A method for tracking outer segment protein trafficking in vivo

Abstract

The present invention relates to a method for tracking outer segment protein trafficking in vivo. Using the photoconvertible fluorescent protein Dendra2, the process and turnover dynamics of outer segment protein trafficking in mice are dynamically tracked. By tracing the source and destination of ectopically accumulated Rhodopsin, the pathways and paths that trigger cell death are revealed. Theoretically, the results will contribute to the development of theories of Rhodopsin trafficking and membrane turnover, further elucidate the processes and modes of abnormal Rhodopsin trafficking in retinal degenerative disease models, and potentially identify new modes of transport. Clinically, the results may reveal cell death pathways in patients with late-stage retinal degenerative diseases, providing new targets and directions for drug development.

Description

Technical Field

[0001] The present invention relates to the field of biological imaging technology, in particular to a method for tracking the transport of outer segment proteins in vivo. Background Art

[0002] Visual information is essential for survival for humans and animals, with 80% of external information coming from vision. The retina is the first point of contact for receiving visual information. Light is focused on the retina by the refractive system composed of the cornea, lens, and vitreous humor. Photons are captured by photoreceptor cells, which convert the light signals into electrical signals that are transmitted through the optic nerve to the visual cortex of the brain, ultimately forming vision. Consequently, degeneration of retinal photoreceptor cells is a major cause of blindness. For example, hereditary retinitis pigmentosa has a global incidence of approximately 1:4000, affecting nearly 300,000 people in my country, and currently lacks effective treatment.

[0003] To ensure the efficiency, sensitivity, and specificity of photosensitivity, a tightly stacked membrane disc structure, relatively isolated from the cell body, forms at the top of the photoreceptor cell to receive photons. This structure is called the outer segment. Since the outer segment itself does not have the ability to synthesize proteins and lipids, they need to be synthesized by the inner segment and input through the connecting cilium. The connecting cilium is a narrow microtubule structure that not only relatively isolates the outer and inner segment structures, preventing the free diffusion of proteins, but also provides a microtubule channel for the transport of proteins and lipids synthesized in the inner segment. The connecting cilium has a complex transport-related ciliary protein that is involved in the transport of various outer segment proteins and lipids. Any genetic mutation that affects the transport of outer segment proteins will eventually lead to various retinal degenerative diseases.

[0004] Abnormal transport of outer segment proteins prevents membrane disc renewal, leading to the accumulation of outer segment proteins and other proteolipids in the inner segment, which overwhelms the photoreceptor cells and triggers cell necrosis. Due to the lack of appropriate tools to track the source and destination of the ectopically accumulated outer segment proteins caused by transport obstruction, further understanding of the signaling pathways that trigger cell death is hampered.

[0005] Most of the existing research and development of technologies for tracking Rhodopsin localization and transport have stopped at static observation of its transport process, and are still unable to effectively monitor the initiation and dynamic tracking of renewal. For example, fusion GFP markers can show the basic time and direction required for renewal of mature membrane discs, but cannot observe the various processes at each time point of transport in real time. Immunoelectron microscopy can show the basic routes of Rhodopsin localization and migration in membrane discs, but can only be observed statically. Although isotope labeling can achieve in vivo tracking, there are safety risks and real-time tracking cannot be achieved. In addition, compared with the advantage of easy observation of the retina in animal models such as African clawed frogs and zebrafish, the eye structure and retina of mice are more consistent with those of humans, but are also more complex and difficult to observe. In vivo observation of the mouse retinal structure often requires the integration of various technologies for trial. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for tracking the transport of outer segment proteins in vivo, which can effectively solve the problems raised in the above background technology.

[0007] To solve the above problems, the technical solution adopted by the present invention is: a method for tracking the transport of outer segment proteins in vivo, comprising the following steps:

[0008] S1. Select a target gene and clone the target gene into a plasmid vector, wherein the target gene includes a gene encoding a target protein, wherein the target protein is an outer membrane protein, and the target protein is in the form of a fusion tag protein for observation and tracking by fluorescence microscopy;

[0009] S2. transfecting the plasmid vector into a host cell to achieve expression of the target protein, wherein the host cell is a mammalian cell capable of expressing the target protein;

[0010] S3. packaging the target gene into a viral vector using a viral vector packaging system, and using the viral vector to infect retinal cells in a mammal, so as to achieve expression of the target protein in the mammal;

[0011] S4. Observe and record the localization and dynamic distribution of the target protein in the cell using a fluorescence microscope, and track the target protein in real time using a fluorescent labeling technique, wherein the fluorescent labeling technique is Dendra2 fluorescent labeling;

[0012] S5. Collect and analyze the dynamic expression data of the target protein, and perform quantitative analysis using image processing software to evaluate the distribution of the target protein inside and outside the host cell and its dynamic changes.

[0013] As a further preferred embodiment of the present invention, the target protein is Rhodopsin protein, and the target protein is fused with Dendra2 fluorescent marker protein to track the dynamic transport process of the target protein.

[0014] As a further preferred embodiment of the present invention, the plasmid vector is a PRK5 plasmid vector, and the PRK5 plasmid vector is transfected into host cells to express the target protein.

[0015] As a further preferred embodiment of the present invention, the viral vector is an adeno-associated virus AAV vector, and the AAV vector is of AAV2 / 8 serotype, which is used for efficiently transducing mammalian retinal cells.

[0016] As a further preferred embodiment of the present invention, the observation step includes performing dynamic observation using a living imaging system after injecting the viral vector into the mammal to obtain the distribution of the target protein in the mammal in real time.

[0017] As a further preferred embodiment of the present invention, the dynamic changes include protein transport speed, distribution range, aggregation and localization changes in different cells, and data analysis includes calculation of protein transport rate, measurement of distribution density and identification of aggregation points.

[0018] As a further preferred embodiment of the present invention, the distribution range includes the cell membrane, endoplasmic reticulum, and Golgi apparatus regions, and the aggregation situation is evaluated as the number and size of protein aggregation points.

[0019] Compared with the prior art, the present invention provides a method for tracking the transport of outer segment proteins in vivo, which has the following beneficial effects:

[0020] This study utilizes the photoconvertible fluorescent protein Dendra2 to dynamically track the transport and turnover dynamics of outer segment proteins in mice. By tracing the source and destination of ectopically accumulated rhodopsin, the researchers reveal the pathways and paths through which it triggers cell death. Theoretically, these findings will contribute to the development of theories of rhodopsin transport and membrane turnover, further elucidate the processes and modes of abnormal rhodopsin transport in models of retinal degenerative diseases, and potentially identify new transport pathways. Clinically, these findings may reveal cell death pathways in patients with late-stage retinal degenerative diseases, providing new targets and directions for drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a diagram showing the photoconversion process of Rho-Dendra2 activated at 405 nm.

[0022] Figure 2 Tracing Rho trafficking maps for Rho-Dendra2 expression;

[0023] Figure 3 This is a diagram showing that Rho-Dendra2 can complete photoconversion after activation at 405nm;

[0024] Figure 4 This is a picture showing that Rho-Dendra2 in mice can complete fluorescence photoconversion at 405nm;

[0025] Figure 5 A map of Rhodopsin transport was used to track Dendra2 photoconversion;

[0026] Figure 6 Diagram of the basic transport process of Rhodopsin in mice tracked by Rho-Dendra2; DETAILED DESCRIPTION

[0027] If "and / or" or "and / or" appears in the full text, its meaning includes three parallel options. Taking "A and / or B" as an example, it includes option A, or option B, or options in which A and B are satisfied at the same time.

[0028] In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in this field can implement them. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0029] The present invention provides a method for tracking outer segment protein transport in vivo, comprising:

[0030] 1. Construct the C-terminal Rho-Dendra2 to PRK5 vector of Rhodopsin. After sequencing verification, select the plasmid with the correct sequence for amplification and extraction.

[0031] 2. Activate Rho-Dendra2 with light of different wavelengths to verify photoconversion efficiency. "Rho" stands for "Rhodopsin," an outer membrane protein. The Rho-Dendra2 plasmid constructed above was transiently transfected into 293T cells using PEI for in vitro validation. During transfection, observe Rho-Dendra2 fluorescence in the cells. Two days after transfection, photoconversion efficiency was measured using a fluorescence microscope using a mercury lamp at different wavelengths (600 nm, 555 nm, 488 nm, and 405 nm).

[0032] 3. The successfully transformed Rho-Dendra2 was repackaged into the AAV-hRK vector to construct the AAV-hRK-Rho-Dendra2 plasmid. After sequencing verification and amplification, the AAV2 / 8 serotype, which has a high efficiency in infecting retinal photoreceptors, was selected for packaging. The virus was then transfected, amplified, purified, and concentrated into a high-titer AAV virus.

[0033] 4. The AAV-hRK-Rho-Dendra2 virus was injected subretinaally to infect rod photoreceptors. Using different microscopes combined with simulated lenses to focus on the mouse fundus, a certain amount of virus was injected subretinaly into the retina of adult mice.

[0034] 5. Mice injected with Rho-Dendra2 were housed for 10 days. Rho-Dendra2 essentially completes a membrane disc renewal cycle within 10 days, filling the entire outer segment. Based on the in vitro validation described above, the 405 nm wavelength used in the in vitro validation was selected to activate the fluorescence photoconversion of Rho-Dendra2 in mouse photoreceptor cells in vivo.

[0035] 6. By continuously optimizing conditions such as the wavelength and intensity of the excitation laser, the efficiency of Rho-Dendra2 fluorescence photoconversion was evaluated through real-time recording and other systems. Immunofluorescence (IF) and rhodopsin co-staining were used to confirm the feasibility and efficiency of Rho-Dendra2 tracking rhodopsin transport. Considering the potential for damage to photoreceptor cells from viruses, UV light, and surgery, the safety of the technology was ensured through functional and structural analysis. Optical coherence tomography (OCT), fundus photography (FP), and electroretinography (ERG) were used to track functional and structural changes in the retina after illumination. IF, ultrastructural expansion microscopy, and quantitative PCR were used to observe changes in photoreceptor cells and RPE.

[0036] 7. Using the optimal conditions described above, describe the dynamic transport of rhodopsin in mice with different gene mutations. After Dendra2 expression has reached the entire outer membrane segment, photoconversion is performed to eliminate green fluorescence, allowing precise tracking of rhodopsin transport using the newly converted red fluorescence. Dynamic transport is observed at different time intervals (6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 5 days, 7 days, and 10 days). Calculate transport rates, directionality, and localization to illustrate membrane renewal. Tracking is performed using sections and IF.

[0037] As a specific embodiment 1 of the present invention:

[0038] (1) Rho-Dendra2 is activated at 405nm to complete the photoconversion process

[0039] We have completed the construction of Rho-Dendra2 plasmid in the early stage, packaged it into lentivirus, and transfected 293T cells for in vitro verification experiments. Two days after transfection, Rho-Dendra2 fluorescence was visible on the cell membrane. The efficiency of photoconversion was detected by fluorescence microscopy using different wavelengths of mercury lamp (600nm→555nm→488nm→405nm). It was found that under 405nm ultraviolet light, the conversion of green to red fluorescence increased with the illumination time. After 50s of irradiation, Rho-Dendra2 was basically completely converted. Prolonging the illumination time did not increase its red fluorescence intensity. Figure 1 At the same intensity and time, no photoconversion of Dendra2 was observed at other wavelengths.

[0040] exist Figure 1A shows the basic components of the Rho-Dendra2 plasmid and the basic in vitro validation process. B-F'' show the photoconversion process of Dendra2 after 405 nm illumination. B' to F' show the changes in green fluorescence with illumination time, and B'' to F'' show the changes in red fluorescence with illumination time. G and H show the trends of fluorescence intensity over time, respectively. n = 4 (cells from four confocal culture dishes).

[0041] (2) Rho-Dendra2 expression tracks Rho transport

[0042] In order to improve the infection efficiency of Rho-Dendra2 in mice and target rod photoreceptor cells. We selected the serotype AAV2 / 8 and hRK promoter with high efficiency in infecting retinal photoreceptors, and reconstructed the plasmid of the AAV vector. After AAV virus packaging, purification and concentration, a total of 5*108GC of AAV-hRK-Rho-Dendra2 virus was injected subretinaly to infect rod photoreceptor cells. It can be seen that Rho-Dendra2 is co-stained with Rho fluorescent staining, which can mark Rho. The position of its expression is consistent with the self-expressed Rhodopsin, and no mislocalization is observed. Ten days after the injection of Rho-Dendra2, Rho-Dendra2 basically completed a membrane disk renewal cycle and basically filled the entire outer segment. Based on the localization of Rho-Dendra2 in the outer segment, it is preliminarily estimated that the time of Rho transport is about 3.85um / day, referring to Figure 2 .

[0043] exist Figure 2 A shows the basic components of the Rho-Dendra2 plasmid and the basic workflow for in vivo expression tracking. B-G'' show the localization of Dendra2 expression at different times after injection. H shows the length of Dendra2-labeled Rho. I shows the rate of Dendra2-labeled Rho outer segment transport over time (length / day). n = 38 (Dendra2 outer segment is shown). J shows a simplified diagram of Rho-Dendra2 tracking of Rho transport.

[0044] (3) Rho-Dendra2 in mice can complete fluorescence photoconversion at 405nm

[0045] After confirming that the AAV-hRK-Rho-Dendra2 virus could specifically infect rod photoreceptors, we began experiments investigating Rho-Dendra2 fluorescence photoconversion in vivo. Photoconversion in mice presents several challenges. First, the mouse eyeball is small, lacking a suitable lens to transmit light to the fundus. Second, the area of ​​successful infection in the mouse must be visualized, which requires an instrument equipped for fluorescence excitation. Third, the intensity and range of the transduced light focused on the retina. Considering the effectiveness of the excitation light transmission route, we prioritized using fiber optic cables to transmit light of varying wavelengths, directly illuminating the retina through the vitreous cavity. However, subsequent imaging revealed that direct in vivo illumination resulted in severe retinal inflammation and low photoconversion efficiency. Considering in vitro conversion, direct photoactivation under a fluorescence microscope was considered to mitigate the photodamage caused by intravitreal illumination. Based on our experience with subretinal injections, after repeated attempts, we employed a method in which PBS droplets were directly applied to the mouse ocular surface to form a lens, focusing the lens on the fundus. Dendra2 green fluorescence was then excited with 488 nm wavelength light, focusing on the successfully infected area. After focusing on the area where dendra2 infection was successful, we selected the strongest 405nm light intensity for preliminary photoconversion experiments, referring to Figure 3 .

[0046] exist Figure 3 A shows the photoconversion process of Rho-Dendra2 activated by 405 nm light. B shows the area of ​​successful infection, focused on by fluorescence microscopy, using 488 nm light to excite Dendra2 green fluorescence. C shows illumination with 405 nm light. D shows the photoconversion image after 15 minutes of 405 nm illumination. E shows an image taken 2 hours after 405 nm illumination.

[0047] After confirming that Rho-Dendra2 can complete the fluorescence photoconversion process in mice, the conditions were further optimized to find the lowest intensity and shortest time to reduce light damage. Figure 4 As shown in the figure, four gradients were set: 40 lux; 5 μV / cm², 66 lux; 18 μV / cm²; 120 lux; 31 μV / cm², 390 lux; and 96 μV / cm². The illumination time varied (5 minutes, 10 minutes, and 15 minutes) to find the most suitable illumination conditions. The fluorescence conversion efficiency trend graph shows that at laser intensities from level 2 to level 3, photoconversion increases with intensity and duration.

[0048] exist Figure 4Center A shows the process of photoconversion in mice 10 days after Rho-Dendra2 injection, using four gradients of 405 nm light. The intensities were 40 lux, 5 μv / cm², 66 lux, 18 μv / cm², 120 lux, 31 μv / cm², 390 lux, and 96 μv / cm², with illumination durations of 5, 10, and 15 minutes. The left panel shows fundus images before illumination (A-A'), with A' representing a single red fluorescence channel. The right panel shows the converted fundus images after illumination for the corresponding durations and intensities (B-B'). The bottom panel shows the trend of fluorescence intensity over time and intensity. n = 4 mouse eyes.

[0049] (4) Rho-Dendra2 can track the Rho transport process

[0050] After determining the conditions under which mouse Rho-Dendra2 can be efficiently converted in vivo, we used Rho-Dendra2 to illuminate the outer segment membrane discs of adult wild-type mice to define the starting point of Rho renewal and re-track its trafficking. Figure 5 As shown, a conversion zone was visible in samples taken 6 hours after illumination, with all Green-Dendra2 converted to Red-Dendra2. Two days later, Red-Dendra2 was observed transporting to the outer segments, followed closely by newly generated Green-Dendra2. No accumulation in the cell body was observed, and a clear boundary between Red-Dendra2 and Green-Dendra2 was observed. This suggests that Dendra2 can accurately track the localization and trafficking of Rhodopsin. Of course, to ensure the safety and efficiency of subsequent experiments, we will continue to conduct a series of experiments to further optimize the conditions.

[0051] exist Figure 5 A shows the process for collecting samples after 405nm photoconversion in mice injected with Rho-Dendra2. 120 lux; 31 μV / cm², illumination duration 5 minutes. BF show images of photoconversion 2 hours and 2 days after illumination. E shows a simplified diagram of Rho-Dendra2 transport via photoconversion.

[0052] In summary, our preliminary work not only supports the scientific questions and hypotheses of this project, but also provides technical support and a theoretical foundation for subsequent research. Therefore, it can be applied to track the rate, directionality, and turnover dynamics of Rhodopsin transport in different diseases. Furthermore, by using indicators such as intracellular phagocytosis, we can observe the abnormal localization and accumulation of Rhodopsin in the inner segment and cell body, hoping to identify the pathways by which Rhodopsin accumulation triggers cell death.

[0053] refer to Figure 6 , as a specific embodiment 2 of the present invention:

[0054] 1. Construct the C-terminal Rho-Dendra2 to PRK5 vector of Rhodopsin, verify it by sequencing, amplify and extract the plasmid.

[0055] 2. The Rho-Dendra2 plasmid constructed above was transiently transfected into 293T cells using PEI for in vitro experiments. The photoconversion efficiency was verified by activating Rho-Dendra2 using light of different wavelengths.

[0056] 3. The in vitro validated Rho-Dendra2 was repackaged into the AAV-hRK vector to construct the AAV-hRK-Rho-Dendra2 plasmid. After sequencing verification and amplification, the AAV2 / 8 serotype, which has a high efficiency in infecting retinal photoreceptors, was selected for packaging. The AAV virus was then transfected, amplified, purified, and concentrated to a high-titer.

[0057] 4. The AAV-hRK-Rho-Dendra2 virus was injected subretinaally to infect rod photoreceptors. Using a surgical microscope (SZX7, Olympus, Japan) coupled with a simulated lens to focus on the mouse fundus, a predetermined amount of virus was injected subretinaly into the retina of adult mice.

[0058] 5. The mice injected with Rho-Dendra2 were housed for 10 days. Rho-Dendra2 essentially completed a membrane disc renewal cycle within 10 days, essentially filling the entire outer segment.

[0059] 6. 405 nm wavelength activated the fluorescence photoconversion of Rho-Dendra2 in mouse photoreceptor cells in vivo. Activation and photoconversion were performed using a fluorescence microscope (Imager.Z2, Zeiss, Germany) equipped with a stage suitable for placing mice and an X-Cite fluorescent illumination system (X-Cite 120Q, EXCELITAS, USA). This system uses lasers with different wavelengths (380 nm to 680 nm) to capture images of Dendra2 in different fluorescent states. 488 nm fluorescent light excites green Dendra2, while 568 nm excites red Dendra2. To convert Dendra2 from green to red, 405 nm fluorescent light was used to illuminate the retina. The power of the 405 nm illumination ranged from 20 to 100 µW, and the duration of illumination was 5 to 15 minutes.

[0060] 7. Using the optimal conditions described above, describe the dynamic transport of rhodopsin in mice with different gene mutations. After Dendra2 expression has reached the entire outer membrane segment, photoconversion is performed to eliminate green fluorescence, allowing precise tracking of rhodopsin transport using the newly converted red fluorescence. Dynamic transport is observed at different time intervals (6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 5 days, 7 days, and 10 days). Calculate transport rates, directionality, and localization to illustrate membrane renewal. Tracking is performed using sections and IF.

[0061] Figure 6 The following figures show 1. an ophthalmic surgical microscope; 2. an adult wild-type mouse; 3. the fundus of the mouse focused on through a microscope under a dilated pupil; 4. the virus is injected subretinaally into the mouse; 5. a schematic diagram of the surgical site for virus injection; 6-8. fluorescence photoconversion of Rho-Dendra2 photoreceptor cells activated by fluorescence microscopy at a wavelength of 405 nm in mice 10 days after virus injection; 9. a schematic diagram of the conversion of green fluorescence to red fluorescence in photoreceptor rod cells infected with the dendra2 virus after photoconversion.

[0062] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. All equivalent structural transformations made by using the contents of the present invention description and drawings under the inventive concept of the present invention, or direct / indirect application in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A method for tracking outer segment protein trafficking in vivo, characterized in that The following steps are involved: S1. Select a target gene and clone the target gene into a plasmid vector. The target gene includes a gene for a target protein, which is an outer membrane protein Rhodopsin, and the target protein is in the form of a fusion-tagged protein for observation and tracking using a fluorescence microscope. S2. transfecting the plasmid vector into a host cell to achieve expression of the target protein, wherein the host cell is a mammalian cell capable of expressing the target protein; S3. Using a viral vector packaging system, packaging the target gene into a viral vector, wherein the viral vector is an adeno-associated virus (AAV) vector, and using the viral vector to infect retinal cells in a mammal to construct an AAV-hRK-Rho-Dendra2 plasmid; thereby achieving expression of the target protein in the mammal; S4. Observe and record the localization and dynamic distribution of the target protein in the cell using a fluorescence microscope, and track the target protein in real time using a fluorescent labeling technique, wherein the fluorescent labeling technique is Dendra2 fluorescent labeling; S5. Collect and analyze the dynamic expression data of the target protein, and perform quantitative analysis using image processing software to evaluate the distribution of the target protein inside and outside the host cell and its dynamic changes; The target protein is fused with the Dendra2 fluorescent marker protein to track the dynamic transport process of the target protein; Rhodopsin-Dendra2 in the host cell completes fluorescence light conversion under an excitation wavelength of 405 nm.

2. The method for tracking outer segment protein transport in vivo according to claim 1, characterized in that The plasmid vector is a PRK5 plasmid vector, which is transfected into host cells to express the target protein.

3. The method for tracking outer segment protein transport in vivo according to claim 1, characterized in that The AAV vector is of AAV2 / 8 serotype and is used for efficiently transducing mammalian retinal cells.

4. The method for tracking outer segment protein transport in vivo according to claim 1, characterized in that The observation step includes performing dynamic observation using a living imaging system after injecting the viral vector into the mammal to obtain the distribution of the target protein in the mammal in real time.

5. The method for tracking outer segment protein transport in vivo according to claim 1, characterized in that The dynamic changes include the transport speed, distribution range, aggregation of proteins and changes in their localization in different cells.

6. The method for tracking outer segment protein transport in vivo according to claim 5, characterized in that The distribution range includes the cell membrane, endoplasmic reticulum, and Golgi apparatus regions, and the aggregation status is assessed by the number and size of protein aggregation points.

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