotubes. This method not only traces particles smaller than the optical diffraction limit by confocal rapid fluorescence imaging, but also uses optical tweezers to accurately capture the particles on the basis of tracing. The present invention can be widely used in the biological field to study the mechanical mechanism and information of transporting substances between cells through membrane nanotubes.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical tweezer manipulation and also belongs to the technical field of single particle tracing, which includes cross-technical fields such as biophysics, molecular biology, nanobiotechnology and optical imaging. Background Art

[0002] Optical tweezers were first invented by American scientist Ashkin in 1986. It is a laser beam generated by focusing a Gaussian beam through a lens, which generates two forces on particles near the optical trap. One force that moves along the direction of light propagation is called scattering force, and the other force that pushes the particles to the position where the light field gradient is the strongest is called gradient force. When the gradient force is much larger than the scattering force, the particles will be firmly bound near the center of the optical trap. Compared with other force measurement methods, optical tweezers have the advantages of long distance, non-contact, and non-destructive. The models of optical tweezers force are divided into geometric optical model, electromagnetic model and Mie model according to the size of the particles. If the size of the particles captured by the optical tweezers is much smaller than the laser wavelength, they are Rayleigh particles, and the applicable force model is the electromagnetic model. When the particles are fluorescently labeled with inorganic luminescent materials such as quantum dots, the refractive index and polarizability can be increased while ensuring biological activity, thereby increasing the stability of particle capture.

[0003] Single particle tracking is a powerful tool for real-time, in situ study of biological dynamic processes in living cells using fluorescence microscopy. Currently, single particle tracking technology has been widely used in the fields of cell membrane structure and function, molecular motor transport mechanism, virus infection of host cells, etc. By detecting single particles in time and space and analyzing them through software, the mechanism and conditions of virus infection can be revealed. At the same time, this method can also be used to dynamically track the particles and motor proteins when transporting cargo in membrane nanotubes, so as to gain a deeper understanding of the mechanical mechanism driving cargo transport in membrane nanotubes.

[0004] Membrane nanotubes (MNTs) are long membrane actin extensions that connect one cell to another to allow the exchange of organelles and signaling molecules between the two connected cells. Membrane continuity can be maintained over long distances between mammalian cells, representing a new biological principle for intercellular communication. MNTs differ from the well-known filopodia in that they are longer (up to several cell diameters in length) and are suspended in the cell culture medium without being attached to a substrate. Recent studies have shown that MNTs can transport substances such as viruses, quantum dots, and organelles, and the transport of cargo within MNTs is mainly driven by motor proteins such as myosin, kinesin, or dynein to move along microfilaments or microtubules. There is some research on the mechanical information and movement mechanism of extracellular motor proteins, but the mechanical information of some motor proteins in living cells is quite different from that outside the cell. This is because the cytoplasm, nucleus, and various organelles in the cell have a great impact on the transport of motor proteins.

[0005] At present, there is still a lack of a method that can accurately measure the mechanical parameters of motor proteins in real time within cells and further obtain the mechanical mechanism of motor proteins. Summary of the invention

[0006] In view of the fact that the existing research on the transport behavior of substances in membrane nanotubes and the working mechanism of motor proteins in membrane nanotubes are still unclear, and it is possible that several different linear molecular motors are involved, the present invention combines single particle tracing technology with optical tweezers technology to study the force information of the transport process of particles in membrane nanotubes and then reflect the mechanical mechanism of motor proteins. Therefore, the present invention provides a method for combining optical tweezers with confocal rapid fluorescence imaging to measure the mechanical parameters of motor proteins in the process of particle transport in membrane nanotubes. By capturing the transport particles in membrane nanotubes with optical tweezers, the mechanical parameters of motor proteins can be accurately measured in real time, and the mechanical mechanism of motor proteins can be further obtained.

[0007] The technical solution provided by the present invention is as follows:

[0008] A method for real-time determination of mechanical parameters of motor proteins by capturing transport particles in membrane nanotubes through optical tweezers, comprising the following steps:

[0009] (1) Fluorescently label the microfilaments, microtubules, and motor proteins of host cells;

[0010] (2) Selecting particles modified with quantum dots or fluorescent dyes as transport particles;

[0011] (3) regulating the density of host cells to control the generation of intercellular membrane nanotubes, and observing the position of intercellular membrane nanotubes under a bright field microscope;

[0012] (4) Infecting host cells with transport particles, which are transported along microfilaments or microtubules in membrane nanotubes between host cells through motor proteins;

[0013] (5) Use confocal imaging technology to image the transport process of transport particles in membrane nanotubes in real time, and simultaneously track the motor proteins near the transport particles in real time;

[0014] (6) Optical tweezers are used to capture transport particles in membrane nanotubes, and based on dynamic tracking and real-time force spectrum information, combined with the optical trap stiffness of the transport particles, the mechanical parameters of the motor protein during cargo transport are obtained.

[0015] Furthermore, the host cells are all cell lines that can form membrane nanotubes between cells and achieve the transport of transport particles therein.

[0016] Furthermore, the host cells include African green monkey kidney cells (Vero cells), human cervical cancer (HeLa) cells, human lung cancer A549 cells, human bronchial epithelial cells (HBE cells), and dog kidney epithelial cells (MDCK cells).

[0017] Furthermore, the motor protein is an intracellular linear motor protein molecule.

[0018] Furthermore, the motor protein includes kinesin, dynein and actin.

[0019] Furthermore, in step (1), the fluorescent labeling is performed by fluorescent protein or dye.

[0020] Furthermore, in step (2), the particles include virus particles, wheat germ agglutinin and polystyrene microspheres. Preferably, the virus particles are H9N2 viruses and are modified with quantum dots.

[0021] Furthermore, in step (6), the force spectrum information is the relative position of the transport particle deviating from the center of the light trap due to the action of the motor protein.

[0022] Furthermore, in step (6), the light trap stiffness is obtained using a power spectral density method.

[0023] Furthermore, in step (6), the mechanical parameters include force magnitude, step length and movement speed.

[0024] The beneficial effects of the present invention are as follows:

[0025] 1. The method provided by the present invention combines an optical tweezers device with a confocal rapid fluorescence imaging device, which can dynamically track captured particles in real time, and by fluorescently labeling the particles, transport particles with a size smaller than the optical diffraction limit can be observed.

[0026] 2. The method provided by the present invention uses optical tweezers to achieve the capture and manipulation of transport particles in membrane nanotubes, which has the advantages of long distance, non-contact, and no damage to cells.

[0027] 3. The present invention uses quantum dots to label single particles (such as viruses), which have relatively strong photostability and resistance to photosynthesis, and can achieve long-term dynamic tracking and optical tweezers capture of particles during transport within membrane nanotubes.

[0028] 4. The present invention uses the power spectral density method to obtain the optical trap stiffness of a single particle, and further obtains the mechanical parameters (such as force size, step size and movement speed) of the motor protein that drives the movement of the single particle. At the same time, this method can ignore the influence of the shape, size and absolute position of the target particle.

[0029] 5. The method provided by the present invention achieves the purpose of accurately measuring the mechanical parameters of motor proteins in real time within cells, and can further obtain the mechanical mechanism of motor proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0031] Figure 1 It is a schematic diagram of the structure of the device of the present invention;

[0032] Figure 2 Schematic diagram of optical tweezers capturing free quantum dots to label avian influenza virus;

[0033] Figure 3 Schematic diagram of optical tweezers capturing labeled avian influenza viruses inside membrane nanotubes. DETAILED DESCRIPTION

[0034] The force measurement process of virus transport in membrane nanotubes is described in detail below in conjunction with specific examples, but the content of the present invention is not limited thereto at all.

[0035] Example

[0036] A method for real-time determination of mechanical parameters of motor proteins by capturing transport particles in membrane nanotubes through optical tweezers, comprising the following steps:

[0037] (1) Vero cells are selected as host cells. The host cells are labeled with fluorescent proteins. The GFP-MAP plasmid and the Tdtomato-lifeact plasmid are transfected into the host cells two days in advance, so that the microfilaments and microtubules can be fluorescently labeled during the experiment; at the same time, actin is labeled with a dye.

[0038] (2) Preparation of quantum dot-labeled H9N2 virus: First, the virus membrane is biotinylated and then coupled with quantum dot-labeled streptavidin, so that the quantum dots can be modified on the surface of the virus membrane to be labeled.

[0039] (3) regulating the density of host cells to control the generation of intercellular membrane nanotubes, and observing the position of intercellular membrane nanotubes under a bright field microscope;

[0040] (4) Infecting the Vero cells with the labeled virus. Under the culture conditions of 5% CO2 and 37°C, the quantum dot-labeled virus will infect the Vero cells. The labeled virus will be transported along microfilaments or microtubules inside the host cells through motor proteins.

[0041] (5) Using a confocal imaging system for fluorescence imaging, we can see the real-time process of virus transport along microtubules inside Vero cells. At this time, we can also see the formation of membrane nanotubes between cells, and the virus is transported along the microfilaments or microtubules in the membrane nanotubes under the action of motor proteins.

[0042] (6) Determine the relative position of the virus particles transported along the microfilaments in the membrane nanotubes and deviate from the center of the light trap under the action of the motor protein, and then calculate the mechanical parameters (force magnitude, step length, and movement speed, etc.) of the virus particles under the action of the motor protein based on the light trap stiffness of the virus particles in the culture medium. The light trap stiffness is obtained using the power spectral density method.

[0043] The above description is only a preferred specific implementation manner of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention should be included in the scope of protection of the invention.

Claims

1. A method for real-time determination of the mechanical parameters of motor proteins by capturing transport particles in membrane nanotubes using optical tweezers, characterized in that: The steps include: (1) Fluorescently label the microfilaments, microtubules, and motor proteins of host cells; (2) Selecting particles modified with quantum dots or fluorescent dyes as transport particles; (3) regulating the density of host cells to control the generation of intercellular membrane nanotubes, and observing the position of intercellular membrane nanotubes under a bright field microscope; (4) Infecting host cells with transport particles, which are transported along microfilaments or microtubules in membrane nanotubes between host cells through motor proteins; (5) Use confocal imaging technology to image the transport process of transport particles in membrane nanotubes in real time, and simultaneously track the motor proteins near the transport particles in real time; (6) Optical tweezers are used to capture transport particles in membrane nanotubes, and based on dynamic tracking and real-time force spectrum information, combined with the optical trap stiffness of the transport particles, the mechanical parameters of the motor protein during cargo transport are obtained.

2. The method according to claim 1, characterized in that: The host cells are all cell lines that can form membrane nanotubes between cells and achieve the transport of transport particles therein.

3. The method according to claim 2, characterized in that: The host cells include African green monkey kidney cells (Vero cells), human cervical cancer (HeLa) cells, human lung cancer A549 cells, human bronchial epithelial cells (HBE cells), and dog kidney epithelial cells (MDCK cells).

4. The method according to claim 1, characterized in that: The motor protein is an intracellular linear motor protein molecule.

5. The method according to claim 1, characterized in that: The motor proteins include kinesin, dynein and actin.

6. The method according to claim 1, characterized in that: In the step (1), the fluorescent labeling is performed by fluorescent protein or dye.

7. The method according to claim 1, characterized in that: In the step (2), the particles include virus particles, wheat germ agglutinin and polystyrene microspheres.

8. The method according to claim 1, characterized in that: In the step (6), the force spectrum information is the relative position of the transport particle deviating from the center of the light trap due to the action of the motor protein.

9. The method according to claim 1, characterized in that: In the step (6), the light trap stiffness is obtained by using a power spectral density method.

10. The method according to claim 1, characterized in that: In step (6), the mechanical parameters include force magnitude, step length and movement speed.

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