A programmable light-controlled organelle autophagy regulation tool based on optogenetics and application thereof

Abstract

This invention discloses a programmable, light-controlled organelle autophagy regulation tool based on optogenetics and its applications. Based on an enhanced photosensitive dimer system, this tool uses blue light to precisely control the rapid localization of effector molecule modules, achieving dynamic regulation of autophagy in target organelles. Functional validation results show that this tool can specifically recruit effector proteins in a short time and significantly reduce the mass of target organelles under continuous light irradiation. Compared with traditional drug-induced methods, this invention not only has higher temporal and spatial resolution but also eliminates the need for additional chemical reagents, avoiding drug-induced nonspecific effects and cytotoxicity, and greatly improving experimental controllability and reproducibility. This platform provides a novel approach for studying organelle homeostasis, metabolic regulation, and their roles in diseases, and lays a solid technical foundation for subsequent applications in high-throughput drug screening, metabolic disease modeling, aging research, and precision medicine.

Description

Technical Field

[0001] This invention belongs to the fields of biomedical engineering and cell biology, specifically relating to a programmable, light-controlled organelle autophagy regulation tool based on optogenetics and its applications. This tool can achieve specific autophagy regulation of various organelles (including but not limited to mitochondria and lipid droplets) and has the potential to be extended to areas such as high-throughput drug screening and disease modeling. Background Technology

[0002] The structural integrity and functional stability of organelles are fundamental to cellular survival and response to changes in the external environment. Organelle-specific autophagy plays a crucial role in clearing damaged organelles, maintaining intracellular homeostasis, and regulating metabolism. Depending on the target organelle, organelle autophagy includes mitophagy, peroxisome autophagy, endoplasmic reticulum autophagy, lipid droplet autophagy, lysosomal autophagy, and ribosome autophagy. By selectively clearing damaged or dysfunctional organelles, organelle autophagy plays a vital role in maintaining intracellular homeostasis and preventing pathological damage.

[0003] Studies have shown that abnormalities in organelle autophagy are closely related to the development of many human diseases. For example, in neurodegenerative diseases, impaired clearance of damaged mitochondria leads to the accumulation of reactive oxygen species and energy metabolism imbalances within neurons, inducing neuronal dysfunction and death. In metabolic diseases, such as non-alcoholic fatty liver disease and obesity, abnormal lipid droplet autophagy leads to lipid metabolism imbalances and insulin resistance. In cardiovascular diseases, mitochondrial autophagy damage is closely related to pathological processes such as myocardial injury and heart failure. Therefore, organelle autophagy not only has important value in basic biological research but is also considered a potential intervention target for neurodegenerative diseases, metabolic diseases, and cardiovascular diseases.

[0004] Despite ongoing research into organelle autophagy, existing autophagy regulation techniques still have significant limitations. Current methods primarily include chemical drug induction, gene knockout or overexpression, and small molecule intervention (e.g., invention patents with publication numbers CN101392007A and CN119219708A). However, these methods mostly act on the global autophagy pathway, lacking selectivity for specific organelles and struggling to achieve precise temporal and spatial control. Furthermore, drug induction is often accompanied by cytotoxicity and non-specific effects, while gene manipulation frequently leads to irreversible pathway activation or inhibition, making dynamic and reversible regulation difficult. Due to the lack of high spatiotemporal resolution, reversibility, and organelle-specific regulatory tools, existing methods not only fail to deeply elucidate the role of organelle autophagy in disease development but also face bottlenecks in drug development and preclinical validation.

[0005] Therefore, there is an urgent need to develop a novel regulatory platform that is programmable, has high spatiotemporal resolution, is reversible, and is organelle-specific, enabling dynamic and controllable regulation of organelle autophagy. This platform can be combined with automated drug screening systems, disease models, and multidimensional pharmacodynamic evaluation technologies to provide strong technical support for basic research, drug screening, and preclinical validation. Summary of the Invention

[0006] The purpose of this invention is to develop a programmable, light-controlled organelle autophagy regulation tool based on optogenetics. By designing a programmable light-controlled recruitment system, combined with organelle targeting and effector molecule modules, real-time, reversible, and precise regulation of the autophagy process of target organelles is achieved. This addresses the shortcomings of existing autophagy regulation technologies in terms of spatiotemporal resolution, reversibility, and organelle specificity, laying a technical foundation for subsequent applications in drug screening, disease model construction, and metabolic research.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: This invention provides a programmable light-induced cleaner for targeted organelles via reprogrammed autophagy (LICTOR) based on optogenetics, comprising: a localization module containing a first light-sensitive protein unit and a localization signal sequence capable of binding to a specific organelle; and an effector molecule module containing a second light-sensitive protein unit and an effector domain capable of inducing autophagy; wherein the first and second light-sensitive protein units can specifically dimerize under irradiation with light of a specific wavelength, thereby recruiting the effector molecule module to the surface of the target organelle, and thereby inducing the target organelle to undergo specific autophagy.

[0008] Preferably, the localization signal sequence is selected from one of the following: mitochondrial targeting sequence, lipid droplet targeting sequence, endoplasmic reticulum resident signal sequence, lysosomal membrane protein targeting sequence, or peroxisome targeting signal sequence. When the programmable light-controlled organelle autophagy regulation tool of the present invention is extended to other organelles, the localization signal sequence can be replaced, and LICTOR can be applied to the endoplasmic reticulum, lysosomes, peroxisomes, etc.

[0009] Furthermore, the localization signal sequence is a mitochondrial targeting sequence, and the tool is used for light-controlled induction of mitophagy; The amino acid sequence of the mitochondrial targeting sequence is the amino acid sequence of 413-456 aa shown in SEQ ID NO.1.

[0010] Furthermore, the positioning signal sequence is a lipid droplet targeting sequence, and the tool is used for photo-induced lipid droplet autophagy; The amino acid sequence of the lipid droplet targeting sequence is the amino acid sequence of 319-618 aa shown in SEQ ID NO.4.

[0011] Preferably, the first photosensitive protein unit is eMagA, with an amino acid sequence of 1-152 aa as shown in SEQ ID NO.1; the second photosensitive protein unit is eMagB, with an amino acid sequence of 252-403 aa as shown in SEQ ID NO.2; The specific wavelength of light is blue light.

[0012] Preferably, the effector domain capable of inducing autophagy is the Fc domain, which recruits the endogenous E3 ubiquitin ligase TRIM21 to trigger the ubiquitination signaling pathway, and then mediates autophagy through the p62 (ubiquitin-binding protein p62)-LC3 (microtubule-associated protein 1 light chain 3, LC3) pathway.

[0013] Furthermore, the effector domain is a human IgG1 Fc fragment, whose amino acid sequence is the amino acid sequence of 418-650 aa shown in SEQ ID NO.2.

[0014] In the programmable light-controlled organelle autophagy regulation tool of the present invention, the localization module and / or effector molecule module are further connected to fluorescent protein tags.

[0015] Furthermore, the fluorescent protein tag is EGFP, mCherry, or miRFP.

[0016] Specifically, this optogenetic-based programmable light-controlled organelle autophagy regulation tool consists of a light-controlled localization module and an effector molecule module, with its core relying on the blue light-induced enhanced photosensitive dimer system (eMags). Different organelle-specific localization sequences are introduced into the localization module (eMagA-EGFP-Mito (amino acid sequence as shown in SEQ ID NO.1) or eMagA-miRFP-6×HP (amino acid sequence as shown in SEQ ID NO.4)), enabling the photosensitive protein eMagA carrying a fluorescent tag (EGFP or miRFP) to precisely anchor on the surface of target organelles such as mitochondria and lipid droplets.

[0017] In the effector module (mCh-eMagB-Fc, amino acid sequence as shown in SEQ ID NO.2), eMagB carries an mCherry fluorescent tag and an Fc domain, and is diffusely distributed in the cytoplasm under dark conditions. Upon blue light irradiation, eMagA and eMagB undergo heterodimerization, and mCh-eMagB-Fc is rapidly recruited to the surface of the target organelle. Thereupon, it binds to the endogenous E3 ubiquitin ligase TRIM21 protein, triggering ubiquitination modification, and then achieves selective autophagy of the target organelle through the p62-LC3 signaling pathway. By replacing different localization sequences, this invention can achieve programmable regulation of autophagy in various organelles, exhibiting good versatility and scalability.

[0018] In the specific implementation of this invention, two application systems based on LICTOR were designed and constructed: LICTOR-M (photocontrolled mitochondrial autophagy regulation system) and LICTOR-L (photocontrolled lipid droplet autophagy regulation system).

[0019] In the LICTOR-M system, by introducing a mitochondrial targeting sequence (MITO) into the targeting module, eMagA-EGFP-MITO is precisely anchored to the mitochondrial outer membrane surface. Under dark conditions, mCh-eMagB-Fc is diffusely distributed in the cytoplasm, while under blue light irradiation, eMagA and eMagB dimerize, and mCh-eMagB-Fc is rapidly recruited to the mitochondrial surface. This binds to TRIM21-mediated ubiquitination signals, activating downstream autophagy pathways and promoting selective mitochondrial clearance. Functional validation revealed that LICTOR-M significantly regulates mitochondrial quality under blue light stimulation, manifested as a change in mitochondrial morphology from filamentous to perinuclear aggregates, and a decrease in the expression of the mitochondrial outer membrane protein TOMM20.

[0020] In the LICTOR-L system, the eMagA-miRFP-6×HP lipid droplet localization sequence (six tandem repeats of Hp motif, 6×HP) is precisely localized to the lipid droplet surface by introducing a lipid droplet localization sequence into the localization module. Under dark conditions, mCh-eMagB-Fc diffuses in the cytoplasm, while upon blue light irradiation, eMagA and eMagB rapidly heterodimerize, and mCh-eMagB-Fc is recruited to the lipid droplet surface and binds to TRIM21 to trigger ubiquitination modification, activating the p62-LC3-mediated lipid droplet autophagy pathway, thereby promoting the selective clearance of lipid droplets. Functional validation results show that LICTOR-L significantly reduces the number and total volume of lipid droplets in single cells under blue light stimulation, while also reducing the expression of the lipid droplet marker protein PLIN2.

[0021] The present invention also provides the application of the above-mentioned programmable light-controlled organelle autophagy regulation tool in the preparation of cell models or kits for drug screening, disease modeling, metabolic regulation or aging research.

[0022] Compared to existing methods for regulating organelle autophagy that rely on chemical drugs or gene editing, the programmable light-controlled organelle autophagy regulation tool (LICTOR) proposed in this invention offers significant advantages in terms of regulatory precision, response speed, organelle specificity, and reversibility. Traditional methods for mitophagy and lipid droplet degradation often rely on drug induction, but these methods suffer from problems such as uncontrollable stimulus intensity, difficulty in precisely limiting the duration of action, and significant cytotoxicity. Furthermore, most rely on endpoint detection, making it difficult to achieve real-time monitoring and dynamic regulation of organelle autophagy. This invention combines optogenetics with organelle-specific localization strategies to achieve efficient, reversible, and programmable control of organelle autophagy, providing a novel technical approach for organelle function research.

[0023] This invention constructs a LICTOR based on an enhanced photodimer system (eMags), which uses blue light to precisely control the rapid localization of effector molecule modules, thereby achieving dynamic regulation of autophagy in target organelles. LICTOR-M targets mitochondria, and LICTOR-L targets lipid droplets. Functional validation results show that LICTOR can achieve specific recruitment of effector proteins in a short time and significantly reduce the mass of target organelles under continuous light irradiation. Compared with traditional drug-induced methods, this invention not only has higher temporal and spatial resolution but also requires no additional chemical reagents, avoiding drug-induced nonspecific effects and cytotoxicity, and greatly improving the controllability and reproducibility of experiments.

[0024] Based on its modular design, LICTOR can be applied not only to mitochondria and lipid droplets, but also to other organelles such as endoplasmic reticulum, lysosomes, and peroxisomes by changing the positioning module, and has strong scalability and versatility.

[0025] In summary, this invention achieves light-controlled regulation of mitochondrial mass and lipid droplet degradation through LICTOR-M and LICTOR-L, overcoming the limitations of existing technologies in terms of spatiotemporal precision, reversibility, specificity, and scalability. This platform provides a novel approach for studying organelle homeostasis, metabolic regulation, and their roles in diseases, and lays a solid technical foundation for subsequent applications in high-throughput drug screening, metabolic disease modeling, aging research, and precision medicine. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the LICTOR-M structure.

[0027] Figure 2 This is a schematic diagram of the LICTOR-M working principle.

[0028] Figure 3 To verify the localization of LICTOR-M: (a) shows the staining of mitochondria with mitochondrial tracer (MitoTracker DeepRed, MTDR) and confocal microscopy imaging; (b) shows the relative fluorescence intensity changes of mitochondrial tracer, EGFP, and mCherry at the locations corresponding to the lines drawn in cells overexpressing eMagA-EGFP-Mito and mCh-eMagB-Fc; (c) shows the relative fluorescence intensity changes of mitochondrial tracer, EGFP, and mCherry at the locations corresponding to the lines drawn in control cells overexpressing Fc mutants (eMagA-EGFP-Mito, mCh-eMagB-Fc(H433A)). This indicates that eMagA-EGFP-Mito is localized on mitochondria, and both mCh-eMagB-Fc and mCh-eMagB-Fc(H433A) are rapidly recruited to the mitochondrial surface under blue light induction.

[0029] Figure 4 Validation of the light response of LICTOR-M: (a) are confocal microscopy images at stimulation times of 15 seconds and 60 seconds; (b) is the immunofluorescence colocalization index, which quantitatively analyzes the colocalization of mCh-eMagB-Fc with mitochondria. **** indicates p<0.0001, and scatter plots represent different samples.

[0030] Figure 5 Verification of the reversibility of LICTOR-M: (a) confocal microscopy images of light followed by darkness and darkness followed by light; (b) colocalization index of cells overexpressing eMagA-EGFP-Mito and mCh-eMagB-Fc; (c) colocalization index of control cells expressing Fc mutant light control tools (eMagA-EGFP-Mito, mCh-eMagB-Fc(H433A)); **** indicates p<0.0001, ns indicates p>0.05, and scatter plots represent different samples.

[0031] Figure 6To quantitatively analyze the role of LICTOR-M in regulating mitochondrial quality using Western blot, HEK293T cells were divided into experimental groups. Cells overexpressing LICTOR-M (eMagA-EGFP-Mito, mCh-eMagB-Fc) served as experimental groups, and cells overexpressing the Fc mutant light-controlled tool (eMagA-EGFP-Mito, mCh-eMagB-Fc(H433A)) served as control groups. The two groups were further divided into dark and light groups. Among them, (a) is the Western blot band diagram, which detects red fluorescent protein mCherry, mitochondrial outer membrane protein TOMM20, and internal reference protein GAPDH; (b) is the relative content of red fluorescent protein mCherry; (c) is the relative content of mitochondrial outer membrane protein TOMM20; * indicates p<0.05, ** indicates p<0.01, ns indicates p>0.05, and scatter points represent different samples.

[0032] Figure 7 To verify the function of LICTOR-M in mitochondrial morphology changes; (a) shows confocal micrographs of mitochondrial morphology under four conditions, with perinuclear aggregated mitochondrial morphology representing the process of mitophagy in cells; (b) shows the statistics of cells with perinuclear aggregated mitochondrial morphology; (c) shows the statistics of cells with normal mitochondrial morphology; **** indicates p<0.0001, ns indicates p>0.05, and scatter points represent different samples.

[0033] Figure 8 To verify the LICTOR-M-mediated autophagy pathway using immunofluorescence (IF), LICTOR-M (eMagA-EGFP-Mito, mCh-eMagB-Fc) was overexpressed in HEK293T cells, and the cells were divided into dark and light groups. Among them, (a) is an immunofluorescence staining image of anti-TRIM21 antibody; (b) is an immunofluorescence staining image of anti-ubiquitin-binding protein p62 antibody; and (c) is an immunofluorescence staining image of anti-microtubule-associated protein 1 light chain 3 beta (LC3β) antibody.

[0034] Figure 9 This is a schematic diagram of the LICTOR-L structure.

[0035] Figure 10To verify the localization of LICTOR-L: (a) cells in the experimental group overexpressing LICTOR-L (eMagA-miRFP-6×HP, mCh-eMagB-Fc) were stained with boron-dipyrromethene (BODIPY) and subjected to confocal microscopy; (b) cells in the control group overexpressing the Fc mutant light control tool (eMagA-miRFP-6×HP, mCh-eMagB-Fc(H433A)) were stained with BODIPY and subjected to confocal microscopy, showing that eMagA-miRFP-6×HP was localized on the lipid droplets, and both mCh-eMagB-Fc and mCh-eMagB-Fc(H433A) were rapidly recruited to the surface of the lipid droplets under blue light induction.

[0036] Figure 11 Validation of the light response of LICTOR-L: (a) is a confocal microscopy image of HeLa cells overexpressing eMagA-miRFP-6×HP and mCh-eMagB-Fc with stimulation times of 15 seconds and 60 seconds; (b) is a confocal microscopy image of HeLa cells overexpressing eMagA-miRFP-6×HP and mCh-eMagB-Fc (H433A) with stimulation times of 15 seconds and 60 seconds.

[0037] Figure 12 To verify the reversibility of LICTOR-L: HeLa cells overexpressing eMagA-miRFP-6×HP, mCh-eMagB-Fc and eMagA-miRFP-6×HP, mCh-eMagB-Fc (H433A) were subjected to confocal microscopy imaging after light exposure followed by darkness and after darkness followed by light exposure.

[0038] Figure 13 To verify the LICTOR-L function for lipid droplet morphology changes; (a) shows confocal micrographs of BODIPY-stained lipid droplets under four conditions; (b) shows the statistical results of the total lipid droplet area of ​​each cell; (c) shows the statistical results of the number of lipid droplets in each cell; **** indicates p<0.0001, ns indicates p>0.05, and scatter points represent different samples.

[0039] Figure 14To quantitatively analyze the role of LICTOR-L in regulating lipid droplet quality using Western blotting, A549 cells were used as the experimental group (overexpressing LICTOR-L (eMagA-miRFP-6×HP, mCh-eMagB-Fc) and the control group (overexpressing the Fc mutant light-controlled tool (eMagA-miRFP-6×HP, mCh-eMagB-Fc(H433A))). The two groups were further divided into a dark group and a light group. (a) shows the Western blotting bands, detecting red fluorescent protein mCherry, lipid droplet marker protein PLIN2, and internal reference protein GAPDH; (b) shows the relative content of red fluorescent protein mCherry; (c) shows the relative content of lipid droplet marker protein PLIN2. * indicates p < 0.05, *** indicates p < 0.001, ns indicates p > 0.05, and scatter plots represent different samples. Detailed Implementation

[0040] Example 1: Construction of LICTOR-M and Validation of Light-Controlled Mitochondrial Localization To achieve light-controlled regulation of mitophagy, we constructed LICTOR-M based on the enhanced photodimer system (eMags), as shown in the schematic diagram below. Figure 1 As shown, the system consists of two core fusion proteins: eMagA-EGFP-Mito (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence of the encoding gene as shown in SEQ ID NO.5) and mCh-eMagB-Fc (amino acid sequence as shown in SEQ ID NO.2, nucleotide sequence of the encoding gene as shown in SEQ ID NO.6). eMagA-EGFP-Mito contains the light-sensitive protein eMagA, fused with an EGFP tag and localized to the outer mitochondrial membrane; mCh-eMagB-Fc contains an mCherry fluorescent tag, an eMagB light-sensitive module, and an Fc domain. Upon blue light irradiation, it binds to eMagA, thereby recruiting TRIM21 protein on the target mitochondria, mediating ubiquitination, and initiating mitophagy. Figure 2 To verify the specificity of the system, an H433A mutation was introduced into the Fc domain (IgG1 antibody) to construct the mCh-eMagB-Fc(H433A) mutant (amino acid sequence as shown in SEQ ID NO.3, nucleotide sequence of the encoding gene as shown in SEQ ID NO.7) as a negative control.

[0041] HEK293T cells were co-transfected with eMagA-EGFP-Mito and mCh-eMagB-Fc or mCh-eMagB-Fc(H433A) plasmids. After staining with the mitochondrial tracer MitoTracker Deep Red (MTDR), confocal microscopy was performed to verify the localization accuracy and controllability of LICTOR-M.

[0042] The eMagA-EGFP-Mito plasmid was purchased from Addgene's website; its plasmid number is 162244.

[0043] mCh-eMagB-Fc was synthesized by myself: the vector was purchased from Beijing Qingke Biotechnology Co., Ltd., and was pcDNA3.1. mCherry, eMagB, and hIgG-Fc were all purchased from Addgene, with plasmid numbers 66839, 162252, and 105576, respectively.

[0044] The mCh-eMagB-Fc(H433A) plasmid was synthesized by introducing mutations into the mCherry-eMagB-Fc plasmid.

[0045] Under dark-field conditions, mCh-eMagB-Fc diffuses in the cytoplasm and does not colocalize with mitochondria. Figure 1 ); when at a wavelength of 470nm, 1 mW / cm 2 After 15 seconds of stimulation with intense blue light (mode: 1 second stimulation every 6 seconds), mCh-eMagB-Fc is rapidly recruited to the mitochondrial surface and co-localizes with eMagA-EGFP-Mito. Figure 3 Quantitative analysis showed that the PCC (Pearson correlation coefficient) increased from 0.27±0.02 in the dark field to 0.58±0.02 after 15 seconds of illumination, and reached a plateau at 60 seconds. Figure 4 The negative control group mCh-eMagB-Fc(H433A) also rapidly localized to the mitochondrial surface under light conditions. Figure 5 This result demonstrates that LICTOR-M enables rapid and reversible recruitment of effector proteins under blue light stimulation, with good localization specificity.

[0046] Example 2: LICTOR-M regulates mitochondrial quality To evaluate the regulatory effect of LICTOR-M on mitochondrial quality, we used HEK293T cells overexpressing LICTOR-M (eMagA-EGFP-Mito, mCh-eMagB-Fc) as the experimental group and cells overexpressing the Fc mutant (eMagA-EGFP-Mito, mCh-eMagB-Fc(H433A)) as the control group. Both groups were further divided into a dark group and a light group.

[0047] Confocal microscopy results showed that under blue light irradiation, the mitochondrial morphology changed from a normal filamentous structure to fragmentation (and...). Figure 7 (The result in (c) is the same), and they accumulate in large quantities around the cell nucleus, forming a typical perinuclear aggregation morphology. Figure 7 (a) in the middle.

[0048] Quantitative analysis showed that the proportion of positive cells with perinuclear aggregate morphology of mitochondria significantly increased from 12.45±0.95% in the dark field control group to 66.88±2.97% in the light-illuminated group. Figure 7 (b) in the middle.

[0049] Further analysis using Western blot revealed that the expression level of the mitochondrial outer membrane protein TOMM20 decreased by 15.59 ± 1.41% in the LICTOR-M light-exposed group, while no significant change was observed in the negative control group. Figure 6 These results indicate that LICTOR-M can significantly reduce mitochondrial mass under blue light activation.

[0050] Example 3: Construction of LICTOR-L and Verification of Photocontrolled Lipid Droplet Localization To achieve photocontrolled degradation of lipid droplets, we designed LICTOR-L based on LICTOR (structural schematic shown in figure). Figure 8 As shown in the figure, this system constructs eMagA-miRFP-6×HP by fusing the lipid droplet localization sequence 6×HP (amino acid sequence as shown in SEQ ID NO. 4, nucleotide sequence of the encoding gene as shown in SEQ ID NO. 8; miRFP and 6xHP were synthesized by Beijing Qingke Biotechnology Co., Ltd., the vector is pLvx-puro; eMagA is from Addgene, plasmid number 162244), thereby specifically targeting the lipid droplet surface and cooperating with mCh-eMagB-Fc to achieve light-controlled lipid droplet regulation. Similarly, we constructed the mCh-eMagB-Fc(H433A) mutant as a negative control.

[0051] To verify the localization ability of LICTOR-L, eMagA-miRFP-6×HP and mCh-eMagB-Fc were co-transfected into HeLa cells. After 24 hours, lipid droplets were detected by BODIPY staining, and the results were observed by confocal microscopy. It was observed that eMagA-miRFP-6×HP specifically localized to lipid droplets. Figure 9 ).

[0052] Under dark conditions, mCh-eMagB-Fc diffused in the cytoplasm and did not accumulate on the lipid droplet surface; after 15 s of blue light irradiation, mCh-eMagB-Fc was rapidly recruited to the lipid droplet surface and completely colocalized with eMagA-miRFP-6×HP. Figure 10 (a) In the control group, mCh-eMagB-Fc(H433A) also responded rapidly under light conditions and localized to the lipid droplet surface. Figure 10 (b) in the middle.

[0053] The results show that LICTOR-L can achieve efficient, reversible, and specific localization of lipid droplets under blue light conditions.

[0054] Example 4: LICTOR-L regulates lipid droplet quality Furthermore, we evaluated the regulatory effect of LICTOR-L on lipid droplet quality. After transfecting HeLa cells with LICTOR-L and irradiating them with light for 24 hours, BODIPY staining results showed a significant reduction in both the number of lipid droplets and the intracellular lipid droplet area. Figure 12 Western blot analysis of the lipid droplet marker protein PLIN2 showed that the PLIN2 protein level decreased by 48.43 ± 14.5% in the light-exposed group, while no significant change was observed in the negative control group. Figure 13 These results indicate that LICTOR-L can significantly reduce the mass of intracellular lipid droplets under blue light activation.

[0055] In summary, this invention, through the construction of LICTOR-M and LICTOR-L, successfully achieved precise light-controlled regulation of mitochondrial mass and lipid droplet degradation at the cellular level, validating the significant advantages of LICTOR in organelle specificity, reversibility, and high efficiency. These results lay a solid technical foundation for future applications of LICTOR, such as extending it to other organelle autophagy regulation, high-throughput drug screening, and disease modeling.

Claims

1. A programmable, light-controlled organelle autophagy regulation tool based on optogenetics, characterized in that, include: The localization module includes a first light-sensitive protein unit and a localization signal sequence capable of binding to specific organelles; The effector molecular module contains a second light-sensitive protein unit and an effector domain capable of inducing autophagy; The first and second photosensitive protein units can undergo specific dimerization under irradiation with light of a specific wavelength, thereby recruiting the effector molecular module to the surface of the target organelle and inducing the target organelle to undergo specific autophagy. The effector domain that can induce autophagy is the human IgG1 Fc domain, which triggers the ubiquitination signaling pathway by recruiting the endogenous E3 ubiquitin ligase TRIM21, and then mediates autophagy through the p62-LC3 pathway. The localization signal sequence is selected from either a mitochondrial targeting sequence or a lipid droplet targeting sequence. When the localization signal sequence is a mitochondrial targeting sequence, the tool is used for light-controlled induction of mitophagy; The amino acid sequence of the mitochondrial targeting sequence is the amino acid sequence of 413-456 aa shown in SEQ ID NO.1; When the positioning signal sequence is a lipid droplet targeting sequence, the tool is used for photo-induced lipid droplet autophagy. The amino acid sequence of the lipid droplet targeting sequence is the amino acid sequence of 319-618 aa shown in SEQ ID NO.

4.

2. The programmable light-controlled organelle autophagy regulation tool according to claim 1, characterized in that, The first photosensitive protein unit is eMagA, with an amino acid sequence of 1-152 aa as shown in SEQ ID NO.1; the second photosensitive protein unit is eMagB, with an amino acid sequence of 252-403 aa as shown in SEQ ID NO.2; The specific wavelength of light is blue light.

3. The programmable light-controlled organelle autophagy regulation tool according to claim 1, characterized in that, The amino acid sequence of the human IgG1Fc fragment is as shown in SEQ ID NO.2, which is the amino acid sequence of 418-650 aa.

4. The application of the programmable light-controlled organelle autophagy regulation tool according to any one of claims 1-3 in the preparation of cell models or kits for drug screening, disease modeling, metabolic regulation or aging research.

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