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Pressure sensing of lysosomes enables control of TFEB responses in macrophages

Nature Cell Biology volume 26, pages 1247–1260 (2024)Cite this article

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Abstract

Polymers are endocytosed and hydrolysed by lysosomal enzymes to generate transportable solutes. While the transport of diverse organic solutes across the plasma membrane is well studied, their necessary ongoing efflux from the endocytic fluid into the cytosol is poorly appreciated by comparison. Myeloid cells that employ specialized types of endocytosis, that is, phagocytosis and macropinocytosis, are highly dependent on such transport pathways to prevent the build-up of hydrostatic pressure that otherwise offsets lysosomal dynamics including vesiculation, tubulation and fission. Without undergoing rupture, we found that lysosomes incurring this pressure owing to defects in solute efflux, are unable to retain luminal Na+, which collapses its gradient with the cytosol. This cation ‘leak’ is mediated by pressure-sensitive channels resident to lysosomes and leads to the inhibition of mTORC1, which is normally activated by Na+-coupled amino acid transporters driven by the Na+ gradient. As a consequence, the transcription factors TFEB/TFE3 are made active in macrophages with distended lysosomes. In addition to their role in lysosomal biogenesis, TFEB/TFE3 activation causes the release of MCP-1/CCL2. In catabolically stressed tissues, defects in efflux of solutes from the endocytic pathway leads to increased monocyte recruitment. Here we propose that macrophages respond to a pressure-sensing pathway on lysosomes to orchestrate lysosomal biogenesis as well as myeloid cell recruitment.

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Fig. 1: Solute efflux supports lysosomal tubulation, volume homeostasis and prevents inflammation.
Fig. 2: Fragmented HA is macropinocytosed and processed in the endocytic pathway of macrophages.
Fig. 3: SLC transporters involved in the efflux of lysosomal sugars.
Fig. 4: TFEB/TFE3 are activated in SLC17A5−/− macrophages, which causes the release of MCP-1.
Fig. 5: High hydrostatic pressure on lysosomes activates TFEB/TFE3 via a lysosomal Na+ leak.
Fig. 6: TMEM63A is a mechanosensitive Na+ leak pathway responsible for activating TFEB/TFE3.
Fig. 7: A lysosome-to-cytosol gradient for Na+ that maintains mTORC1 activity is collapsed in lysosomes under high hydrostatic pressure.

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Data availability

Mass spectrometry data have been deposited in the MassIVE database as MSV000094949 (https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=327454621fae453d9df1e5e21bfa972e). Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank A. Belkacemi and V. Flockerzi for helpful discussions and Y. Li and B. Fubara at the NINDS Proteomics Core Facility for technical assistance. S.A.F. and R.J.B. are recipients of Canada Research Chairs. S.A.F. was supported by grants PJT-169180 and PJT-190244 from the Canadian Institutes of Health Research (CIHR). R.C. is the recipient of a CIHR fellowship. S.U. is supported by Deutsche Forschungsgemeinschaft (DFG) grants (448121430, 405969122 and 447268119), the Hightech Agenda Bavaria and by an ERC starting grant (101039438). R.J.B. is supported by CIHR PJT-166047 and research support by Toronto Metropolitan University.

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Authors and Affiliations

  1. Program in Cell Biology and Peter Gilgan Centre for Research and Learning, Hospital for Sick Children, Toronto, Ontario, Canada

    Ruiqi Cai, Ori Scott, Gang Ye, Trieu Le, Ekambir Saran, Whijin Kwon, Blayne A. Sayed & Spencer A. Freeman

  2. Department of Chemistry and Biology, Toronto Metropolitan University, Toronto, Ontario, Canada

    Subothan Inpanathan & Roberto J. Botelho

  3. Molecular Science Graduate Program, Toronto Metropolitan University, Toronto, Ontario, Canada

    Subothan Inpanathan & Roberto J. Botelho

  4. Neurosciences and Cellular and Structural Biology Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA

    Amra Saric

  5. Program in Neurosciences and Mental Health, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children, Toronto, Ontario, Canada

    Amra Saric

  6. Department of Internal Medicine, Rheumatology and Immunology, Universitätsklinikum Erlangen, Friedrich-Alexander University Erlangen, Erlangen, Germany

    Stefan Uderhardt

  7. Deutsches Zentrum für Immuntherapie, Universitätsklinikum Erlangen, Friedrich-Alexander University Erlangen, Erlangen, Germany

    Stefan Uderhardt

  8. Exploratory Research Unit, Optical Imaging Centre Erlangen, Friedrich-Alexander University Erlangen, Erlangen, Germany

    Stefan Uderhardt

  9. Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

    Spencer A. Freeman

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Contributions

R.C., A.S., S.U. and S.A.F. designed the study. B.A.S., R.J.B., A.S., S.U. and S.A.F. supervised the experiments. R.C., O.S., G.Y., T.L., E.S., W.K., S.I., A.S., S.U. and S.A.F. performed the experiments. R.C. and S.A.F. wrote the paper; all authors reviewed and edited the paper.

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Correspondence to Spencer A. Freeman.

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Extended data

Extended Data Fig. 1 The slow accumulation of solutes in lysosomes impairs their tubulation and motility without rupture.

a) BMDM pulsed for 4 h with 3 kDa TMR-Dextran and chased for >4 h in normal medium then given a hypotonic solution for 5 min. b-c) BMDM control or those treated with 200 μM GPN for 10 min were evaluated for their tubulation and scored. > 200 cells. d-f) BMDM as in a, with or without 20 mM sucrose or trehalose overnight, then chased for >2 h followed by incubation with 0.5 mg/mL invertase for 30 min. Lysosome volume determined using confocal microscopy. Cell volume for >10000 cells per n determined by Coulter Counter. In e, p = 2.8 × 10−18 and 2.6 × 10−20. In f, p = 1.1 × 10−10. n = 4 independent experiments. g-j) RAW264.7 cells incubated with 20 mM sucrose for indicated times or 5 mM LLOMe for 15 min, pulsed for 5 min with lysotracker red and imaged live or fixed and immunostained as indicated. In h, the lysotracker intensity was determined for individual fields of >5 cells, p = 4.0 × 10−9. n = 3 independent experiments. For i and j, quantification of puncta is graphed in Fig. 1. k-m) HeLa cells expressing tubulin-GFP and LAMP-1-RFP. LAMP-1-RFP compartments were tracked over 60 s and analyzed for motion type (see Methods). Cells were then given 200 μM GPN for 10 min and LAMP-1-RFP compartments were tracked again. The % of tracks identified as being directed in their motion (c) and the diffusion coefficient (d) is shown for >20 cells, >1000 lysosomes. Data are presented as mean values ± SD (c, e, f, h, l, m). ns, not significant. For panels c, e, f, h, l, and m, the statistical significance was determined by two-sided Student’s t-test. Source numerical data are available in source data. n = 3 independent experiments.

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Extended Data Fig. 2 Organic solutes are taken up by macropinocytosis and fluxed into the cytosol.

a) 10 μM 2-NBDG and 70 kDa TMR-dextran given to BMDM for 10 min before staining their surface with AlexaFluor647-wheat germ agglutin for 1 min to indicate the plasma membrane (PM). b) 10 μM 2-NBDG and FM4-64 given to BMDM for 10 min before washing 5 times and imaging live. c) TMR-High molecular weight HA was treated with 1 units/mL hyaluronidase for 30 min then incubated with BMDM together with 70 kDa FITC-dextran for 20 min. n = 3 independent experiments. d) Cells treated as in a, but chased for 5 min and then imaged. The fluorescence intensity of the 2-NBDG compared to the TMR-dextran for individual macropinosomes is shown. >100 macropinosomes. e) BMDM pulsed with 10 μM 2-NBDG for 10 min imaged immediately or after 15 min chase. f) RAW264.7 cells pulsed with 10 μM 2-NBDG and 70 kDa TMR-dextran for 10 min imaged immediately (0 min) or after 15 min chase before staining their surface with AlexaFluor647-WGA. g) BMDM incubated with 70 kDa dextran together with fragmented TMR-hyaluronan for 10 min.

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Extended Data Fig. 3 GLUT localization in macrophages.

a) QPCR. n = 3 independent experiments. Data are presented as mean values ± SD. b) BMDM incubated with 3 kDa TMR-dextran and 20 mM sucrose overnight, then chased for > 2 h followed by incubation with 0.5 mg/mL invertase for 30 min ± BAY876 (10 μM). Quantitation as in b. p = 2.6 × 10−10. n = 3 independent experiments. Data are presented as mean values ± SD. c) Western blotting of BMDM with or without fragmented HA overnight. d) immunostaining of BMDM. e) Lysosomes of indicated cells for 1 h ± BAY876 (10 μM). f) RAW264.7 macrophages expressing SLC2A6-GFP (cyan) with or without LAMP1-RFP challenged with 20 mM sucrose overnight or C. albicans expressing RFP. g-h) BMDM challenged with heat-killed TMR-conjugated C. albicans for 6 h and 0.5 mg/mL zymolase for the last 3 h. The % of phagosomes undergoing tubulation is graphed in h. Each dot represents one field, >15 fields, p = 3.0 × 10−7, n = 3 independent experiments. Data are presented as mean values ± SD (a, h). For panels a, b, and h, the statistical significance was determined by two-sided Student’s t-test. Source numerical data and unprocessed blots are available in source data.

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Extended Data Fig. 4 Solute accumulation in lysosomes activates TFEB dependent on TMEM63A.

a) MCP-1 immunostaining of liver tissues in WT and SLC17A5−/− mice. Scale bar, 20 μm. b) MCP-1 collected from culture medium of indicated BMDM with or without 20 mM sucrose overnight. n = 3 independent experiments. c) TFEBexplorer software (https://tfeb.tigem.it) to determine coordinated lysosomal expression and regulation (CLEAR) elements upstream of the MCP-1/CCL2 promotor region. d) Western blotting as indicated. e) WT and SLC17A5 KO RAW264.7 cells immunostained for galectin-3 or CHMP-3. The number of puncta per cell (numbers indicate individual cells) was quantified. n = 3 independent experiments. f) Example images of TFE3 and DAPI in BMDM with or without 20 mM sucrose overnight followed by the TRPML antagonist ML-SI3 for 2 h. g) QPCR for TMEM63A, n = 3 independent experiments. h-i) WT or SLC17A5 KO RAW264.7 cells expressing scramble or siRNA against TMEM63A, challenged with 20 mM sucrose overnight and stained for TFE3 and quantified. Individual cells are plotted. In h, p = 5.5 × 10−33. In i, p = 4.0 × 10−44. n = 3 independent experiments. j-k) Re-expression of TMEM63A-OFP in TMEM63A KO cells. Individual cells quantified in k (dots). p = 5.2 × 10−33. n = 3 independent experiments. l) Lysosomes from indicated RAW264.7 cells isolated using magnetic nanoparticles. [Na+] was determined using atomic absorption spectroscopy (AAS) and normalized to LAMP-1 protein from the isolates (below). p = 3.5 × 10−4. n = 3 independent experiments. m-n) Lysosomal Natrium Green/cresyl violet with sucrose for indicated cells. Dots represent individual cells for >150 cells. p = 1.3 × 10−36. n = 3 independent experiments. o-q) RAW264.7 cells loaded with sucrose overnight, dialyzed, and analyzed by AAS. p = 6.6 × 10−5 (o), p = 2.4 × 10−4 (p), p = 3.5 × 10−2 (q). n = 3 independent experiments. r) ELISA. n = 3 independent experiments. s) Filipin staining of U18666A-treated cells before and after 5 min shift to hypertonic medium. All scale bars unless otherwise indicated, 10 μm. Data are presented as mean values ± SD (b, e, g, h, i, k, l, n-r). For these panels, the statistical significance was determined by two-sided Student’s t-test. Source numerical data and unprocessed blots are available in source data. The data are presented as mean values ± SD for panels k-m and o-r.

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Extended Data Fig. 5 Lysosomes in Salla fibroblasts are swollen and less motile.

a-c) Primary fibroblasts pulsed with 3 kDa TMR-dextran for 4 h, chased for 4 h, then imaged. In b, the volume of the lysosome compartment is determined by z stacks for single cells (dots) > 20, for 2 controls and 3 patient cultures. p = 2.3 × 10−15 (control 1 vs patient 1), p = 4.7 × 10−16 (control 2 vs patient 1), p = 1.1 × 10−14 (control 1 vs patient 2), p = 3.1 × 10−15 (control 2 vs patient 2), p = 3.3 × 10−18 (control 1 vs patient 3), p = 6.0 × 10−19 (control 2 vs patient 3). In c, the position of the lysosomes can be observed relative to the nuclei. n = 3 independent experiments. d-g) Tracking of single lysosomes. The % of tracks identified as being confined to linear trajectories (e; ****, p = 5.2 × 10−15) and directed in their motion (f; ****, p = 3.9 × 10−11) and the diffusion coefficient (c; ****, p = 1.9 × 10−11) is shown for >20 cells, >1000 lysosomes. n = 3 independent experiments. Data are presented as mean values ± SD (b, e-g). For these panels, the statistical significance was determined by two-sided Student’s t-test. Source numerical data are available in source data.

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Extended Data Fig. 6 Sucrosomes recruit functional motor complexes.

a) Model of lysosomal anterograde and retrograde transport. b) iPSC-expressing LAMP-1-APEX cells were given 40 mM sucrose overnight followed by invertase treatment (‘invertase’) or no treatment (‘sucrose’), biotin-phenol was added for 30 minutes and pulsed for 30 s with hydrogen peroxide. Biotinylated proteins were isolated on Streptavidin beads and identified by mass spectrometry (see Methods). n = 3 independent experiments. c-e) HeLa cells expressing indicated fusion proteins given 3 kDa TMR-dextran overnight together with 20 mM sucrose where indicated. Cells were chased for >4 h before imaging. Invertase or hypertonic medium were added where indicated.

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Cai, R., Scott, O., Ye, G. et al. Pressure sensing of lysosomes enables control of TFEB responses in macrophages. Nat Cell Biol 26, 1247–1260 (2024). https://doi.org/10.1038/s41556-024-01459-y

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