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A lysosome membrane regeneration pathway depends on TBC1D15 and autophagic lysosomal reformation proteins

Nature Cell Biology volume 25, pages 685–698 (2023)Cite this article

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Abstract

Acute lysosomal membrane damage reduces the cellular population of functional lysosomes. However, these damaged lysosomes have a remarkable recovery potential independent of lysosomal biogenesis and remain unaffected in cells depleted in TFEB and TFE3. We combined proximity-labelling-based proteomics, biochemistry and high-resolution microscopy to unravel a lysosomal membrane regeneration pathway that depends on ATG8, the lysosomal membrane protein LIMP2, the RAB7 GTPase-activating protein TBC1D15 and proteins required for autophagic lysosomal reformation, including dynamin-2, kinesin-5B and clathrin. Following lysosomal damage, LIMP2 acts as a lysophagy receptor to bind ATG8, which in turn recruits TBC1D15 to damaged membranes. TBC1D15 interacts with ATG8 proteins on damaged lysosomes and provides a scaffold to assemble and stabilize the autophagic lysosomal reformation machinery. This potentiates the formation of lysosomal tubules and subsequent dynamin-2-dependent scission. TBC1D15-mediated lysosome regeneration was also observed in a cell culture model of oxalate nephropathy.

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Fig. 1: Cellular response to LLOMe-mediated acute lysosomal damage.
Fig. 2: ATG8-dependent recruitment of TBC1D15 to damaged lysosomes.
Fig. 3: TBC1D15 is necessary for lysosomal regeneration.
Fig. 4: Proximity labelling of TBC1D15 identifies LC3B-binding proteins and ALR machinery near damaged lysosomal membranes.
Fig. 5: Lysosomes regenerate through TBC1D15-dependent tubulation.
Fig. 6: TBC1D15 regulates lysosomal tubulation in PTECs treated with calcium oxalate.

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Data availability

MS data have been deposited into ProteomeXchange with the primary accession code PXD040191. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank staff at Abberior Instruments, specifically D. Luchtman, for performing STED imaging; O. Florey (Babraham Institute, UK) for his gift of ATG16L plasmids (WT and K490A); and staff at the quantitative proteomics Unit of IBC2 for the use of their proteomics platform and the Frankfurt Center for Advanced Microscopy (FCAM) for access to microscopes. I.D. acknowledges funding from the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation) project number 259130777–SFB 1177, the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement number 742720), Else Kröner Fresenius Stiftung, Dr Rolf M. Schwiete Stiftung, and the Ernst Jung Prize for Medicine. A.B. acknowledges funding from LYSOFOR2625 (project identifier 323732846-LYSFOR2625). R.M. received funding through an Alexander von Humboldt Stiftung postdoctoral fellowship.

Author information

Authors and Affiliations

  1. Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt, Germany

    Anshu Bhattacharya, Rukmini Mukherjee, Santosh Kumar Kuncha, Melinda Elaine Brunstein, Rajeshwari Rathore, Christian Münch & Ivan Dikic

  2. Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt, Germany

    Anshu Bhattacharya, Rukmini Mukherjee, Santosh Kumar Kuncha & Ivan Dikic

  3. Max Planck Institute of Biophysics, Frankfurt, Germany

    Rukmini Mukherjee, Stephan Junek & Ivan Dikic

  4. Max Planck Institute for Brain Research, Frankfurt, Germany

    Stephan Junek

  5. Fraunhofer Institute for Translational Medicine and Pharmacology (ITMP), Frankfurt, Germany

    Ivan Dikic

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Contributions

A.B. and R.M. conceptualized and performed all cell biology and proteomics experiments. S.K.K. performed structural and sequence analysis. R.R. handled the MS machine runs, and M.E.B. and C.M. established the MS protocol for the analysis of proximity labelling assays. R.M. and S.J. performed Airyscan confocal imaging, and A.B., R.M. and I.D. analysed the data. I.D. supervised the project. A.B., R.M. and I.D. wrote the manuscript.

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Correspondence to Ivan Dikic.

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Extended data

Extended Data Fig. 1 Role of TFEB, TFE3, and mTOR in lysosomal regeneration following LLOMe-mediated damage.

(a) HeLa cells were treated with siRNA to deplete endogenous TFEB/TFE3. Lysosomal damage was induced with 1mM LLOMe treatment for 2 h followed by washout for the indicated durations. Cells were loaded with LysoTracker Red for 30 min, fixed, and stained with a TFEB/TFE3 specific antibody. Data from 3 experiments was used to plot the graph in Figure 1A. Scale bar:10µm. (b) HeLa cells were treated with siRNA to deplete endogenous TFEB and LAMP1-RFP was transfected to mark lysosomes. These cells were then subjected to LLOMe treatment (1mM, 2 h) followed by washout for indicated timepoints. DQ-BSA was loaded for the last 30 minutes of different treatment conditions prior to imaging the samples by confocal microscopy. TFEB knockdown efficiency was checked by western blotting. Scale bar:10µm. (c) DQ-BSA signal intensities were compared between control cells and cells depleted for endogenous TFEB as a percentage of the signal observed in control siRNA treated cells without LLOMe. n=31 cells taken from 3 experiments. For the box plot, center lines: medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers: dots. (d) mTOR activity was assessed by monitoring the phosphorylation status of p70-S6K by western blotting for indicated treatments. LLOMe:1mM, 2 h, EBSS: 4 h, Torin1: 500nM, 6 h. The experiment was repeated 3 times with similar results. Source numerical data and unprocessed blots are available in Source data_extended data figure1(part 1 and 2).

Source data

Extended Data Fig. 2 TBC1D15 recruitment to damaged lysosomes is specific for LLOMe treatment; and does not occur under other forms of stress.

(a) HeLa cells expressing LC3-BFP and GAL3-GFP were treated with 1mM LLOMe for 2 h to induce lysosomal damage. Endogenous TBC1D15 was stained to evaluate its recruitment to the damaged lysosomes. n= 53 cells taken from 3 experiments (***, p = 1.81E-20). Scale bar: 5µm, scale bar in inset:2µm. (b) HeLa cells expressing HA-ubiquitin were treated with LLOMe and endogenous TBC1D15 was stained with an HA-specific antibody to determine the the number of Ub+ TBC1D15+ puncta/cell. n= 51 cells taken from 3 experiments (***, p = 2.57E-11). Scale bar: 5µm, scale bar in inset:2µm. (c) HeLa cells expressing TBC1D15-GFP and mCherry-LC3B were treated with indicated drugs. TBC1D15 did not form puncta under these conditions. The experiment was repeated 3 times with similar results. Scale bar: 5µm. (d) HEK 293T cells were transfected with wild-type or LIR1 mut variant of HA tagged TBC1D15. Cells were treated with LLOMe, HA immunoprecipitation was performed to assess the binding with LC3B by western blotting. The experiment was repeated 3 times with similar results. (e) HEK 293T cells were transfected with different HA tagged variants of TBC1D15, as indicated in the figure. HA immunoprecipitation was performed after LLOMe treatment to assess the binding with GABARAPL2 by western blotting. The lysates used here are the same as those shown in Figure 2g. The experiment was repeated 3 times with similar results. (f) HeLa cells were treated with TBC1D15 specific siRNA for 48 h followed by reconstitution with HA-tagged wild-type or mutant TBC1D15 for 24 h. The cells were then treated with LLOMe followed by lysis. GTP-bound Rab7 was immunoprecipitated from lysates by incubating with GST-RILP. Total Rab7 levels were assessed in cell lysates by western blotting. Data represents mean ± standard deviation from 3 independent experiments. Data points from 3 experiments are marked. For the box plots, center lines: medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers: dots. p values are calculated using 2-tailed Student’s t-test. Source numerical data and unprocessed blots are available in Source data_extended data figure2.

Source data

Extended Data Fig. 3 Macroautophagy is important for the recruitment of TBC1D15 to damaged lysosomes.

(a) HeLa cells were treated with specific siRNA for 48 h to knockdown different autophagy related genes followed by transfection of LC3B-BFP for another 18 hours. Cells were then treated with 1mM LLOMe for 2 h, fixed and stained for LAMP1 and TBC1D15 followed by confocal imaging. Images were analysed in FIJI to determine the number of cells where TBC1D15 is localised on lysosomes. n=111, 91, 98, 102, 102, 104 cells respectively taken from 3 independent experiments [***, p = 1.53E-6 (control si versus ATG3 si); p = 5.91E-6 (ATG12si versus control); p = 6.04E-8 (ATG13si versus control)]. Knockdown efficiency for each gene was checked by western blotting. Scale bar: 10µm. (b) HeLa cells were treated with siRNA to knockdown ATG16L followed by reconstituting with either wild-type or K490A mutant variants. Cells were then transfected with LC3B-BFP for 18 h, followed by 1mM LLOMe treatment for 2 h and stained for LAMP1 and TBC1D15 to quantitate the recruitment of TBC1D15 to damaged lysosomes. n=90,83,86,87 cells taken from 3 independent experiments (***p = 4.02E-5). Expression levels of ATG16L was checked by western blotting. Scale bar: 10µm. For the box plots, center lines: medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers: dots. p values are calculated using 2-tailed Student’s t-test. Source numerical data and unprocessed blots are available in Source data_extended data figure3(part 1and 2).

Source data

Extended Data Fig. 4 Pharmacological inhibition of autophagy reduces recruitment of TBC1D15 to damaged lysosomes and reduces lysosomal regeneration flux.

(a) HeLa cells were transfected with HA-TBC1D15 and LC3B-BFP followed by treating them with only 1mM LLOMe for 2 h or LLOMe in combination with different known chemical agents to block autophagy. Cells were then fixed and stained for LAMP1 to check the recruitment of TBC1D15 to damaged lysosomes. Scale bar: 10µm. (b) Recruitment efficiency of TBC1D15 from experiment in panel (a) n=83, 81, 72, 73,79 cells taken from 3 independent experiments (***, p = 2.72E-6 (LLOMe versus Wortmannin), p = 1.42E-5 (LLOMe versus MRT68921), **, p = 0.0136 (LLOMe versus SAR405). (c) HEK 293T cells were transfected with HA tagged wild-type TBC1D15 followed by treating them with 1mM LLOMe for 2 h or LLOMe in combination with indicated inhibitors. Binding efficiency of TBC1D15 with LC3B under these conditions were assessed by HA immunoprecipitation followed by western blotting. The experiment was repeated 3 times with similar results. (d) Lysosomal regeneration flux assay was performed using HeLa cells stably expressing the tfGal3 reporter after 1mM LLOMe treatment for 2 h followed by LLOMe washout. The indicated drugs were added to the medium 2 h prior to LLOMe treatment, during LLOMe treatment and washout. Scale bar: 10µm. (e) Efficiency of lysosomal regeneration was compared between treatments as shown in experiment in panel (d) by measuring number of red galectin3 puncta/cell. n=51 cells taken from 3 experiments. ***, p = 6.76E-13 (control versus Wortmannin), p = 3.52E-13 (control versus MRT68921), p = 1.16E-11 (control versus SAR405). Drug treatments used: Wortmannin (300nM, 6 h), MRT68921 (10µM, 6 h), SAR405 (10µM, 6 h). For the box plots, center lines: medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers: dots. p values are calculated using 2-tailed Student’s t-test. Source numerical data and unprocessed blots are available in Source data_extended data figure4(part 1and 2).

Source data

Extended Data Fig. 5 Role of ATG8 interactors in TBC1D15 recruitment and lysosomal regeneration.

(a) Endogenous ATP6V1B2 was immunoprecipitated from LLOMe treated cells; binding with ATG8 proteins were assessed by western blotting. The experiment was repeated 3 times with similar results. (b) HA-tagged WT and LIR mutant TMEM192 constructs were transfected in HEK 293T cells. Cells were treated with LLOMe, GABARAPL2 binding was assessed by HA-immunoprecipitation and western blotting. The experiment was repeated 3 times with similar results. (c) HeLa cells depleted of LIMP2, TMEM192 or both were treated with LLOMe, fixed and imaged by confocal imaging. Scale bar: 5µm, scale bar in inset:2µm. (d) TBC1D15 puncta formation was compared between control cells and cells depleted of both markers as described in panel (c). n=107, 108, 100, 101 cells taken from 3 experiments. n.s, p = 0.26 (control versus TMEM192 si), *, p = 0.018 (control versus LIMP2 si), ***, p = 1.25E-6 (control versus double knockdown). (e) LIMP2 and TMEM192 knockdown efficiency was checked by western blotting. (f) Lysosomal regeneration flux assay in cells treated with LIMP2 and TMEM192 siRNA. Scale bar:10µm. (g) Number of red Gal3 puncta/cell in different samples from experiment in panel (f). n= 50 cells taken from 3 experiments. (n.s, p = 0.47 (control versus TMEM192 si), ***, p = 1.62E-6 (control versus LIMP2 si), p = 1.21E-9 (control versus double knockdown), *, p = 0.015 (LIMP2 si versus double knockdown). (h) HeLa cells expressing LC3-BFP were treated with 50 µM MLSA1 for 1 h followed by confocal imaging. Scale bar:10µm, scale bar in inset: 3µm. The experiment was repeated 3 times with similar results. (i) HeLa cells expressing APEX2-TRPML1 were treated with 1mM LLOMe or 50µM MLSA1 for 2 h followed by proximity labelling. Lysates were used in a streptavidin pulldown assay followed by western blotting with the indicated antibodies. The experiment was repeated 3 times with similar results. or the box plots, center lines: medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers: dots. p values are calculated using 2-tailed Student’s t-test. Source numerical data and unprocessed blots are available in Source data_extended data figure5(part 1and 2).

Source data

Extended Data Fig. 6 ALR proteins interact with TBC1D15 in LLOMe-treated cells.

(a) Metascape network analysis of proteins significantly enriched in the proximity labelling experiment shown in Fig. 4a and 4b. (b) HA-TBC1D15 was immunoprecipitated from cells after 1mM LLOMe treatment for 2 h followed by MS analysis. Proteins related to ALR (KIF22, CLTC and DNM2) were enriched by LLOMe treatment. Control = lysates from cells treated with LLOMe but not expressing HA-TBC1D15. p values are calculated using 2-tailed Student’s t-test. (c) Cells expressing LAMP1-RFP, TBC1D15-GFP and LC3-BFP were treated with 1 mM LLOMe for 2 h. Before fixing the cells, 0.05% digitonin was added to reduce the cytosolic background of TBC1D15 staining. Digitonin-treated cells show the presence of TBC1D15 in LAMP1-RFP+ LC3-BFP+ membrane mass. The experiment was repeated 3 times with similar results. Scale bar:10µm. (d) TBC1D15-GFP in lysates from cells treated with LLOMe and digitonin similar to panel(c) was immunoprecipitated using anti-GFP beads followed by MS analysis. ALR proteins were enriched by LLOMe treatment. p values are calculated using 2-tailed Student’s t-test. Source data available in Source data_extended data figure6.

Source data

Extended Data Fig. 7 TBC1D15 and DNM2 are important for lysosomal regeneration.

(a) AlphaFold predicted structure of TBC1D15 showing N-terminal domain (NTD) in green, the linker in yellow and the C-terminal domain (CTD) in blue. (b) GST-tagged deletion constructs of TBC1D15 were incubated with cell lysates from HEK 293T cells in a GST pulldown assay and followed by western blotting. The experiment was repeated 3 times with similar results. (c) GST-tagged N-terminal deletion constructs of TBC1D15 were incubated with HEK 293T cell lysates in a GST pulldown assay and detected by western blotting with the indicated antibodies. The experiment was repeated 3 times with similar results. (d) AlphaFold predicted structure showing the N-terminal loop required for DNM2 binding. (e) GFP-tagged wild-type TBC1D15 or the loop mutant were enriched from LLOMe-treated cells using anti-GFP beads followed by western blotting with the indicated antibodies. The experiment was repeated 3 times with similar results. (f) HeLa cells stably expressing the tfGal3 flux reporter were subjected to a lysosomal regeneration flux assay in cells with or without 20 µM Dynasore added in LLOMe-free medium during washout. The number of red tGal3 puncta per cell was counted. n=45,47 cells from three independent experiments (***, p = 2.00E-15). Scale bar:10µm. (g) HeLa cells expressing the tfGal3 flux reporter were treated with DNM2 siRNA and then were subjected to a lysosomal regeneration flux assay. The number of red tfGal3 puncta per cell was counted. n=49cells from three independent experiments (***, p = 1.32E-18). Scale bar:10µm. (h) HeLa cells stably expressing tfGal3 were depleted of endogenous TBC1D15 for 48 h followed by transfection of wild-type TBC1D15 or the loop mutant. Cells were treated with 1mM LLOMe for 2h followed by LLOMe washout and imaged by confocal microscopy. The number of red tGal3 puncta per cell was counted. n=50 cells from three independent experiments (***, p = 2.03E-13). Scale bar:10µm. For the box plots, center lines: medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers: dots. p values are calculated using 2-tailed Student’s t-test. Source numerical data and unprocessed blots are available in Source data_extended data figure7(part 1and 2).

Source data

Extended Data Fig. 8 Lysosomal regeneration from damaged membranes occurs via tubulation and successive scission in a TBC1D15 and DNM2 dependent manner.

(a) HeLa cells were treated with LLOMe and were allowed to recover for the indicated timepoints. Cells were fixed and stained for LAMP1 and imaged by STED microscopy. Scale bar:5µm. (b) HeLa cells were treated with siRNA to deplete either TBC1D15 or DNM2 individually. These cells were the allowed to recover for 2hr after LLOMe treatment and were then fixed and stained for LAMP1 to assess the effect of the TBC1D15 and DNM2 on lysosomal tubulation events by STED microscopy. Efficiency of knockdown was assessed by western blotting. Scale bar:5µm. (c) Different lysosomal structures, from experiments in panel (a) and (b) were analysed in FIJI and classified into closed rings, interconnected tubules and tubules Data are means ± SEM of 33 cells from three independent experiments. p values were calculated using two-sided Students t-test. ***, p = 3.93E-16 (control versus LLOMe), p = 1.93E-8 (LLOMe versus LLOMe recovery-30min), p = 8.36E-20 (control versus TBC1D15 siRNA), p = 1.65E-5 (control versus DNM2siRNA). Source numerical data and unprocessed blots are available in Source data_extended data figure8.

Source data

Extended Data Fig. 9 Lysosomal regeneration occurs via tubulation and successive scission in a TBC1D15 and DNM2 dependent manner.

(a) Effect of 20µM dynasore treatment for 2 h in the absence of LLOMe. n=53 cells taken from 3 experiments. For the box plot, center lines: medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers: dots. Scale bar:5µm, scale bar in inset:2µm. (b) 3D projections made from Z-stacks of airy-scan images as seen in Fig. 5B. Scale bar: 2µm. (c) Time-lapse imaging of 1mM LLOMe treated cells expressing LAMP-RFP and TBC1D15-GFP. Scale bar: 2µm. Source numerical data are available in Source data_extended data figure9.

Source data

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Supplementary Video 1

3D projection of the image in Fig. 5c (LLOMe followed by LLOMe washout).

Supplementary Video 2

3D projection of the image in Fig. 5c (LLOMe followed by LLOMe washout in presence of dynasore).

Supplementary Video 3

Time-lapse imaging of lysosomal regeneration shown in Fig. 5e.

Supplementary Video 4

Time-lapse imaging of lysosomal regeneration shown in Extended Data Fig. 9c.

Source data

Source Data Fig. 1

Numerical source data for graphs.

Source Data Fig. 1

Source data for unprocessed blots.

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Numerical source data for graphs.

Source Data Fig. 2

Source data for unprocessed blots.

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Numerical source data for graphs.

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Source data for unprocessed blots.

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Numerical source data for graphs

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Source data for unprocessed blots.

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Numerical source data for graphs.

Source Data Fig. 5

Source data for unprocessed blots.

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Numerical source data for graphs.

Source Data Fig. 6

Source data for unprocessed blots.

Source Data Extended Data Fig. 1

Numerical source data for graphs.

Source Data Extended Data Fig. 1

Source data for unprocessed blots.

Source Data Extended Data Fig. 2

Numerical source data for graphs.

Source Data Extended Data Fig. 2

Source data for unprocessed blots.

Source Data Extended Data Fig. 3

Numerical source data for graphs.

Source Data Extended Data Fig. 3

Source data for unprocessed blots.

Source Data Extended Data Fig. 4

Numerical source data for graphs.

Source Data Extended Data Fig. 4

Source data for unprocessed blots.

Source Data Extended Data Fig. 5

Numerical source data for graphs.

Source Data Extended Data Fig. 5

Source data for unprocessed blots.

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MS data used to produce the volcano plot.

Source Data Extended Data Fig. 7

Numerical source data for graphs.

Source Data Extended Data Fig. 7

Source data for unprocessed blots.

Source Data Extended Data Fig. 8

Numerical source data for graphs.

Source Data Extended Data Fig. 8

Source data for unprocessed blots.

Source Data Extended Data Fig. 9

Numerical source data for graphs.

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Bhattacharya, A., Mukherjee, R., Kuncha, S.K. et al. A lysosome membrane regeneration pathway depends on TBC1D15 and autophagic lysosomal reformation proteins. Nat Cell Biol 25, 685–698 (2023). https://doi.org/10.1038/s41556-023-01125-9

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