A fluorescent probe pair for monitoring the interaction between endocytosis and mitophagy in real time and application thereof

Abstract

The application discloses a fluorescent probe pair for monitoring the interaction between endocytosis and mitochondrial autophagy in real time and application thereof. The probe pair is composed of a fluorescent probe G-CM and a fluorescent probe R-Mito. The G-CM can be specifically anchored to a cell membrane, delivered to a lysosome through an endocytosis pathway and used as a FRET donor; the R-Mito can be covalently anchored to a mitochondrion, enter a lysosome along with a damaged mitochondrion in a mitochondrial autophagy process and used as a FRET acceptor. When endocytosis and mitochondrial autophagy converge in the same lysosome, the spatial distance between the two probes is close, and a specific FRET signal (405 nm excitation, 665-735 nm emission) is generated. The application realizes in-situ and real-time visual monitoring of functional crosstalk of the two key cellular processes, and has wide application prospects in the fields of cell biology, anticancer drug screening and cancer diagnosis.

Description

Technical Field

[0001] This invention belongs to the fields of fluorescence imaging technology and chemical biology, specifically relating to a fluorescent probe pair for real-time monitoring of the interaction between endocytosis and mitophagy and its application. Background Technology

[0002] Endocytosis and mitophagy are two crucial physiological processes in eukaryotic cells. Endocytosis is a key pathway for cells to take up nutrients, regulate membrane proteins, repair cell membranes, and transduce signals; mitophagy is an important way for cells to selectively degrade damaged or excess mitochondria through autophagy, maintaining mitochondrial quality control and metabolic homeostasis. Numerous studies have shown that endocytosis and mitophagy are functionally closely related: both are highly dependent on membrane fusion and share the terminal organelle—lysosome. Endocytosis delivers vesicles from the cell membrane to lysosomes for degradation, while mitophagy encapsulates damaged mitochondria and delivers them to lysosomes. Therefore, under specific physiological or pathological conditions, endocytosis and mitophagy may converge within the same lysosome, i.e., there is "crosstalk" between them. Revealing the existence, extent, and regulatory mechanisms of this crosstalk is of significant scientific importance for understanding cellular metabolism, signal transduction, cancer development, and neurodegenerative diseases such as Parkinson's disease.

[0003] Currently, the main methods for studying endocytosis and mitophagy include electron microscopy, Western blotting, and fluorescence imaging. Electron microscopy and Western blotting require cell fixation or lysis, making dynamic observation of live cells impossible. In the field of fluorescence imaging, some commercial or laboratory probes are available for monitoring endocytosis (e.g., pH-sensitive membrane probes) or mitophagy (e.g., mitochondrial-targeted pH probes) separately. However, these probes are single-function, reporting only the occurrence of a single process and unable to simultaneously report whether the two processes converge in the same time and space. In particular, no molecular tool can currently directly visualize the functional crosstalk between endocytosis and mitophagy. Therefore, developing a dual-probe system capable of simultaneously tracking endocytosis and mitophagy and generating a specific signal when they synergize is a pressing technical challenge in this field. Summary of the Invention

[0004] To address the gaps in existing technologies, this invention provides for the first time a pair of fluorescent probes (G-CM and R-Mito) based on the FRET principle for real-time, in-situ monitoring of crosstalk between endocytosis and mitophagy. The probe pair of this invention can specifically label the cell membrane (for tracking endocytosis) and mitochondria (for tracking mitophagy), respectively, and generate a FRET signal when both processes converge at the same lysosome, thus directly reporting the occurrence of crosstalk.

[0005] This invention provides a set of fluorescent probe pairs, characterized in that the fluorescent probe pairs are formed by the interaction of probe G-CM and fluorescent probe R-Mito, and the structural formulas of probe G-CM and probe R-Mito are as follows: .

[0006] The preparation methods of probe G-CM and probe R-Mito provided by this invention are as follows: Synthesis of probe G-CM: Intermediate 1: Dissolve 4-methylpyridine and 1-iodooctadecane in anhydrous ethanol, reflux, cool and filter, wash with cold ethanol to obtain a white solid, i.e., intermediate 1; Synthesis of G-CM: Intermediate 1 and 9-ethyl-9H-carbazole-3-carbaldehyde were dissolved in ethanol, piperidine was added dropwise, the mixture was refluxed, cooled and filtered, and washed with cold ethanol to obtain a pale yellow solid, namely probe G-CM. Intermediate 2: 4-methylquinoline, 4-(dimethylamino)benzaldehyde, and p-toluenesulfonic acid were dissolved in DMF and refluxed for 12 h. After cooling, water was added to precipitate the solid, which was then filtered and purified by column chromatography to obtain a purple solid, i.e., intermediate 2. Synthesis of R-Mito: Intermediate 2 and p-dichlorobenzyl were dissolved in toluene, refluxed, cooled and filtered, and recrystallized from toluene to obtain purple crystals, namely probe R-Mito.

[0007] This invention provides the application of the fluorescent probe pair in monitoring endocytosis and mitochondrial autophagy crosstalk and its detection method.

[0008] The probe design principle of this invention is based on the FRET mechanism and makes full use of the biological characteristics that both endocytosis and mitophagy end at the lysosome.

[0009] The donor probe G-CM was designed using a pyridinium salt modified with a long alkyl chain (C18) as a membrane anchoring group, enabling it to stably insert into the phospholipid bilayer of the cell membrane and remain there for extended periods without endocytosis or free diffusion. Simultaneously, a carbazole-vinylpyridine fluorophore with a large Stokes shift was introduced, emitting green pseudo-color fluorescence (500-550 nm). During endocytosis, G-CM invaginates into the cell membrane and is ultimately delivered to the lysosomes.

[0010] The receptor probe R-Mito is designed using a quinolineonium cation as a mitochondrial targeting group, responding to the mitochondrial membrane potential. Simultaneously, a benzyl chloride group is introduced, which undergoes an irreversible covalent reaction with thiol groups on mitochondrial membrane proteins, thus anchoring the probe to the mitochondria and preventing detachment even after a subsequent decrease in mitochondrial membrane potential (inevitable during mitophagy). Its fluorophore emits deep red fluorescence (665-735 nm), showing good overlap with the emission spectrum of G-CM, making it an ideal FRET receptor.

[0011] FRET crosstalk detection mechanism: In normal cells, G-CM is located on the cell membrane, while R-Mito is located in the mitochondria. The spatial distance between them is too great for FRET to occur. When endocytosis and mitophagy occur simultaneously, G-CM enters the lysosome via endocytosis, while R-Mito enters the lysosome via autophagy along with the damaged mitochondria. Only when both processes converge on the same lysosome does the distance between the two probes shorten to less than 10 nm. At this point, when G-CM is excited with 405 nm excitation light, the emitted green light energy is absorbed by the neighboring R-Mito, generating a deep red FRET emission signal (detected via the 665-735 nm channel). Therefore, the appearance of the FRET signal directly and specifically reports the functional crosstalk between endocytosis and mitophagy.

[0012] This invention also provides a pair of fluorescent probes, G1 and G2, with the following chemical structures. G1 and G2 serve as control compounds. Although they possess good spectral properties as FRET donors, due to subtle structural differences, they cannot anchor to the plasma membrane as stably as G-CM. It was through comparative experiments with G1 and G2 that the unique advantages of G-CM were fully demonstrated: it can bind stably to the cell membrane for a long time without undergoing non-specific internalization, which is a key characteristic for its successful use in monitoring endocytosis. Ultimately, G-CM was selected as the official probe due to its excellent membrane anchoring ability, while G1 and G2 were discarded.

[0013] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: (1) This invention realizes real-time, in situ, two-color FRET monitoring of functional crosstalk between endocytosis and mitophagy, filling the technological gap in this field.

[0014] (2) G-CM has an ultra-long cell membrane residence capacity (no internalization for at least 4 hours), ensuring accurate tracking of endocytic events; R-Mito is covalently anchored and does not detach during the entire process of mitochondrial autophagy, ensuring the reliability of mitochondrial labeling.

[0015] (3) The FRET signal is generated only when the two processes converge in the same lysosome, avoiding non-specific background interference and having a high signal-to-noise ratio.

[0016] (4) Using the probe pair of the present invention, it was revealed that the level of endocytosis-mitochondrial autophagy crosstalk in various cancer cells (HeLa, A549, MCF-7) was significantly higher than that in normal cells (L02, 293T, HUVEC), providing new visual evidence for cancer metabolic reprogramming and potentially providing new targets for tumor diagnosis and treatment. Attached Figure Description

[0017] Figure 1 The image shows the hydrogen NMR spectrum of the probe G-CM. Figure 2 The image shows the carbon NMR spectrum of the probe G-CM. Figure 3 This is a high-resolution mass spectrum of the probe G-CM; Figure 4 The hydrogen NMR spectrum of the probe R-Mito; Figure 5 The carbon NMR spectrum of the probe R-Mito; Figure 6 This is a high-resolution mass spectrum of the probe R-Mito; Figure 7 Spectral characterization spectra of G-CM and R-Mito; Figure 8 This is a diagram from an experiment testing R-Mito targeting mitochondrial colocalization. Figure 9 Mapping of mitochondrial autophagy for R-Mito monitoring; Figure 10 A diagram visualizing the cell endocytosis process using the probe G-CM; Figure 11 Visualize the crosstalk between endocytosis and mitophagy for probes G-CM and R-Mito; Figure 12 Visualize autophagy maps for inducing probes G-CM and R-Mito; Figure 13 Diagram of endocytosis-mitochondrial autophagy interactions in cancer cells; Detailed Implementation To make the objectives and technical solutions of this invention clearer, the following embodiments are provided for further explanation. However, the scope of protection of this invention is not limited to these embodiments; the embodiments are merely for illustrative purposes. Those skilled in the art should understand that any changes or equivalent substitutions that do not depart from the concept of this invention are included within the scope of protection of this invention. The invention is further described below with reference to embodiments, but this invention is not limited to the embodiments.

[0018] The fluorescent probe pair described in this invention is also known as a fluorescent probe composition.

[0019] Synthesis of compound G1: First, 4-methylpyridine and 1-iodooctadecane were refluxed in anhydrous acetonitrile to prepare an intermediate of 1-octadecyl-4-methylpyridine iodide; this intermediate was then reacted with 4-(methylthio)benzaldehyde in a reflux system of ethanol under piperidine catalysis to undergo a Knoevenagel condensation reaction, followed by cooling and crystallization, and purification by recrystallization with ethanol to obtain product G1.

[0020] Synthesis of compound G2: First, 4-methylpyridine and 1-iodooctadecane were refluxed in anhydrous acetonitrile to obtain an intermediate of 1-octadecyl-4-methylpyridine iodide; this intermediate was then reacted with 6-methoxy-2-naphthaldehyde in a Knoevenagel condensation reaction in anhydrous ethanol reflux system under piperidine catalysis. After cooling and crystallization, the product was purified by recrystallization with ethanol to obtain G2.

[0021] Example 1: Synthesis of probe G-CM: Intermediate 1: 4-Methylpyridine (5.0 mmol) and 1-iodooctadecane (5.5 mmol) were dissolved in 5 mL of anhydrous ethanol and refluxed at 85 °C for 10 h. After cooling, the mixture was filtered and washed with cold ethanol to give a white solid, namely intermediate 1, with a yield of 86%.

[0022] Synthesis of G-CM: Intermediate 1 (0.5 mmol) and 9-ethyl-9H-carbazole-3-carbaldehyde (0.5 mmol) were dissolved in 5 mL of ethanol, and 3 drops of piperidine were added. The mixture was refluxed for 18 h. After cooling, the mixture was filtered and washed with cold ethanol to give a pale yellow solid, namely probe G-CM, with a yield of 78%. 1H NMR (600 MHz, Chloroform-d) δ 8.83 (d, J = 6.3 Hz, 2H), 8.33 (s,1H), 8.14 (d, J = 7.7 Hz, 1H), 8.01 (d, J = 6.3 Hz, 2H), 7.87 (d, J = 16.0Hz, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.41 – 7.36 (m,2H), 7.30 (t, J = 7.4 Hz, 1H), 7.15 (d, J= 16.0 Hz, 1H), 4.56 (t, J = 7.4 Hz,2H), 4.31 (q, J = 7.2 Hz, 2H), 1.97 – 1.91(m, 2H), 1.43(t, J = 7.3 Hz, 3H), 1.26 (dt, J = 35.3, 11.0 Hz, 30H), 0.87 (t, J = 7.0 Hz, 3H). 13C NMR (151MHz, DMSO) δ 152.87, 143.34, 142.05, 140.39, 139.56, 125.60, 122.47, 122.14,121.52, 120.63, 119.88, 119.32, 117.89, 109.31, 108.68, 58.86, 30.67, 28.43,24.22, 21.48, 13.35, 13.18. HRMS (ESI): m / z, for [C 39 H 55 N2 + ], calcd, 551.4360; found, 551.4370. See attachment. Figure 1 Appendix Figure 2 Appendix Figure 3 .

[0023] Intermediate 2: 4-Methylquinoline (13.56 mmol), 4-(dimethylamino)benzaldehyde (11.3 mmol), and p-toluenesulfonic acid (16.95 mmol) were dissolved in 6 mL of DMF and refluxed at 120 °C for 12 h. After cooling, water was added to precipitate the solid, which was then filtered and purified by column chromatography to give a purple solid, i.e., intermediate 2, with a yield of 63%.

[0024] Synthesis of R-Mito: Intermediate 2 (1.82 mmol) and p-dichlorobenzyl (2.18 mmol) were dissolved in 5 mL of toluene and refluxed at 120 °C for 12 h. After cooling, the mixture was filtered and recrystallized from the toluene to give purple crystals, i.e., probe R-Mito, in 65% yield. 1 H NMR(600 MHz, Chloroform-d) δ 10.48 (d, J = 6.7 Hz, 1H), 8.56 (d, J = 8.3 Hz,1H), 8.25 (d, J = 6.7 Hz, 1H), 8.08 (d, J = 8.8 Hz, 1H), 7.94 (d, J = 7.3 Hz,1H), 7.92 – 7.90 (m, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.69 – 7.65 (m, 4H), 7.33(q, J = 8.3 Hz, 4H), 6.76 (d, J = 8.9 Hz, 2H), 6.44 (s, 2H), 4.52 (s, 2H), 3.13 (s, 6H). 13 C NMR (151 MHz, DMSO) δ 153.81, 147.33, 138.36, 131.63, 129.95, 128.93, 127.66, 119.15, 114.69, 112.09, 59.02, 56.95, 45.33, 19.03. HRMS(ESI): m / z, calcd for: [C 27 H 26 ClN2 + ], 413.1779; found 413.1787. See attachment. Figure 4 Appendix Figure 5 Appendix Figure 6 .

[0025] Example 2: Spectroscopic testing of probes G-CM and R-Mito R-Mito (5 μM) was dissolved in solvents of different polarities (toluene, dioxane, dichloromethane, DMF, DMSO, and ethanol), and its absorption and emission spectra were measured. The results showed that its absorption peak was in the range of 520-600 nm, and its emission peak was in the range of 650-710 nm, exhibiting a significant solvation-induced color change effect.

[0026] When G-CM (5 μM) is dissolved in the above solvent, its absorption peak is at 430-440 nm and its emission peak is at 580-600 nm, and it is less affected by the polarity of the solvent.

[0027] The normalized emission spectrum of G-CM was superimposed with the normalized absorption spectrum of R-Mito, and the three spectra (G-CM, G1, G2) overlapped well with the R-Mito spectrum. The Förster distance (R0) was calculated: R0 = the distance between G-CM and R-Mito. o The values ​​are 45 Å for G1, 60 Å for G2, and 58 Å for G3, indicating that efficient FRET can occur between G-CM and R-Mito. (See appendix) Figure 7 (A) Absorption and emission spectra of R-Mito in different solvents; (B) Absorption and emission spectra of G-CM in different solvents; (C) Overlay of normalized emission spectrum of G-CM and normalized absorption spectrum of R-Mito.

[0028] Example 3: R-Mito targeting of mitochondria and monitoring of mitophagy Cell culture and staining: HeLa cells were incubated with 2 μM R-Mito for 30 min and then subjected to confocal imaging (λ_ex=561nm, λ_em=665-735 nm). The results showed clear filamentous mitochondrial morphology. After treatment with 10 μM CCCP for 10 min, there was no significant change in fluorescence intensity and distribution, indicating that R-Mito is anchored to the mitochondria and does not detach due to a decrease in membrane potential.

[0029] Colocalization experiment: R-Mito was co-stained with MitoTracker Green (MTG), and the Pearson coefficients were 0.91 (untreated) and 0.92 (CCCP treated), demonstrating its mitochondrial targeting.

[0030] Mitophagy induction: Cells were co-stained with R-Mito and LysoTracker Green (LTG) and then treated with D-Hanks (complete starvation) for 1–2 h or with serum-free DMEM for 6–12 h. Results showed that the filamentous fluorescence of R-Mito gradually changed to punctate fluorescence and co-localized with LTG, confirming that R-Mito can monitor mitophagy. (See appendix) Figure 8 , including Figure 8 (A) Images of live cells stained with 2 µM R-Mito for 30 minutes, followed by / without processing with 10 µM CCCP for 10 minutes; (B) Images of live cells stained with 2 µM MR-Mito and 200 nM MTG for 30 minutes, followed by / without processing with 10 µM CCCP for 10 minutes; (C) Intensity distribution of R-Mito and MTG along the arrows in B; Scale bar = 20 µm; Appendix Figure 9 , including Figure 9(A) Live-cell fluorescence image after co-staining with 2 µM R-Mito and 200 nM LTG for 30 minutes; (B) Intensity distribution of R-Mito and LTG along the arrow in A; (C) Live-cell fluorescence images after co-staining with 2 µM R-Mito and 200 nM LTG for 30 minutes, followed by incubation in D-Hanks for 1 hour and 2 hours; (D) Intensity distribution of R-Mito and LTG along the arrow in C; (E) Live-cell fluorescence images after co-staining with 2 µM R-Mito and 200 nM LTG for 30 minutes, followed by incubation in DMEM for 6 hours and 12 hours; (F) Intensity distribution of R-Mito and LTG along the arrow in E; Scale bar = 20 µm.

[0031] Example 4: G-CM targeting of cell membrane and endocytosis tracing Fixed cell staining: HeLa cells were incubated with 2 μM G-CM for 0-4 h. Only the cell membrane outline was visible, with no intracellular signal, indicating that G-CM was stably anchored on the membrane (compared to probes G1 and G2, which were internalized within 2 h).

[0032] Live cell endocytosis tracing: Live cells were incubated with 2 μM G-CM for 0-2 h, and green fluorescence was observed to gradually transfer from the cell membrane to intracellular punctate structures, proving that G-CM was endocytosed.

[0033] Low-temperature inhibition experiment: After incubation at 4℃ for 2 h, G-CM remained localized on the membrane, proving that endocytosis is energy-dependent.

[0034] Lysosomal colocalization: After incubation with G-CM for 4 h, LysoTracker Deep Red (LTDR) staining revealed numerous punctate colocalizations (which turned white after merging), indicating that the endocytic endpoint was the lysosome. See Appendix. Figure 10 , including Figure 10 (A) Images of fixed cells incubated with 2 µM G-CM for 0–4 hours, and intensity distribution along the arrow; (B) Images of fixed cells incubated with 2 µM G-CM for 2 hours, and intensity distribution along the arrow; (C) Images of fixed cells incubated with 2 µM G-CM for 2 hours, and intensity distribution along the arrow; (D) Fluorescence images of live cells incubated with 2 µM G-CM for 0–2 hours; (E) Fluorescence images of live cells incubated with 2 µM G-CM for 4 hours, then stained with 200 nM LTDR for 30 minutes, and intensity distribution of G-CM and LTDR along the arrow path; Scale bar = 20 µm.

[0035] Example 5: FRET monitoring of endocytosis and mitophagy crosstalk Starvation-induced crosstalk: Cells were first incubated with G-CM for 30 min, washed off, and cultured for another 24 h to allow for full G-CM internalization; then incubated with R-Mito for 30 min, washed off, and starved in D-Hanks for 0-3 h. Results: At 0 h of starvation, there was no FRET signal (405 nm excitation, 665-735 nm reception); at 1-2 h of starvation, a significant FRET signal appeared, and the intensity increased with starvation time; at 3 h of starvation, FRET no longer increased. In the group treated with 10 μM chloroquine (CQ), the FRET signal almost disappeared, confirming that crosstalk occurred in functional lysosomes.

[0036] Rapamycin-induced crosstalk: Cells were simultaneously incubated with G-CM and R-Mito for 30 min, then cultured in complete medium containing 5 μM rapamycin for 24 h, or in D-Hanks containing 5 μM rapamycin for 3 h. Significant FRET signals were detected in both cases, with stronger signals in the combined treatment group.

[0037] Quantitative analysis: FRET channel fluorescence intensity quantification results showed that the starvation or rapamycin treatment groups had significantly higher fluorescence intensity than the control group, while the CQ treatment group showed a significant decrease. (See appendix) Figure 11 and attached Figure 12 , including Figure 11 (A) Cells were stained with 2 µM G-CM for 30 minutes, washed, incubated in culture medium for 24 hours, then stained with 2 µM R-Mito for 30 minutes, washed, and incubated in D-Hanks for different times (0-3 hours); (B) Cells incubated in D-Hanks for 0-3 hours; (C) Cells incubated in D-Hanks containing 10 µM CQ for 3 hours; Scale bar = 20 µm; Among them, the appendix Figure 12 (A) Fluorescence image of live cells stained with 2 µM G-CM and 2 µM R-Mito for 30 min and then incubated in medium containing 5 µM rapamycin for 24 h; (B) Fluorescence image of live cells stained with 2 µM G-CM and 2 µM R-Mito for 30 min and then incubated in D-Hanks containing 2 µM rapamycin for 3 h; (C) Quantitative emission intensity of FRET channels in untreated cells, cells treated with D-Hanks with / without CQ, or cells treated with rapamycin in medium and D-Hanks; Scale bar = 20 µm; Example 6: Endocytosis-mitochondrial autophagy crosstalk in cancer cells Cell lines: Three cancer cells (HeLa, A549, MCF-7) and three normal cells (L02, 293T, HUVEC) were selected.

[0038] Procedure: Each cell was incubated with G-CM for 30 min, washed, and cultured for 24 h; then incubated with R-Mito for 30 min; and finally starved in PBS for 2 h.

[0039] Results: Significant FRET signal (dark red dots) was observed in all cancer cell groups; almost no FRET signal was observed in the normal cell group. Quantitative FRET intensity analysis showed that cancer cells had significantly higher FRET levels than normal cells (P<0.01).

[0040] Conclusion: The probe of this invention significantly upregulated the level of crosstalk between endocytosis and mitophagy in cancer cells, suggesting it may serve as a biomarker for abnormal cancer metabolism. (See appendix) Figure 13 , including Figure 13 Cancer cells (A) and normal cells (B) were stained with G-CM for 30 minutes, cultured in fresh medium for 24 hours, stained with R-Mito for 30 minutes, and starved in PBS buffer for 2 hours; (C) Quantitative emission intensity of FRET channel; scale bar = 20 µm.

[0041] The fluorescent probe provided by this invention is reliable for the preparation methods of G-CM and R-Mito, exhibits excellent optical properties and good biocompatibility, and can achieve real-time, two-color FRET monitoring of live-cell endocytosis and mitophagy crosstalk under a conventional confocal microscope. This probe has significant scientific research value and clinical application potential for basic cell biology research (such as metabolic regulation and signal transduction), anticancer drug screening (targeting autophagy or endocytosis pathways), and the development of early cancer diagnostic biomarkers.

[0042] In summary, this invention constructed a fluorescent probe pair based on the FRET mechanism, successfully visualized the functional crosstalk between endocytosis and mitophagy in live cells, and revealed the phenomenon of significant upregulation of this crosstalk in cancer cells.

Claims

1. A fluorescent probe pair for monitoring the interaction between endocytosis and mitophagy, characterized in that, The fluorescent probe pair consists of probe G-CM and probe R-Mito, and the structural formulas of probe G-CM and probe R-Mito are as follows: 。 2. A method for preparing the fluorescent probe pair according to claim 1, characterized in that, The preparation methods for probe G-CM and probe R-Mito of the fluorescent probe pair are as follows: Synthesis of probe G-CM: 4-Methylpyridine and 1-iodooctadecane were dissolved in anhydrous ethanol, refluxed, cooled and filtered, and washed with cold ethanol to obtain intermediate 1. Intermediate 1 and 9-ethyl-9H-carbazole-3-carbaldehyde were dissolved in ethanol, piperidine was added dropwise, the mixture was refluxed, cooled and filtered, and washed with cold ethanol to obtain probe G-CM. 4-Methylquinoline, 4-(dimethylamino)benzaldehyde and p-toluenesulfonic acid were dissolved in DMF, refluxed, cooled and then water was added to precipitate the solid. The solid was filtered and purified by column chromatography to obtain intermediate 2. Intermediate 2 and p-dichlorobenzyl were dissolved in toluene, refluxed, cooled, filtered, and recrystallized from toluene to obtain probe R-Mito.

3. The fluorescent probe pair according to claim 1, characterized in that, The probe G-CM is a donor probe based on the FRET mechanism, which can specifically bind to the cell membrane and anchor to membrane phospholipids for a long time, and is used to monitor cell membrane dynamics and endocytosis. The R-Mito is a receptor probe based on the FRET mechanism, which forms a covalent bond with the thiol group on the mitochondrial membrane protein through its benzyl chloride group, and is used to monitor mitochondrial dynamics and mitophagy.

4. The fluorescent probe pair according to claim 1, characterized in that, The excitation wavelength of the probe G-CM is 405 nm, and the emission wavelength is 500-550 nm; the excitation wavelength of the probe R-Mito is 561 nm, and the emission wavelength is 665-735 nm; the absorption spectrum of the probe R-Mito overlaps with the emission spectrum of the probe G-CM, and the Förster distance between them is 45-60 Å.

5. A method for detecting crosstalk between endocytosis and mitophagy using the fluorescent probe of claim 1, characterized in that, The detection method includes the following steps: (a) Incubate the probe G-CM and probe R-Mito as described in claim 1 with the live cell sample to be tested simultaneously or in stages; (b) The incubated cell samples were irradiated with excitation light at 405 nm and 561 nm using a confocal microscope, respectively; (c) Collect signals from three sets of fluorescence channels: donor channel, which is excited at 405 nm and emits light at 500–550 nm; acceptor channel, which is excited at 561 nm and emits light at 665–735 nm; and FRET channel, which is excited at 405 nm and emits light at 665–735 nm. (d) FRET efficiency is calculated by the ratio of the fluorescence signal intensity of the FRET channel to the signal intensity of the donor and acceptor channels.

6. The use of the fluorescent probe of claim 1 in real-time monitoring of functional crosstalk between endocytosis and mitochondrial autophagy in living cells.

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