Method for monitoring lysosome acidification process mediated by mitochondrial-lysosome interaction

Abstract

The invention belongs to the field of biomedicine, and discloses a method and a kit for monitoring a lysosome acidification process mediated by mitochondrial-lysosome interaction. According to the method, an LYSO-PZ probe with a specific structure is used for marking cell lysosome contents, a pH rodo dye and a lysosome membrane marker sfGFP-LAMP1-mCherry are combined for monitoring the pH value and membrane positioning of the lysosome respectively, then a three-channel fluorescence image is obtained through a structural illumination microscope (SIM), and the fluorescence intensity ratio is quantitatively analyzed. In addition, the method further comprises a step of forming mitochondrial-lysosome contact (MLC) through optogenetics regulation, related protein binding is induced through blue light, fluorescence intensity changes before and after MLC formation are compared, and MLC-mediated proton transport is verified. A complete monitoring system is established for the first time, lysosome acidification can be accurately monitored, information can be obtained in multiple dimensions, and the method has important clinical application value and wide market prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a method for monitoring a lysosome acidification process mediated by mitochondria-lysosome interaction. Background Art

[0002] In the fields of cell biology and biomedical research, lysosomes, as crucial "digestive plants" within cells, play a central role in the degradation and recycling of substances. Lysosomes maintain a specific acidic environment, with a pH typically stable between 4.5 and 5.5. This acidic condition is crucial for lysosomal enzymes to remain active and function properly. Proton pumps, such as vacuolar ATPase (V-ATPase), are distributed across the lysosomal membrane. These proton pumps, through active transport and other means, participate in maintaining the acidic environment within the lysosome, ensuring that the lysosome can efficiently perform its physiological functions.

[0003] However, the specific source of protons required for lysosomal acidification has long been a key scientific question in this field. In-depth research on this issue is crucial for a comprehensive understanding of lysosomal function and the mechanisms of acidification. Crucially, dysregulated lysosomal acidification is closely linked to the development and progression of a variety of serious diseases. For example, lysosomal storage diseases are a group of inherited metabolic disorders caused by the deficiency or dysfunction of certain hydrolases in lysosomes, leading to the accumulation of substrates within lysosomes. Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease also involve abnormal lysosomal acidification in their pathogenesis, affecting the normal degradation and metabolism of substances such as proteins within nerve cells. The development of non-alcoholic fatty liver disease is also associated with dysregulated lysosomal acidification. Therefore, in-depth research on the mechanisms of lysosomal acidification is of vital importance for the diagnosis and treatment of these related diseases.

[0004] Mitochondrial-lysosomal contact (MLC) has become a research hotspot in recent years in the study of organelle interactions involving lysosomes. MLC plays an important role in various cellular physiological processes, including regulation of cellular metabolism, calcium signaling, lipid metabolism, and organelle quality control. For example, in cellular metabolic regulation, MLC may coordinate mitochondrial energy production and lysosomal degradation to maintain intracellular metabolic balance. In calcium signaling, MLC may serve as a channel or regulatory site for calcium ion transfer, influencing intracellular signal transduction. In lipid metabolism, MLC may participate in the transport and metabolism of lipids between lysosomes and mitochondria. In organelle quality control, MLC may help identify and remove damaged organelles.

[0005] However, at present, when studying the relationship between MLC and lysosomal acidification, the existing monitoring technology has many obvious limitations and cannot meet the needs of in-depth research.

[0006] In terms of monitoring capabilities, although traditional fluorescent dyes can label lysosomes or mitochondria, they lack the ability to specifically monitor key parameters in the lysosomal acidification process. For example, traditional fluorescent dyes cannot effectively monitor proton flux, a key parameter reflecting the flow of protons during lysosomal acidification, or the dynamic changes in content digestion, a key indicator reflecting the functional state of lysosomes. This makes it impossible to clearly present the dynamic relationship between MLC and lysosomal acidification, making it difficult for researchers to accurately grasp the interaction mechanism between the two.

[0007] In terms of research methods, most studies have only analyzed lysosomes or mitochondria from a single perspective. This single-dimensional approach lacks multidimensional, systematic monitoring methods, making it difficult to fully and deeply explore the molecular mechanisms and dynamic processes of MLC-mediated lysosomal acidification. For example, studying only from the perspective of lysosomes may overlook the role of mitochondria, while studying only from the perspective of mitochondria may not accurately understand the specific conditions of lysosomal acidification.

[0008] Furthermore, existing technologies lack effective means to regulate and monitor the molecular mechanisms of MLC formation and its effects on lysosomal acidification. This makes it difficult for researchers to precisely control MLC formation and accurately monitor its effects on lysosomal acidification in real time. These limitations severely restrict in-depth understanding of the relevant physiological and pathological mechanisms, and in turn hinder the development of therapeutic strategies for diseases associated with dysregulated lysosomal acidification.

[0009] In summary, developing a method that can effectively monitor the lysosomal acidification process mediated by mitochondria-lysosome interactions has important practical significance for in-depth understanding of the functional mechanism of lysosomes, revealing the pathogenesis of related diseases, and developing effective treatment strategies. Summary of the Invention

[0010] To address the above technical issues, the present inventors designed and synthesized the molecular probe LYSO-PZ. LYSO-PZ exhibits excited-state intramolecular proton transfer (ESIPT) properties. Its lysosomal targeting group, the piperazine group, allows precise localization of lysosomes. Fluorescence intensity changes reflect the digestion status of lysosomal contents in real time, thereby monitoring lysosomal acidification. Simultaneously, the combined use of pHrodo and sfGFP-LAMP1-mCherry tools allows for multi-dimensional monitoring of lysosomal acidification at mitochondrial-lysosomal interaction sites. This represents the first comprehensive methodology for monitoring lysosomal acidification mediated by mitochondrial-lysosomal interactions, filling a gap in this field and providing important technical support for related research in cell biology.

[0011] In one aspect, the present invention provides a probe for monitoring mitochondrial-lysosomal interaction-mediated lysosomal acidification, wherein the probe is 1-[[4-[(4-methylpiperazin-1-yl)benzylidene]-hydrazino]-methyl]-naphthalen-2-ol, also known as LYSO-PZ, and its structure is shown in Formula (I):

[0012] In another aspect, the present invention further provides a method for monitoring lysosomal acidification mediated by mitochondria-lysosome interaction, comprising the following steps: Step 1: labeling the lysosomal contents of cells with a LYSO-PZ probe, wherein the LYSO-PZ probe structure is 1-[[4-[(4-methylpiperazin-1-yl)benzylidene]-hydrazino]-methyl]-naphthalen-2-ol; Step 2: Combined use of pHrodo dye and lysosomal membrane marker sfGFP-LAMP1-mCherry to monitor lysosomal pH and membrane localization, respectively; Step 3: Synchronously acquire three-channel fluorescence images of LYSO-PZ, pHrodo, and sfGFP-LAMP1-mCherry using structured illumination microscopy (SIM); Step 4: Quantitatively analyze the fluorescence intensity ratio of the interaction site to the non-interaction site, where the interaction site meets the following requirements: the pHrodo / sfGFP-LAMP1-mCherry ratio increases and the LYSO-PZ fluorescence intensity increases.

[0013] Furthermore, in the method, the detection conditions of the LYSO-PZ probe include: an emission wavelength of 480-520 nm.

[0014] Furthermore, the method further comprises the step of optogenetic regulation of MLC formation: Through blue light-induced binding of the mitochondrial targeting protein TOM20-CIB-GFP and the lysosomal targeting protein LAMP-mCherry-CRY2; After blue light irradiation, the fluorescence intensity changes of lysosomal pHrodo and LYSO-PZ were compared before and after MLC formation.

[0015] Furthermore, in the method, in the optogenetic regulation step, when blue light induces MLC formation, the lysosomal pH value at the interaction site decreases by 0.5-1.0 units, and the LYSO-PZ fluorescence intensity increases by 20%-50%.

[0016] Furthermore, the method further includes verifying MLC-mediated proton transport through the interaction between the HOPS complex and syntaxin17, specifically comprising: VPS39 knockout or STX17 knockout cells were used; The positive correlation between MLC formation rate and lysosomal acidification degree was detected.

[0017] Furthermore, the method is applied to the study of mitochondria-lysosome interactions in cells.

[0018] Furthermore, in the method, the cells are HeLa, PC12, MCF-7 or N418 cells.

[0019] In addition, a kit for implementing the method of the present invention is provided, comprising: LYSO-PZ probe; pHrodo dye; lysosomal membrane marker plasmid sfGFP-LAMP1-mCherry; Optogenetic control plasmid combination TOM20-CIB-GFP and LAMP-mCherry-CRY2.

[0020] Furthermore, the kit also includes an MLC molecular mechanism verification component, comprising: VPS39 siRNA; STX17 siRNA; HOPS complex antibody.

[0021] Compared with the prior art, the present invention has at least the following advantages or beneficial effects: A breakthrough in precise monitoring: To address the significant shortcomings of traditional fluorescent dyes in specifically monitoring key parameters of lysosomal acidification, this study innovatively designed and synthesized the molecular probe LYSO-PZ. Leveraging its unique excited-state intramolecular proton transfer (ESIPT) properties and a targeting moiety that precisely localizes to lysosomes, LYSO-PZ accurately reflects the digestion status of lysosomal contents in real time through dynamic changes in fluorescence intensity, thereby precisely monitoring the degree of lysosomal acidification. This breakthrough provides a novel, highly accurate monitoring method for lysosomal acidification research.

[0022] Multidimensional Monitoring System: Utilizing a combination of advanced tools, including pHrodo and sfGFP-LAMP1-mCherry, we have established a comprehensive, multidimensional monitoring system for in-depth monitoring of lysosomal acidification at mitochondrial-lysosomal interaction sites. This multi-tool collaborative monitoring approach overcomes the limitations of single monitoring methods, enabling comprehensive and accurate monitoring of lysosomal acidification from multiple perspectives.

[0023] Significantly Improved Sensitivity: Rigorous experimental validation has demonstrated that LYSO-PZ, when used to monitor HeLa cells, can more sensitively capture fluorescence changes during lysosomal degradation, compared to traditional dyes, increasing detection sensitivity by approximately 30%. This significant improvement enables researchers to detect subtle changes in lysosomal acidification earlier and more accurately, providing strong support for in-depth research into the lysosomal acidification process.

[0024] For the first time, a comprehensive and systematic methodology for monitoring lysosomal acidification mediated by mitochondrial-lysosomal interactions has been successfully established. This innovation fills a gap in monitoring technology in this field, provides important technical support for related research in cell biology, and is expected to further advance lysosomal acidification research.

[0025] Innovative Application of Optogenetic Systems: Recognizing the shortcomings of existing research in multidimensional, systematic monitoring methods, this study innovatively employs an optogenetic system (LAMP-mCherry-CRY2 and TOM20-CIB-GFP) to induce CRY2-CIB binding under blue light, effectively promoting the formation of mitochondrial-lysosomal interactions. This innovative application provides a new experimental approach for studying mitochondrial-lysosomal interactions, enabling researchers to more precisely control the interaction process and laying the foundation for in-depth research on the mechanisms of lysosomal acidification.

[0026] Dynamic monitoring capabilities are enabled: Utilizing a variety of advanced detection and imaging technologies, the system dynamically monitors changes in lysosomal acidity and digestive function as mitochondrial-lysosomal interactions increase. This dynamic monitoring provides real-time insights into the dynamics of lysosomal acidification, helping researchers gain a deeper understanding of the regulatory mechanisms of lysosomal acidification and providing key data for uncovering the molecular mechanisms of lysosomal acidification.

[0027] Promoting the development of disease diagnosis and treatment: The monitoring methods and technical means provided by this invention can accurately screen for drug targets related to lysosomal acidification. This addresses the technical problem that the development of treatment strategies for diseases related to lysosomal acidification disorders in existing technologies is limited by insufficient research on the MLC-mediated lysosomal acidification process. This provides a powerful tool for the development of targeted drugs for lysosomal storage diseases, neurodegenerative diseases, and other diseases. In clinical applications, it is expected to shorten the drug development cycle by approximately 20%-30% and reduce R&D costs, thus possessing significant clinical application value and broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1The LYSO-PZ probe monitors the digestion characteristics of lysosomal contents. (a) The structure of LYSO-PZ; (b) The normalized absorption and fluorescence spectra of 5 μM LYSO-PZ in DMSO; (c) The PCC (Pearson's correlation coefficient) values of LTR and LYSO-PZ in HeLa cells, PC12 cells, and MCF-7 cells; (d) The fluorescence intensity of LYSO-PZ in Gly and BSA solutions; (e) The fluorescence intensity of LYSO-PZ in dNTPs and ct-DNA solutions; (f) The fluorescence intensity of LYSO-PZ in sugar and glycogen solutions; (g) LYSO-PZ-stained HeLa cells treated with sucrose (80 mM, 30 min), U18666A (5 μM, 24 h) and untreated (control group) SIM images; h is the normalized fluorescence intensity graph of LYSO-PZ-stained HeLa cells that were untreated, sucrose-treated, and U18666A-treated; i is the SIM image of HeLa cells stained with MTDR and LYSO-PZ, and the magnified image in the dotted box in the left figure; j is the LYSO-PZ intensity of lysosomes with or without MLC in HeLa cells; k is the SIM image of LYSO-PZ-stained HeLa cells after ER-mCherry transfection, and the magnified image in the dotted box; l is the LYSO-PZ intensity graph of lysosomes in HeLa cells with or without ER contact.

[0029] Figure 2 Figure 1 shows the results of multi-tool combined monitoring of lysosomal acidification at mitochondrial-lysosomal interaction sites. (a) SIM images of mitochondria and lysosomes in live HeLa cells expressing LAMP-mCherry and TOM20-GFP and stained with pHrodo; (b) pHrodo fluorescence intensity statistics; (c) SIM images of mitochondria and lysosomes in live HeLa cells expressing sfGFP-LAMP1-mCherry and stained with MTDR; (d) quantitative analysis of changes in lysosomal acidification in the presence and absence of MLC (*P < 0.05, **P < 0.01).

[0030] Figure 3Diagram of the HOPS complex and syntaxin17 mediating the formation of MLC. a is a schematic diagram of optogenetic control of MLC; b is a SIM super-resolution image of living HeLa cells expressing LAMP1-mCherry-CRY2 (lysosomal marker) and TOM20-CIB1-GFP (mitochondrial marker) under blue light irradiation or dark conditions, and the degree of lysosomal acidification is labeled by the pHrodo fluorescent probe; c is a local magnified image of the rectangular area in b, showing the contact details between mitochondria (green) and lysosomes (red); d is a quantitative analysis of the percentage of MLC and non-MLC in b; e is the normalized quantification of pHrodo fluorescence intensity in b; f is a SIM image of HeLa cells treated with U18666A for 24 hours (inducing lysosomal storage), lysosomes are labeled by LYSO-PZ (green) and mitochondria are labeled by TOM20-CIB1-GFP (red); g is a quantitative analysis of LYSO-PZ fluorescence in lysosomes under blue light regulation conditions; h is a quantitative analysis of LYSO-PZ fluorescence in lysosomes under blue light-free conditions.

[0031] Figure 4 Figure 1 shows the results of the study on the HOPS-syntaxin17 interaction mechanism. (a) Western blot analysis of the knockout efficiency of VPS39 (a key subunit of the HOPS complex) in HeLa cells; (b) Quantitative analysis of MLC formation rate in the VPS39 knockout group and control group Figure 3 shows the results of MLC formation; c is a SIM super-resolution image of co-localization of mitochondria and lysosomes in living HeLa cells; d is a diagram showing the co-immunoprecipitation (Co-IP) of the HOPS complex and STX17; e is a diagram showing the knockout efficiency of STX17 in U2OS cells verified by Western blot; f is a diagram showing the MLC formation rate in the STX17 knockout group and the MLC formation results in the control group; g is a SIM image of the distribution of mitochondria and lysosomes in living HeLa cells; h is a schematic diagram of proton flux from mitochondria to lysosomes through MLCs.

[0032] Figure 5 This is the H-NMR spectrum data of H-LYSO. DETAILED DESCRIPTION

[0033] The technical solutions of the present invention are described below with reference to the following embodiments; however, the present invention is not limited to the following embodiments.

[0034] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below with reference to specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0035] The experimental methods and detection methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0036] Chemical reagents: Hydrazine hydrate, tetrahydrofuran (THF), 2-hydroxy-1-naphthaldehyde, 4-(4-methylpiperazin-1-yl) benzaldehyde, p-toluenesulfonic acid monohydrate, dichloromethane, methanol, n-hexane, triethylamine, etc. with a purity of ≥98% were purchased for probe synthesis and purification; antimycin A, rotenone, oligomycin A, bafilomycin A1, etc. were used for cell treatment; all chemical reagents were purchased from Sigma-Aldrich, and the reagents were confirmed to be free of deterioration and deliquescence before use.

[0037] Cell culture materials: HeLa cells (human cervical cancer cells), PC12 cells (rat adrenal pheochromocytoma cells), MCF-7 cells (human breast cancer cells), and N418 cells (mouse embryonic fibroblasts) were selected as experimental cell lines and obtained from the China Center for Type Culture Collection. The culture medium used was Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 U / mL streptomycin, 100 units / mL penicillin, and 10% fetal bovine serum (FBS). Cell culture consumables, including 10-cm cell culture dishes, glass-bottom culture dishes (for microscopic imaging), pipette tips, and centrifuge tubes, were purchased from Corning.

[0038] Fluorescent dyes and plasmids: LysoTracker Green DND-26 (lysosome-specific fluorescent dye), MitoTracker Green FM (mitochondria-specific fluorescent dye), pHrodo (pH-sensitive lysosomal dye); construct and preserve plasmids such as LAMP1-GFP, ER-GFP, LAMP-mCherry-CRY2, and TOM20-CIB-GFP. LAMP1-GFP is used to mark lysosomal membranes, while LAMP-mCherry-CRY2 and TOM20-CIB-GFP are used for optogenetic experiments to regulate mitochondrial-lysosomal interactions.

[0039] Experimental instruments: 3D-Nikon structured illumination microscope (N-SIM, resolution up to 120 nm), blue light emitting diode (LED) array (wavelength 450 nm, power density 300 μW / cm 2 ), high-speed refrigerated centrifuge (maximum speed 12000 rpm), pipette (range covering 0.5-1000 μL), Synergy Mx microplate reader (for cell viability detection) and other instruments and equipment. All instruments were calibrated and performance tested before the experiment.

[0040] Example 1 This example is the synthesis of LYSO-PZ probe.

[0041] 1.1 Probe synthesis (1) Add 5 g of hydrazine hydrate to a 100 mL round-bottom flask, place it on a magnetic stirrer, and stir at 200 rpm; dissolve 10 g of 2-hydroxy-1-naphthaldehyde in 50 mL of THF and add it dropwise to the round-bottom flask through a constant pressure dropping funnel within 30 minutes. The reaction temperature is 28±2°C; after stirring and reacting for 6 hours, use a Buchner funnel to filter and collect the filtrate; slowly pour the filtrate into 200 mL of deionized water to precipitate a yellow precipitate, filter it again, and dry it in a vacuum drying oven at 60°C for 12 hours to obtain 8 g of crude product of 1-hydrazinomethyl-naphthalene-2-ol (H-LYSO). The yield of crude H-LYSO was calculated to be 78%. It was used directly in subsequent reactions without further purification. After the crude H-LYSO was purified, its structure was detected by nuclear magnetic resonance (NMR). The results are as follows: Figure 5 shown.

[0042] (2) H-LYSO (5 g) and 4-(4-methylpiperazin-1-yl)benzaldehyde (6 g) were dissolved in 80 mL of methanol and transferred to a 250 mL round-bottom flask. Under nitrogen protection, the reaction system was heated to 62±2°C and refluxed for 12 hours. After the reaction, methanol was evaporated at 40°C under reduced pressure using a rotary evaporator. The remaining solid was washed three times with 20 mL of cold ethanol and filtered to obtain a crude product. The product was purified by silica gel column chromatography (silica gel particle size 200-300 mesh, eluent: dichloromethane:methanol = 5:1, v / v). The target eluate was collected, concentrated by rotary evaporation again, and then dried in vacuum to obtain 4.5 g of yellow solid 1-[[4-[(4-methylpiperazin-1-yl)benzylidene]-hydrazino]-methyl]-naphthalen-2-ol (LYSO-PZ). The yield of LYSO-PZ was calculated to be 55%.

[0043] 1.2 Characterization and performance testing of LYSO-PZ probe.

[0044] The structure of the LYSO-PZ probe synthesized in Example 1 was confirmed by nuclear magnetic resonance (NMR) and mass spectrometry (MS). Figure 1 As shown in a. The normalized absorption spectrum and fluorescence emission spectrum of 5 μM LYSO-PZ in DMSO were tested by UV-visible spectrophotometer and fluorescence spectrophotometer. The results are shown in Figure 1 As shown in b.

[0045] Example 2 This example is to monitor the mitochondria-lysosome interaction-mediated lysosomal acidification process.

[0046] 2.1 Cell culture HeLa, PC12, MCF-7, and N418 cells were seeded into 10 cm cell culture dishes at a volume ratio of 1:3 and cultured in a cell culture incubator containing 5% CO2, 95% humidity, and 37°C. The culture medium was replaced every 2-3 days. When the cell density reached 80%-90%, the cells were digested with 0.25% trypsin-EDTA for subsequent experiments.

[0047] 2.2 Monitoring mitochondria-lysosome interactions mediating lysosomal acidosis Cells were seeded in glass-bottomed culture dishes and cultured for 24 hours until the cells adhered. Then, 5 μM LYSO-PZ probe was added and incubated for 30 minutes at 37°C in a 5% CO2 environment. Lysosomal digestion was monitored using structured illumination microscopy (SIM) imaging with an excitation wavelength of 405 nm and an emission detection range of 480-520 nm.

[0048] The cells were plated at 5×10 4 Cells were seeded at a density of 100 μg / mL per well in glass-bottomed culture dishes and cultured for 24 hours until they were completely attached. For staining, 5 μM LYSO-PZ probe was added and incubated for 30 minutes, or 100 μg / mL pHrodo was used for staining for 30 minutes. LysoTracker Green DND-26 (100 nM) and MitoTracker Green FM (100 nM) dyes were used at the same time. After staining, the cells were washed three times with preheated PBS (pH 7.4) for 5 minutes each time. The culture dish was placed on the stage of a 3D-Nikon structured light illumination microscope, and the excitation wavelength was set to 405 nm (LYSO-PZ, pHrodo) or 514 nm (LysoTrackerGreen DND-26, MitoTracker Green FM). The emission wavelength detection range was set according to the characteristics of the dye, such as 480-520 nm for LYSO-PZ. A 0.95NA oil objective was used with a 0.1 Z-axis scanning was performed with a step size of 1 μm to obtain three-dimensional images of the cells. ImageJ software was used to analyze the images, including fluorescence intensity measurement and colocalization analysis (calculation of Pearson correlation coefficient).

[0049] Cell viability assay: Different types of cells were cultured at a rate of 1×10 4Cells were seeded into 96-well plates at a density of 100 μM / well and cultured for 24 hours. Different concentrations of LYSO-PZ probe (0, 1, 5, 10, and 20 μM) were added and cultured for another 24 hours. Four hours before the end of culture, 10 μL of Cell Counting Kit-8 (CCK-8) solution was added to each well and incubated in a cell culture incubator. The absorbance at 450 nm was measured using a Synergy Mx microplate reader, and cell viability was calculated based on the standard curve to evaluate the cytotoxic effects of the probe.

[0050] Immunoprecipitation: HEK293T cells were washed three times with ice-cold PBS, scraped and transferred to a 1.5 mL centrifuge tube; 500 μL lysis buffer (containing 1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 1× protease inhibitor) was added and lysed on ice for 30 minutes, vortexing for 10 seconds every 5 minutes; centrifuged at 12,000 rpm at 4°C for 15 minutes, and the supernatant was collected; 50 μL anti-Flag M2 affinity gel was added to the supernatant and incubated with rotation at 4°C for 12 hours; the immune complexes were washed five times with wash buffer (containing 0.1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1 mM EDTA) for 5 minutes each; 100 μL 3× Flag peptide (200 The eluate was mixed with an equal volume of 2× sample buffer and boiled for 5 minutes to denature the protein for SDS-PAGE and immunoblotting analysis.

[0051] VPS39 knockdown: According to the instructions of Lipofectamine 3000 reagent, universal negative control siRNA, VPS39 siRNA (#1) and esiRNA (#2) were transfected into HeLa cells respectively; the transfection system was as follows: 20 pmol siRNA and 5 μL Lipofectamine 3000 reagent were mixed in 250 μL Opti-MEM medium, incubated at room temperature for 20 minutes, and then added to the cell culture dish (cell density 50-60%). After 6 hours, the culture medium was replaced with complete medium; 48 hours after transfection, the VPS39 protein expression level was detected by Western blotting to confirm the knockdown efficiency.

[0052] Transfection: TOM20-CIB-GFP and LAMP-mCherry-CRY2 plasmids were added to 100 μL DMEM medium at a 1:1 ratio (1 μg each), mixed with 100 μL DMEM medium containing 2 μL TurboFect transfection reagent, and incubated at room temperature for 15 minutes. The mixture was added to a glass-bottom culture dish seeded with cells (cell density 60-70%). After 6 hours, the medium was replaced with DMEM medium containing 10% FBS and cultured for another 24 hours before subsequent experiments.

[0053] Optogenetic experiments: After culturing cells transfected with TOM20-CIB-GFP and LAMP-mCherry-CRY2 plasmids for 24 hours, they were treated with pHrodo (100 μg / mL), LTR (100 ng / mL), LYSO-PZ (5 μM), or Baf A1 (1 μM) for 30 minutes. The cells were then placed under a blue light-emitting diode (LED) array at 300 μW / cm 2 The cells were irradiated with blue light for 20 minutes to initiate CIB-CRY2 binding. Immediately after irradiation, images were acquired using a 3D-Nikon structured light microscope to compare changes in lysosomal acidity (pHrodo fluorescence intensity) and content digestion function (LYSO-PZ fluorescence intensity) before and after blue light irradiation.

[0054] 2.3 Data Analysis S1. Statistical analysis was performed using GraphPad Prism 9.0 software. The Student's t-test was used to compare data between two groups, and one-way LYSOOVA was used to compare data from multiple groups. P < 0.05 was considered statistically significant. Data are expressed as mean ± standard error (SEM).

[0055] S2. Use ImageJ software to analyze microscopic images, including fluorescence intensity measurement, colocalization analysis (calculation of the Pearson correlation coefficient to assess the degree of mitochondrial-lysosome interaction), and area measurement (calculation of the area of lysosomes or mitochondria). Process the time-lapse imaging data and plot the fluorescence intensity versus time curve.

[0056] S3. Use Nikon Elements software to perform three-dimensional reconstruction and visualization of images acquired using structured light microscopy to more intuitively demonstrate mitochondrial-lysosomal interactions and lysosomal acidification.

[0057] S4. Use PowerPoint and Adobe Illustrator software to layout and optimize experimental images and data charts to produce clear and accurate experimental results presentations.

[0058] 2.4 Results Analysis LYSO-PZ probe verifies the digestion status of lysosomal contents Figure 1 As shown, Figure 1 (c) shows the PCC (Pearson's correlation coefficient) between LTR and LYSO-PZ in HeLa, PC12, and MCF-7 cells. Data are mean ± SEM, n = 10 cells. Figure 1 df represents the fluorescence intensity of LYSO-PZ in solutions of small and macromolecules at different concentrations. d represents glucose (Gly) and albumin (BSA), e represents deoxyribonucleotides (dNTPs) and calf thymus DNA (ct-DNA), and f represents sugar and glycogen. Figure 1 Middle g shows SIM images of LYSO-PZ-stained HeLa cells treated with sucrose (80 mM, 30 min), U18666A (5 μM, 24 h), and untreated (control group). Figure 1 Middle (h) shows the normalized fluorescence intensity of LYSO-PZ-stained HeLa cells that were untreated, sucrose-treated, or U18666A-treated. Data are mean ± SEM, n = 5 cells. Figure 1 The middle i is a SIM image of HeLa cells stained with MTDR and LYSO-PZ, and the enlarged image in the dotted box in the left panel. Figure 1 (j) Lyso-PZ intensity in HeLa cells with and without MLC. Data are M ± SEM, n = 10 cells. Figure 1 Middle k is a SIM image of HeLa cells stained with LYSO-PZ after ER-mCherry transfection, and the dotted box enlarges the image. Figure 1 Figure 1 (center) shows the LYSO-PZ intensity in lysosomes of HeLa cells with and without ER contact. Data are M±SEM, n=10 cells. The results demonstrate the specificity of the LYSO-PZ probe, with fluorescence intensity positively correlated with the macromolecular content within lysosomes. In lysosomes (colocalized with LysoTracker, PCC > 0.8), enhanced fluorescence reflects active digestion. Lysosomes with MLC exhibited significantly higher LYSO-PZ fluorescence than those without MLC, indicating that MLC promotes digestion. Lysosomes in contact with the endoplasmic reticulum (ER) exhibited no such effect, demonstrating the specificity of MLC's action.

[0059] Multi-tool combination confirmed that lysosomal acidification at the MLC site was enhanced. Figure 2 shown. Figure 2In figure a, to assess the acidification degree of lysosomes, the commercial pH-sensitive fluorescent probe pHrodo was used, and LAMP-mCherry and TOM20-GFP were co-expressed for imaging. Figure 2 The results in middle b show that the pHrodo fluorescence intensity of lysosomes in contact with mitochondria is significantly enhanced (indicating a stronger acidic environment). In contrast, the fluorescence signal of lysosomes not in contact with mitochondria is weaker. Figure 2 Middle c used another fluorescent marker protein sfGFP-LAMP1-mCherry (i.e., pHLARE) that can dynamically respond to changes in lysosomal pH. Figure 2 Similar results were observed in the experiment in middle d: lysosomes in contact with mitochondria showed higher acidity, and the fluorescence intensity ratio of sfGFP to mCherry in their pHLARE decreased significantly, further supporting the view that MLC can enhance the acidification state of lysosomes.

[0060] The molecular mechanism of HOPS-syntaxin17-mediated MLC Figure 3 As shown, Figure 3 Figure 5 (a) shows that the MLC formation rate was significantly reduced and lysosomal acidification was weakened after VPS39 knockout. Figure 3 Middle (d) shows co-immunoprecipitation confirming that HOPS directly binds to syntaxin17 (STX17). Figure 3 Figures fg and fg show that STX17 knockout also reduces MLC, indicating that HOPS-STX17 is essential for MLC formation. Figure 3 Middle h shows that MLC mediates proton transport from mitochondria to lysosomes through the HOPS-STX17 complex, maintaining an acidified environment.

[0061] The results of optogenetic regulation of MLC are as follows Figure 4 As shown, Figure 4 (a) Schematic diagram of optogenetic control of MLCs. The light-sensitive protein CIB is anchored to mitochondria via its mitochondrial-targeting transmembrane domain, TOM20; the light-sensitive protein CRY2 is anchored to lysosomes via its lysosomal-targeting transmembrane domain, LIGHT. Blue light-induced CRY2-CIB binding promotes MLC formation. GFP is used as a mitochondrial marker, and mCherry is used as a lysosomal marker. Figure 4 (b) Representative SIM images of mitochondria and lysosomes in living HeLa cells expressing LAMP-mCherry-CRY2 (lysosomes) and TOM20-CIB-GFP (mitochondria) and stained with pHrodo under blue light illumination or in the dark. Figure 4 c in the middle is Figure 4Magnified image of the region in b. d Quantification of the percentage of MLCs and those without MLCs in b. e Quantification of the percentage of pHrodo-normalized intensity in b. Data in d and e are expressed as mean ± SEM, n = 100 lysosomes from 10 cells. Figure 4 Middle f shows a representative SIM image of mitochondria and lysosomes in live HeLa cells expressing LAMP-mCherry-CRY2 and TOM20-CIB-GFP, followed by treatment with U18666A for 24 h. Lysosomes were labeled with LYSO-PZ, reflecting enhanced digestion of lysosomal contents. Figure 4 (g) Quantification of LYSO-PZ fluorescence in lysosomes under the control of blue light irradiation with CRY2-CIB. The relative lysosomal intensity is the ratio of the fluorescence intensity of the red fluorescent co-stained lysosomal signal (LYSO-PZ) to the mitochondrial signal, which is used to offset the photobleaching effect caused by blue light irradiation. Figure 4 Middle h is the quantification of LYSO-PZ fluorescence in lysosomes in the absence of blue light control in the absence of CRY2-CIB binding. Figure 4 In middle g and h, data are expressed as mean ± SEM, n = 200 lysosomes from 10 cells. Blue light irradiation increased the rate of MLC formation in cells expressing TOM20-CIB-GFP (mitochondria) and LAMP-mCherry-CRY2 (lysosomes). Following MLC formation, lysosomal pHrodo fluorescence and LYSO-PZ fluorescence increased, indicating enhanced content digestion, an effect that was dependent on blue light activation. These results suggest that MLC formation directly promotes lysosomal acidification and content degradation.

[0062] As described above, the present invention can be better implemented. The above embodiments only describe the preferred implementation methods of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the present invention.

Claims

1. A probe for monitoring mitochondrial-lysosomal interaction-mediated lysosomal acidification, the probe being 1-[[4-[(4-methylpiperazin-1-yl)benzylidene]-hydrazino]-methyl]-naphthalen-2-ol, also known as LYSO-PZ, having a structure as shown in Formula (I): 。 2. A method for monitoring lysosomal acidification mediated by mitochondria-lysosome interaction, characterized in that: The following steps are involved: Step 1: labeling the lysosomal contents of cells with a LYSO-PZ probe, wherein the LYSO-PZ probe structure is 1-[[4-[(4-methylpiperazin-1-yl)benzylidene]-hydrazino]-methyl]-naphthalen-2-ol; Step 2: Combined use of pHrodo dye and lysosomal membrane marker sfGFP-LAMP1-mCherry to monitor lysosomal pH and membrane localization, respectively; Step 3: Synchronously acquire three-channel fluorescence images of LYSO-PZ, pHrodo, and sfGFP-LAMP1-mCherry using a structured light illumination microscope; Step 4: Quantitatively analyze the fluorescence intensity ratio of the interaction site to the non-interaction site, where the interaction site meets the following requirements: the pHrodo / sfGFP-LAMP1-mCherry ratio increases and the LYSO-PZ fluorescence intensity increases.

3. The method according to claim 2, characterized in that The detection conditions of the LYSO-PZ probe include: an emission wavelength of 480-520 nm.

4. The method according to claim 2, characterized in that Also included are steps for optogenetic regulation of MLC formation: Through blue light-induced binding of the mitochondrial targeting protein TOM20-CIB-GFP and the lysosomal targeting protein LAMP-mCherry-CRY2; After blue light irradiation, the fluorescence intensity changes of lysosomal pHrodo and LYSO-PZ were compared before and after MLC formation.

5. The method according to claim 4, characterized in that In the optogenetic regulation step, when blue light induces MLC formation, the lysosomal pH value at the interaction site decreases by 0.5-1.0 units, and the LYSO-PZ fluorescence intensity increases by 20%-50%.

6. The method according to claim 2, characterized in that It also includes the verification of MLC-mediated proton transport through the interaction between the HOPS complex and syntaxin17, including: VPS39 knockout or STX17 knockout cells were used; The positive correlation between MLC formation rate and lysosomal acidification degree was detected.

7. The method according to any one of claims 2 to 6, characterized in that: The method is applied to the study of mitochondria-lysosome interactions in cells.

8. The method according to claim 7, characterized in that The cells are HeLa, PC12, MCF-7 or N418 cells.

9. A kit for implementing the method according to claim 2, characterized in that: Include: LYSO-PZ probe; pHrodo dye; lysosomal membrane marker plasmid sfGFP-LAMP1-mCherry; Optogenetic control plasmid combination TOM20-CIB-GFP and LAMP-mCherry-CRY2.

10. The kit according to claim 9, characterized in that Also included are MLC molecular mechanism verification components, including: VPS39 siRNA; STX17 siRNA; HOPS complex antibody.

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