A method of monitoring the process of lysosomal acidification mediated by mitochondrial-lysosome interaction

Abstract

The application belongs to the field of biomedicine, and discloses a method and a kit for monitoring a process of mitochondrion-lysosome interaction-mediated lysosome acidification. The method is characterized in that a cell lysosome content is first labeled with a LYSO-PZ probe with a specific structure, a pHrodo dye and a lysosome membrane marker sfGFP-LAMP1-mCherry are combined to monitor the pH value and membrane localization of the lysosome, respectively, then three-channel fluorescence images are acquired through a structured illumination microscope (SIM), and the fluorescence intensity ratio is quantitatively analyzed. In addition, the method further comprises a step of optogenetic regulation of mitochondrion-lysosome contact (MLC) formation, the fluorescence intensity change before and after the formation of MLC is compared through blue light induction of related protein binding, and the proton transport mediated by MLC is verified. The application first establishes a complete monitoring system, can accurately monitor lysosome acidification, and has important clinical application value and broad market prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedicine, and particularly relates to a method for monitoring the process of lysosome acidification mediated by mitochondria-lysosome interaction. BACKGROUND

[0002] In the field of cell biology and biomedical technology research, lysosomes play a crucial role in material degradation and recycling as the "digestion workshop" inside cells. The interior of lysosomes maintains a specific acidic environment, with a pH value usually stable between 4.5-5.5, which is the key prerequisite for lysosomal enzymes to remain active and normally exert hydrolytic function. The lysosomal membrane is distributed with proton pumps such as vacuolar ATPase (V-ATPase), which participates in maintaining the acidic environment of lysosomes through active transport and other means, ensuring that lysosomes can efficiently perform their physiological functions.

[0003] However, for a long time, the specific source of protons required in the process of lysosome acidification has been a key scientific problem in this field that needs to be solved. In-depth exploration of this problem is of great significance for a comprehensive understanding of the function and acidification mechanism of lysosomes.

[0004] In the study of organelle interactions related to lysosomes, mitochondria-lysosome contact (MLC) has gradually become a research hotspot in recent years. MLC plays an important role in various physiological processes of cells, including cellular metabolic regulation, calcium signaling, lipid metabolism, and organelle quality control. For example, in terms of cellular metabolic regulation, MLC may participate in coordinating the energy production of mitochondria and the material degradation process of lysosomes to maintain the balance of cellular metabolism; in calcium signaling, MLC may act as a channel or regulatory site for calcium ion transmission, affecting intracellular signal transduction; in lipid metabolism, MLC may be involved 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, in the study of the relationship between MLC and lysosome acidification, the existing monitoring techniques have many obvious limitations, making it difficult to meet the needs of in-depth research.

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

[0007] In terms of research means, most studies can only analyze lysosomes or mitochondria from a single perspective. This single-dimensional research method lacks multi-dimensional and systematic monitoring means, making it difficult to comprehensively and deeply explore the molecular mechanisms and dynamic processes of MLC-mediated lysosome acidification. For example, studying from the perspective of lysosomes alone may overlook the role of mitochondria; and studying from the perspective of mitochondria alone may not accurately understand the specific situation of lysosome acidification.

[0008] In addition, in the study of the molecular mechanism of MLC formation and its influence on lysosome acidification, the existing technology lacks effective regulation and monitoring means. This makes it difficult for researchers to accurately regulate the formation process of MLC and to monitor the influence of MLC on lysosome acidification in real time and accurately. In summary, developing a method that can effectively monitor the process of mitochondria-lysosome interaction-mediated lysosome acidification has important practical significance for in-depth understanding of the functional mechanism of lysosomes. SUMMARY

[0009] To solve the above technical problems, the present application designs and synthesizes a molecular probe LYSO-PZ. LYSO-PZ has the property of excited state intramolecular proton transfer (ESIPT), and its lysosome targeting group-piperazinyl can accurately locate lysosomes. Through the change of fluorescence intensity, it can reflect the digestion situation of lysosome contents in real time, and then monitor the degree of lysosome acidification. At the same time, combined with the use of pHrodo, sfGFP-LAMP1-mCherry tools, the lysosome acidification process at the mitochondria-lysosome interaction site is monitored in multiple dimensions. For the first time, a complete method system for monitoring the process of mitochondria-lysosome interaction-mediated lysosome acidification is established, filling the gap in the monitoring technology in this field and providing important technical support for related research in the field of cell biology.

[0010] In one aspect, the present application provides a probe for monitoring mitochondria-lysosome interaction-mediated lysosome acidification, which 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):

[0011]

[0012] In another aspect, the present application also provides a method for monitoring the process of mitochondria-lysosome interaction-mediated lysosome acidification for non-diagnostic and non-therapeutic purposes, characterized in that it comprises the following steps:

[0013] Step one, using the LYSO-PZ probe of the present application to label the lysosome contents of cells;

[0014] Step two, combined use of pHrodo dye and lysosome membrane marker sfGFP-LAMP1-mCherry to monitor lysosome pH value and membrane localization, respectively;

[0015] Step three, synchronously acquire three-channel fluorescence images of LYSO-PZ, pHrodo and sfGFP-LAMP1-mCherry by structured illumination microscopy (SIM);

[0016] Step four, quantitatively analyze the fluorescence intensity ratio of interaction sites and non-interaction sites, wherein the interaction sites meet the following conditions: the pHrodo / sfGFP-LAMP1-mCherry ratio increases and the LYSO-PZ fluorescence intensity increases.

[0017] Further, in the method, the detection conditions of the LYSO-PZ probe include an emission wavelength of 480-520 nm.

[0018] Further, the method further comprises a step of optogenetically regulating MLC formation.

[0019] Blue light-induced binding of the mitochondrial targeting protein TOM20-CIB-GFP and the lysosome targeting protein LAMP-mCherry-CRY2;

[0020] After blue light irradiation, compare the fluorescence intensity changes of lysosome pHrodo and LYSO-PZ before and after MLC formation.

[0021] Further, in the method, in the step of optogenetic regulation, after blue light induces MLC formation, the lysosome pH value of the interaction site decreases by 0.5-1.0 units, and the LYSO-PZ fluorescence intensity increases by 20%-50%.

[0022] Further, the method further comprises verifying MLC-mediated proton transport through the interaction of HOPS complex and syntaxin 17, specifically comprising:

[0023] Using VPS39 gene knockout or STX17 gene knockout cells;

[0024] Detecting the positive correlation between MLC formation rate and lysosome acidification degree.

[0025] Further, the method is applied to the study of mitochondrial-lysosome interaction in cells.

[0026] Further, in the method, the cells are HeLa, PC12, MCF-7 or N418 cells.

[0027] In addition, a kit for implementing the method of the present application is also provided, comprising:

[0028] LYSO-PZ probe;

[0029] pHrodo dye;

[0030] lysosome membrane marker plasmid sfGFP-LAMP1-mCherry;

[0031] Optogenetic regulation plasmid combination TOM20-CIB-GFP and LAMP-mCherry-CRY2.

[0032] Further, the kit also includes an MLC molecular mechanism verification component, comprising:

[0033] VPS39 siRNA;

[0034] STX17 siRNA;

[0035] HOPS complex antibody.

[0036] Compared with the prior art, the present application has at least the following advantages or beneficial effects:

[0037] Precise monitoring breakthrough: In view of the significant shortcoming of traditional fluorescent dyes in specific monitoring of key parameters of lysosome acidification, the present application innovatively designs and synthesizes a molecular probe LYSO-PZ. LYSO-PZ, with its unique excited-state intramolecular proton transfer (ESIPT) characteristics and precise lysosome-targeting groups, can reflect the digestion of lysosome contents in real time and accurately through the dynamic changes of fluorescence intensity, and thus accurately monitor the degree of lysosome acidification. This breakthrough provides a new and high-precision monitoring method for lysosome acidification research.

[0038] Multi-dimensional monitoring system: By combining the use of pHrodo, sfGFP-LAMP1-mCherry and other advanced tools, a multi-dimensional and all-round monitoring system is constructed to monitor the lysosome acidification process at the mitochondria-lysosome interaction site. This multi-tool collaborative monitoring method overcomes the limitations of single monitoring method and can obtain relevant information of lysosome acidification from multiple angles, greatly improving the comprehensiveness and accuracy of monitoring.

[0039] Significant improvement in sensitivity: Through rigorous experimental verification, when monitoring HeLa cells using LYSO-PZ, compared with traditional dyes, it can more sensitively capture the fluorescence changes in the degradation process of lysosome contents, and the detection sensitivity is improved by about 30%. This significant improvement enables researchers to detect subtle changes in lysosome acidification earlier and more accurately, providing strong support for in-depth study of the lysosome acidification process.

[0040] A complete and systematic method for monitoring the process of lysosome acidification mediated by mitochondrial-lysosome interaction is successfully established for the first time. This innovation fills the gap in the field of monitoring technology and provides important technical support for related research in the field of cell biology, which is expected to promote the further development of lysosome acidification research.

[0041] Innovative application of optogenetic system: In view of the shortcomings of existing research in multidimensional and systematic monitoring methods, the invention innovatively uses the optogenetic system (LAMP-mCherry-CRY2 and TOM20-CIB-GFP) to effectively promote the formation of mitochondrial-lysosome interaction by inducing CRY2-CIB binding through blue light. This innovative application provides a new experimental means for studying mitochondrial-lysosome interaction, enabling researchers to more accurately control the interaction process and laying the foundation for in-depth study of lysosome acidification mechanism.

[0042] Dynamic monitoring function: With the help of various advanced detection and imaging technologies, dynamic monitoring of lysosome acidity and content digestion function changes when mitochondrial-lysosome interaction increases is achieved. This dynamic monitoring can reflect the dynamic changes of lysosome acidification process in real time, helping researchers to better understand the regulation mechanism of lysosome acidification and providing key data for revealing the molecular mechanism of lysosome acidification. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1Characterization of LYSO-PZ for monitoring lysosomal content digestion. a, Structure of LYSO-PZ; b, Normalized absorption and fluorescence spectra of 5 mM LYSO-PZ in DMSO; c, PCC (Pearson’s correlation coefficient) values of LTR and LYSO-PZ in HeLa cells, PC12 cells and MCF-7 cells; d, Fluorescence intensity plots of LYSO-PZ in Gly and BSA solutions; e, Fluorescence intensity plots of LYSO-PZ in dNTPs and ct-DNA solutions; f, Fluorescence intensity plots of LYSO-PZ in sugar and glycogen solutions; g, SIM images of LYSO-PZ-stained HeLa cells treated with sucrose (80 mM, 30 min), U18666A (5 mM, 24 h) and untreated (control); h, Normalized fluorescence intensity plots of LYSO-PZ-stained HeLa cells untreated, treated with sucrose and treated with U18666A; i, SIM images of HeLa cells stained with MTDR and LYSO-PZ, and the magnified images in the dashed boxes in the left panel; j, LYSO-PZ intensity in HeLa cells with or without MLC lysosomes; k, SIM images of LYSO-PZ-stained HeLa cells after ER-mCherry transfection, and the magnified images in the dashed boxes; l, LYSO-PZ intensity plots of lysosomes in HeLa cells with or without ER contact.

[0044] Figure 2 Results of lysosomal acidification for monitoring mitochondrial-lysosome interaction sites with multiple tools. a, SIM images of mitochondria and lysosomes in live HeLa cells expressing LAMP-mCherry and TOM20-GFP and stained with pHrodo; b, Statistical results of pHrodo fluorescence intensity; c, SIM images of mitochondria and lysosomes in live HeLa cells expressing sfGFP-LAMP1-mCherry and stained with MTDR; d, Quantitative analysis of lysosomal acidification under conditions with or without MLC (*P < 0.05, **P < 0.01).

[0045] Figure 3Figure 1. MLC formation by HOPS complex and syntaxin 17. a, Schematic diagram of optogenetic control of MLC; b, SIM super-resolution images of live HeLa cells expressing LAMP1-mCherry-CRY2 (lysosome marker) and TOM20-CIB1-GFP (mitochondria marker) under blue light illumination or dark condition, lysosome acidification degree was labeled by pHrodo fluorescent probe; c, Local magnified image of the rectangular region in b, showing the details of mitochondria (green) and lysosome (red) contact; d, Quantitative analysis of the percentage of MLC and non-MLC in b; e, Normalized quantitative analysis of pHrodo fluorescence intensity in b; f, SIM images of HeLa cells treated with U18666A for 24 hours (inducing lysosomal storage), lysosomes were labeled by LYSO-PZ (green), mitochondria were labeled by TOM20-CIB1-GFP (red); g, Quantitative analysis of LYSO-PZ fluorescence in lysosomes under blue light regulation condition; h, Quantitative analysis of LYSO-PZ fluorescence in lysosomes without blue light condition.

[0046] Figure 4 Figure 2. Mechanism of HOPS-syntaxin 17 interaction. a, Western blot detection of the knockdown efficiency of VPS39 (a key subunit of HOPS complex) in HeLa cells; b Quantitative analysis of MLC formation rate in VPS39 knockout group and Control group MLC formation results; c, SIM super-resolution images of mitochondria and lysosome co-localization in live HeLa cells; d, Co-immunoprecipitation (Co-IP) results of HOPS complex and STX17; e, Western blot verification of the knockdown efficiency of STX17 in U2OS cells; f, MLC formation rate of STX17 knockdown group and control group; g, SIM images of the distribution of mitochondria and lysosome in live HeLa cells; h, Schematic diagram of proton flux through MLCs from mitochondria to lysosome.

[0047] Figure 5 Figure 3. NMR hydrogen spectrum-carbon spectrum data of H-LYSO. DETAILED DESCRIPTION

[0048] Hereinafter, the technical solutions of the present application will be described in conjunction with examples, but the present application is not limited to the following examples.

[0049] In order for those skilled in the art to better understand the technical solutions of the present application and to implement them, the present application will be further described below in conjunction with specific examples and drawings, but the examples are not limiting to the present application.

[0050] The experimental methods and detection methods described in the following examples are all conventional methods unless otherwise specified. The reagents and materials described are commercially available unless otherwise specified.

[0051] 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. were purchased with a purity of ≥98% for probe synthesis and purification; Antimycin A, rotenone, oligomycin A, bafilomycin Al, etc. were used for cell treatment; all chemical reagents were purchased from Sigma-Aldrich Company, and the reagents were confirmed to be free of deterioration and deliquescence before use.

[0052] 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 strains and obtained from the China Typical Culture Collection Center; the culture medium was Dulbecco's modified Eagle's medium (DMEM) containing 100 U / mL streptomycin, 100 units / mL penicillin, and 10% fetal bovine serum (FBS); cell culture consumables included 10 cm cell culture dishes, glass bottom culture dishes (for microscopic imaging), pipette tips, centrifuge tubes, etc., and were purchased from Corning Company.

[0053] Fluorescent dyes and plasmids: LysoTracker Green DND-26 (lysosome-specific fluorescent dye), MitoTracker Green FM (mitochondria-specific fluorescent dye), pHrodo (pH-sensitive lysosome dye); LAMP1-GFP, ER-GFP, LAMP-mCherry-CRY2, TOM20-CIB-GFP, etc. plasmids were constructed and preserved, wherein LAMP1-GFP was used to label lysosome membranes, and LAMP-mCherry-CRY2 and TOM20-CIB-GFP were used for optogenetic experiments to regulate mitochondria-lysosome interactions.

[0054] Experimental instruments: 3D-Nikon structured light 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), etc. All instruments were calibrated and performance tested before the experiment.

[0055] Example 1

[0056] This example is the synthesis of LYSO-PZ probe.

[0057] 1.1 Probe synthesis

[0058] (1) 5 g of hydrazine hydrate was added into a 100 mL round-bottom flask, which was placed on a magnetic stirrer and stirred at 200 rpm; 10 g of 2-hydroxy-1-naphthaldehyde was dissolved in 50 mL of THF and added dropwise into the round-bottom flask through a constant pressure dropping funnel within 30 minutes, with the reaction temperature being 28±2℃; after stirring for 6 hours, the reaction was filtered by a Buchner funnel, and the filtrate was slowly poured into 200 mL of deionized water to precipitate a yellow solid, which was dried in a vacuum drying oven at 60℃ for 12 hours after being filtered again, to obtain 1-hydrazinomethyl-naphthalen-2-ol (H-LYSO) crude product 8 g. The yield of H-LYSO crude product was calculated to be 78%. The crude product was directly used in the subsequent reaction without further purification. After purification of the H-LYSO crude product, nuclear magnetic resonance (NMR) was used to detect the structure, and the results are shown in FIG. 1a. Figure 5

[0059] (2) H-LYSO (5 g) and 4-(4-methylpiperazin-1-yl) benzaldehyde (6 g) were dissolved in 80 mL of methanol and transferred into a 250 mL round-bottom flask; the reaction system was heated to 62±2℃ under nitrogen protection, and refluxed for 12 hours; after the reaction was completed, the methanol was evaporated under reduced pressure at 40℃ by using a rotary evaporator; the remaining solid was washed with 20 mL of cold ethanol for 3 times, and then filtered to obtain a crude product; the crude product was purified by silica gel column chromatography (silica gel particle size 200-300 mesh, eluent dichloromethane:methanol=5:1, v / v), the target eluent was collected, and then concentrated by rotary evaporation and dried in vacuum to obtain 1-[[4-[(4-methylpiperazin-1-yl)benzylidene]-hydrazinyl]-methyl]-naphthalen-2-ol (LYSO-PZ) 4.5 g, and the yield of LYSO-PZ was calculated to be 55%.

[0060] 1.2 Characterization and performance test of LYSO-PZ probe

[0061] The structure of the LYSO-PZ probe synthesized in Example 1 was confirmed by nuclear magnetic resonance (NMR) and mass spectrometry (MS), and the results are shown in FIG. 1a. The normalized absorption spectrum and fluorescence spectrum emission spectrum of 5 μM LYSO-PZ in DMSO were tested by using an ultraviolet-visible spectrophotometer and a fluorescence spectrophotometer, and the results are shown in FIG. 1b. Figure 1 Figure 1

[0062] Example 2

[0063] This example is to monitor the process of lysosome acidification mediated by mitochondrial-lysosome interaction.​​​

[0064] 2.1 Cell culture

[0065] HeLa, PC12, MCF-7 and N418 cells were seeded in 10 cm cell culture dishes at a ratio of 1:3 by volume, and incubated in a cell culture incubator with 5% CO2, humidity 95%, temperature 37 ℃; the culture medium was replaced every 2-3 days, and when the cell density reached 80%-90% density, the cells were digested with 0.25% trypsin-EDTA for subsequent experiments.

[0066] 2.2 Monitoring of lysosome acidification mediated by mitochondrial-lysosome interaction

[0067] Cells were seeded in glass-bottom dishes and incubated for 24 hours until the cells adhered. Then 5 μM LYSO-PZ probe was added and incubated at 37 ℃, 5% CO2 for 30 minutes. The structure light illumination microscopy (SIM) imaging technology was used to monitor the lysosome content digestion, with an excitation wavelength of 405 nm and an emission wavelength detection range of 480-520 nm.

[0068] Cells were seeded in glass-bottom dishes at a density of 5×10 4 cells / well and incubated for 24 hours until the cells adhered completely. Staining was performed by adding 5 μM LYSO-PZ probe for 30 minutes or 100 μg / mL pHrodo for 30 minutes, together with LysoTracker Green DND-26 (100 nM) and MitoTracker Green FM (100 nM) dyes. After staining, the cells were washed with preheated PBS (pH 7.4) for 3 times, 5 minutes each time. The dishes were placed on the 3D-Nikon structure light illumination microscope stage, with an excitation wavelength of 405 nm (LYSO-PZ, pHrodo) or 514 nm (LysoTracker Green DND-26, MitoTracker Green FM), and the emission wavelength detection range was set according to the characteristics of the dyes, such as 480-520 nm for LYSO-PZ. A 0.95 NA oil objective was used to scan the Z axis at a step size of 0.1 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).

[0069] Cell viability assay: Different types of cells were seeded in 96-well plates at a density of 1×10 4Cells were seeded into 96-well plates at a density of 1 x 104cells / well and incubated for 24 h. Then, different concentrations of LYSO-PZ (0, 1, 5, 10, 20 μΜ) were added to the cells, and incubation was continued for another 24 h. Four hours before the end of incubation, 10 μL of Cell Counting Kit-8 (CCK-8) solution was added to each well, and the cells were incubated in the cell incubator. The absorbance value at 450 nm was measured using a Synergy Mx microplate reader. The cell viability was calculated according to the standard curve, and the toxicity of the probe to the cells was evaluated.

[0070] Co-immunoprecipitation: HEK293T cells were washed with ice-cold PBS for 3 times, scraped off and transferred to 1.5 mL centrifuge tubes. Then, 500 μL of lysis buffer (containing 1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1x protease inhibitor) was added, and the cells were lysed on ice for 30 min, with vortexing for 10 s every 5 min. The supernatant was obtained by centrifugation at 4 ℃ and 12000 rpm for 15 min. Then, 50 μL of anti-Flag M2 affinity gel was added to the supernatant, and the mixture was incubated at 4 ℃ for 12 h. The immunocomplex was washed with washing buffer (containing 0.1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1 mM EDTA) for 5 times, with 5 min for each time. Then, 100 μL of 3x Flag peptide (200 μg / mL) was added, and the mixture was incubated at 4 ℃ for 2 h for elution. The eluate was mixed with an equal volume of 2x sample buffer, and boiled for 5 min to denature the proteins, which were subjected to SDS-PAGE and immunoblotting analysis.

[0071] VPS39 knockdown: Universal negative control siRNA, VPS39 siRNA (#1) and esiRNA (#2) were transfected into HeLa cells according to the instructions of Lipofectamine 3000 reagent. The transfection system was as follows: 20 pmol of siRNA was mixed with 5 μL of Lipofectamine 3000 reagent in 250 μL of Opti-MEM medium, and incubated at room temperature for 20 min. Then, the mixture was added to the cell culture dish (cell density 50-60%), and the complete medium was replaced after 6 h. The expression level of VPS39 protein was detected by Western blotting 48 h after transfection, and the knockdown efficiency was confirmed.

[0072] Transfection: TOM20-CIB-GFP and LAMP-mCherry-CRY2 plasmids were added into 100 μL DMEM medium at a ratio of 1:1 (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 the glass-bottomed culture dish inoculated with cells (cell density 60-70%), and after 6 hours, the medium was replaced with DMEM medium containing 10% FBS, and the cells were cultured for another 24 hours before subsequent experiments.

[0073] Optogenetics experiment: after culturing the 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, respectively; the cells were placed under a blue light-emitting diode (LED) array to irradiate them with 300 μW / cm 2 of blue light for 20 minutes to activate CIB-CRY2 binding; immediately after irradiation, a 3D-Nikon structured illumination microscope was used to acquire images to compare the changes in lysosome acidity (pHrodo fluorescence intensity) and content digestion function (LYSO-PZ fluorescence intensity) before and after blue light irradiation.

[0074] 2.3 Data analysis

[0075] S1, statistical analysis was performed using GraphPad Prism 9.0 software, Student's t-test was used for comparison between two groups of data, and one-way ANOVA was used for comparison between multiple groups of data (One-way LYSOOVA), with P<0.05 being considered statistically significant; data were expressed as mean ± standard error (SEM).

[0076] S2, ImageJ software was used to analyze the microscopic images, including fluorescence intensity measurement, colocalization analysis (Pearson correlation coefficient was calculated to evaluate the degree of mitochondria-lysosome interaction), area measurement (lysosome or mitochondria area was calculated), etc.; time-lapse imaging data were processed to generate fluorescence intensity-time curve.

[0077] S3, Nikon Elements software was used to perform three-dimensional reconstruction and visualization processing on the images acquired by the structured illumination microscope, to more intuitively display the mitochondria-lysosome interaction and lysosome acidification process.

[0078] S4, PowerPoint and Adobe Illustrator software were used to layout and optimize experimental images and data charts to produce clear and accurate experimental result display graphs.

[0079] 2.4 Results analysis

[0080] LYSO-PZ probe verified lysosome content digestion status as shown in Figure 1 Figure 1 In c, PCC (Pearson’s correlation coefficient) values of LTR and LYSO-PZ in HeLa cells, PC12 cells and MCF-7 cells. Data are M ± SEM, n = 10 cells. Figure 1 In d-f, fluorescence intensity of LYSO-PZ in different concentrations of small and large molecule solutions. d represents glucose (Gly) and albumin (BSA), e represents deoxyribonucleotides (dNTPs) and calf thymus DNA (ct-DNA), f represents sugar and glycogen. Figure 1 In g, SIM images of LYSO-PZ stained HeLa cells treated with sucrose (80 mM, 30 min), U18666A (5 μM, 24 h) and untreated (control). Figure 1 In h, normalized fluorescence intensity of LYSO-PZ stained HeLa cells without treatment, sucrose treatment or U18666A treatment. Data are M ± SEM, n = 5 cells. Figure 1 In i, SIM images of HeLa cells stained with MTDR and LYSO-PZ, and enlarged images in the dashed box in the left panel. Figure 1 In j, LYSO-PZ intensity in HeLa cells with and without MLC lysosomes. Data are M ± SEM, n = 10 cells. Figure 1 In k, SIM images of LYSO-PZ stained HeLa cells after ER-mCherry transfection, and enlarged images in the dashed box. Figure 1 In l, LYSO-PZ intensity of lysosomes in HeLa cells with and without ER contact. Data are M ± SEM, n = 10 cells. The results show that the LYSO-PZ probe is characterized by its fluorescence intensity positively correlated with the content of macromolecules in lysosomes. In lysosomes (co-localized with LysoTracker, PCC > 0.8), the fluorescence enhancement reflects active content digestion, and the LYSO-PZ fluorescence intensity of lysosomes with MLC is significantly higher than that of lysosomes without MLC, indicating that MLC promotes content digestion. Lysosomes in contact with the endoplasmic reticulum (ER) do not have this effect, proving the specificity of the role of MLC.

[0081] Multi-tool joint verification of MLC site lysosome acidification enhancement, results as shown in Figure 2 Figure 2 ​​a. The pHrodo was used to evaluate the acidification of lysosomes. LAMP-mCherry and TOM20-GFP were co-expressed for imaging. Figure 2 b. The results showed that lysosomes contacting mitochondria had significantly enhanced pHrodo fluorescence intensity (indicating a stronger acidic environment), in contrast to lysosomes not contacting mitochondria. Figure 2 c. Another fluorescently labeled protein, sfGFP-LAMP1-mCherry (pHLARE), was used to dynamically respond to changes in lysosomal pH. Figure 2 d. Similar results were observed in the experiment: lysosomes contacting mitochondria showed higher acidity, with a significantly reduced sfGFP / mCherry fluorescence intensity ratio in pHLARE, further supporting the view that MLC can enhance lysosomal acidification.

[0082] The molecular mechanism of HOPS-syntaxin17-mediated MLC is shown in Figure 3 Figure 3 a-c. The results showed that after VPS39 knockout, the formation rate of MLC was significantly reduced, and lysosomal acidification was weakened. Figure 3 d. Co-immunoprecipitation confirmed the direct binding of HOPS to syntaxin17 (STX17). Figure 3 f-g. STX17 knockout also reduced MLC, indicating that HOPS-STX17 is a necessary condition for MLC formation, Figure 3 h. The results showed that

[0083] MLC mediates the proton transport from mitochondria to lysosomes through the HOPS-STX17 complex, maintaining the acidification environment.

[0084] The results of optogenetic regulation of MLC are shown in Figure 4 Figure 4 a. Schematic diagram of optogenetic control of MLC. The light-sensitive protein CIB is anchored to mitochondria through the mitochondrial targeting transmembrane domain TOM20; the light-sensitive protein CRY2 is anchored to lysosomes through the lysosome targeting transmembrane domain lamp. The regulation of CRY2-CIB binding by blue light promotes the formation of MLC; GFP is expressed as a marker for mitochondria, and mCherry is expressed as a marker for lysosomes. Figure 4 b. Representative SIM images of mitochondria and lysosomes in live HeLa cells expressing LAMP-mCherry-CRY2 (lysosome) and TOM20-CIB-GFP (mitochondria), and stained with pHrodo under blue light or in the dark. Figure 4 c. The results showed that Figure 4 ​​Scale bar, 10 pm. d Quantification of MLCs and MLCs-free percentage in b. e Quantification of pHrodo normalized intensity percentage in b. Data in d and e are presented as M ± SEM, n = 100 lysosomes from 10 cells. Figure 4 f Representative SIM images of mitochondria and lysosomes in live HeLa cells expressing LAMP-mCherry-CRY2 and TOM20-CIB-GFP, then treated with U18666A for 24 h. Lysosomes were labeled with LYSO-PZ, reflecting enhanced content digestion in lysosomes. Figure 4 g Quantification of LYSO-PZ fluorescence in lysosomes under blue light control of CRY2-CIB binding. Relative lysosome intensity is the ratio of lysosome signal (LYSO-PZ) to mitochondrial signal in red fluorescence co-staining, used to offset photobleaching effects caused by blue light illumination. Figure 4 h Quantification of LYSO-PZ fluorescence in lysosomes in the absence of blue light control of CRY2-CIB binding. ​ g and h, Data are presented as M ± SEM, n = 200 lysosomes from 10 cells. Cells expressing TOM20-CIB-GFP (mitochondria) and LAMP-mCherry-CRY2 (lysosomes) showed increased MLC formation rate upon blue light illumination. After MLC formation, lysosome pHrodo fluorescence intensity increased, and LYSO-PZ fluorescence intensity increased, indicating enhanced content digestion function, and this effect was dependent on blue light activation. The results showed that MLC formation directly promoted lysosome acidification and content degradation.

[0085] As above, the application can be well realized, the above-described embodiments are merely to describe the preferred embodiments of the application, and not to limit the scope of the application, under the premise of not departing from the design spirit of the application, various changes and improvements of the technical solutions of the application made by those skilled in the art, should fall within the scope of the application.

Claims

1. A probe for monitoring the mitochondrial-lysosome interaction-mediated lysosome acidification, referred to as LYSO-PZ, having the structure shown in formula (I) : ###0001### Formula (I) 。 2. A method of monitoring the process of lysosomal acidification mediated by the mitochondrial-lysosomal interaction for non-diagnostic and non-therapeutic purposes, characterized by, comprising the following steps: Step 1, labeling the lysosome content of cells with the LYSO-PZ probe of claim 1; Step 2, using the pHrodo dye and the lysosome membrane marker sfGFP-LAMP1-mCherry in combination to monitor the lysosome pH value and membrane localization, respectively; Step 3, synchronously acquiring three-channel fluorescence images of LYSO-PZ, pHrodo and sfGFP-LAMP1-mCherry by structured light illumination microscopy; Step 4, quantitatively analyzing the fluorescence intensity ratio of the interaction sites and non-interaction sites, wherein the interaction sites satisfy the following conditions: the pHrodo / sfGFP-LAMP1-mCherry ratio is increased and the fluorescence intensity of LYSO-PZ is increased.

3. The method of claim 2, wherein, The detection conditions of the LYSO-PZ probe include an emission wavelength of 480-520 nm.

4. The method of claim 2, wherein, Further comprising a step of optogenetically regulating the formation of MLC: Blue light-induced binding of the mitochondrial-targeting protein TOM20-CIB-GFP and the lysosome-targeting protein LAMP-mCherry-CRY2; After blue light irradiation, the fluorescence intensity changes of lysosome pHrodo and LYSO-PZ before and after MLC formation are compared.

5. The method of claim 4, wherein, In the step of optogenetic regulation, after blue light-induced MLC formation, the lysosome pH value of the interaction sites is reduced by 0.5-1.0 units, and the fluorescence intensity of LYSO-PZ is increased by 20%-50%.

6. The method of claim 2, wherein, Further comprising a step of verifying MLC-mediated proton transport through the interaction of the HOPS complex with syntaxin 17, specifically comprising: Using VPS39 gene knockout or STX17 gene knockout cells; Detecting the positive correlation between the MLC formation rate and the degree of lysosome acidification.

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

8. The method of claim 7, wherein, The cells are HeLa, PC12, MCF-7 or N418 cells.

9. A kit for performing the method of claim 2, wherein, Comprising: The LYSO-PZ probe of claim 1; The pHrodo dye; The lysosome membrane marker plasmid sfGFP-LAMP1-mCherry; The optogenetic regulation plasmid combination TOM20-CIB-GFP and LAMP-mCherry-CRY2.

10. The kit of claim 9, wherein Further comprising an MLC molecular mechanism verification component, comprising: VPS39 siRNA; STX17 siRNA; HOPS complex antibodies.

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