A molecular mechanism to regulate lysosome motility for lysosome positioning and tubulation

Abstract

To mediate the degradation of biomacromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca2+ channel TRPML1 cause lysosomal storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca2+-dependent centripetal movement of lysosomes towards the perinuclear region (where autophagosomes accumulate) following autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca2+ sensor that associates physically with the minus-end-directed dynactin–dynein motor, while PtdIns(3,5)P2, a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PtdIns(3,5)P2–TRPML1–ALG-2–dynein signalling is necessary for lysosome tubulation and reformation. In contrast, the TRPML1 pathway is not required for the perinuclear accumulation of lysosomes observed in many LSDs, which is instead likely to be caused by secondary cholesterol accumulation that constitutively activates Rab7–RILP-dependent retrograde transport. Ca2+ release from lysosomes thus provides an on-demand mechanism regulating lysosome motility, positioning and tubulation.

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Figure 1: TRPML1 channel activity is required for acute, minus-end-directed retrograde transport of lysosomes.
Figure 2: Activation of TRPML1 is sufficient to promote Ca2+-dependent retrograde migration of lysosomes.
Figure 3: Cholesterol accumulation causes perinuclear localization of lysosomes in LSDs.
Figure 4: TRPML1 promotes retrograde migration of lysosomes independent of the Rab7–RILP pathway.
Figure 5: ALG-2 interacts with dynein complexes to mediate TRPML1-dependent minus-end-directed transport of lysosomes.
Figure 6: ALG-2 is required for the TRPML1-promoted acute retrograde migration of lysosomes.
Figure 7: The PtdIns(3,5)P2–TRPML1–Ca2+ pathway is required for lysosome tubulation.
Figure 8: TRPML1 regulates the switch between the plus- and minus-end-directed lysosome motility.

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Acknowledgements

This work was supported by NIH grants (NS062792, MH096595 and AR060837 to H.X.). The authors are grateful to R. Puertollano (NHLBI, NIH, USA) for providing the mCherry–ALG-2 construct, K. Verhey (University of Michigan, USA) for providing GFP–DYNIC2-DN, GFP–dynamitin and KIF5B cDNA constructs, R. Botelho (Ryerson University, Toronto, Canada) for providing the RILP–GFP construct, J. Neefjes (Netherlands Cancer Institute, Netherlands) for providing the ORP1L–GFP construct, and D. Rubinsztein (Cambridge Institute for Medical Research, UK) for providing the LC3 stable cell line. The authors thank R. Hume, R. Fuller, K. Verhey and Y. Wang for their suggestions. We appreciate the encouragement and helpful comments from the Xu laboratory colleagues.

Author information

X.L. initiated the project; X.L. and H.X. designed the research; X.L., N.R., A.H., X.Z., J.Y., W.W., Q.G. and X.C. performed the experiments; X.L. generated new reagents; X.L., N.R., A.H., X.Z., J.Y., W.W., Q.G., X.C. and H.X. analysed and interpreted data; X.L. and H.X. wrote the manuscript with input from all authors.

Correspondence to Haoxing Xu.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Lysosomes undergo perinuclear migration during acute starvation to facilitate lysosome-autophagosome fusion.

(a) WT fibroblasts showing the co-localization between Lamp1-GFP and Lyso-Tracker. (b) Lamp1-transfected WT fibroblasts were loaded with dextran-red, with (right) or without (left) starvation. (c,d) WT fibroblasts were transfected with Lamp1-GFP and left untreated (c), or starved for 2 h (d), then fixed and immunolabelled for γ-tubulin to visualize centrosomes (MTOC, white arrows). (e) HeLa cells stably expressing GFP-RFP-LC3 were subjected to starvation for 2 h (lower panel). The GFP channel shows puncta that label autophagosomes specifically, but not autolysosomes. These results show that both lysosomes and autophagosomes are perinuclearly localized under acute starvation. (f) HeLa cells stably expressing GFP-RFP-LC3 were left untreated (upper), starved for 2 h (middle), or starved for 2 h in the presence of 25 μM ML-SI3 (bottom). (g) Quantification of the number of puncta that were GFP- and RFP-positive (autophagosomes, AP) over the number of puncta that were only RFP-positive (autolysosomes, AL) for groups shown in f. Nuclei are labeled with “N”. Graphed data are presented as means ± SEM, the numbers of cells (n) used for quantification were pooled across at least three independent experiments and are shown in the parentheses. p < .05, p < .01 in ANOVA. Scale bar = 10 μm.

Supplementary Figure 2 Effect of cytosolic pH on TRPML1 channel activity and lysosome positioning.

(a) ML-SA1 (10 μM)-activated whole-lysosome TRPML1 currents were inhibited potently by the TRPML1 inhibitor ML-SI3 (2.5 μM). (b) PI(3,5)P2-activated whole-endolysosome TRPML1 currents were modulated by cytosolic pH. (c,d) Lamp1-mCherry-transfected WT fibroblasts were treated with 30 min ammonia Ringer’s solution in the presence (d) or absence (c) of 25 μM ML-SI3. (e) Quantification of groups shown in (c) and (d). (f) Western blot showing the phosphorylated form of S6K (pS6K) in HeLa cells starved for 2 h, treated with 1 μM Torin-1 for 2 h, or treated with ammonia Ringer’s for 30 min. Red lines outline cell boundaries and nuclei are marked with a red “N”. Graphed data are presented as means ± SEM, the numbers of cells (n) used for quantification were pooled across at least three independent experiments and are shown in the parentheses. p < .05, p < .01 in ANOVA. Scale bars = 10 μm. Uncropped western blot images are shown in Supplementary Figure 9.

Supplementary Figure 3 Specific regulation of lysosome distribution by TRPML1.

(a) WT fibroblasts treated with ML-SA3 (25 μM) or ML-SA5 (1 μM), two structurally-independent TRPML1 agonists, for 1 h. (b) In TRPML1-overexpressing cells, the majority of lysosomes were localized in the perinuclear region in the presence or absence of 25 μM ML-SA1. (c) Representative images showing WT fibroblasts overexpressing TRPML2 or TRPML3. (d) Quantifications of groups shown in (b). (e) Quantification of groups shown in (c). (f) The perinuclear localization of lysosomes induced by TRPML1 overexpression (left) was reversed by either a low (1 μM, middle) or high (25 μM, right) dose of ML-SI3. (g) Quantification of groups shown in (f). (h,i) Representative images of Lamp1-GFP-transfected fibroblasts stained with MitoTracker (100 nM) for 1 h (h), or 1 h of MitoTracker in the presence of 25 μM ML-SA1 (i). (j) Quantification of groups shown in (h) and (i). Upon ML-SA1 treatment, lysosomes became more perinuclear, while the distribution of mitochondria was not altered. Red lines outline cell boundaries and nuclei are marked with a red “N”. Graphed data are presented as means ± SEM, the numbers of cells (n) used for quantification were pooled across at least three independent experiments and are shown in the parentheses p < .05, p < .01 in ANOVA. Scale bars = 10 μm.

Supplementary Figure 4 Chronic loss of TRPML1 activity causes cholesterol accumulation and perinuclear localization of lysosomes in fibroblasts.

(a) Lysosome distribution in WT fibroblasts treated with vehicle (0.1% DMSO) (upper right), ML-SI1 25 μM (bottom left), or ML-SI3 25 μM (bottom right) for 18 h in complete medium. (b) Quantification of the groups shown in (a). (c) Time-dependence of TRPML1 inhibition of lysosome distribution in WT fibroblasts in complete medium. (d) Representative images showing lysosome distribution in Rab7-T22N-expressing cells in the presence of ML-SI3 (25 μM) for 18 h. (e) Representative images showing ML1 KO fibroblasts transfected with Lamp1-mCherry, and stained with filipin. Intracellular puncta filipin staining co-localized well with Lamp1. (f) Representative images showing ML1 KO fibroblasts transfected with DynIC2-DN-GFP, and stained with filipin. Graphed data are presented as means ± SEM, the numbers of cells (n) used for quantification were pooled across at least three independent experiments and are shown in the parentheses. p < .05,p < .01 in ANOVA. Scale bars = 10 μm for (a) and (d), and = 50 μm for (e) and (f).

Supplementary Figure 5 Regulation of lysosomes motility by PI(3,5)P2 and dynein motors.

(a) Lysosome distribution in WT fibroblasts treated with 1 μM apilimod for 1 h (upper), 1 μM YM 201636 for 1 h (middle), or 1 μM YM 201636 plus 25 μM ML-SA1 for 1 h (bottom). (b) Lysosome distribution in WT fibroblasts starved for 1 h (upper), or starved for 1 h in the presence of 1 μM apilimod (middle) or YM 201636 (bottom). (c) Lysosome distribution in WT fibroblasts treated with 1 μM Torin-1 for 1 h in the presence of 1 μM apilimod (middle) or YM 201636 (bottom). (d) Lysosome distribution in TRPML1-7Q-transfected cells in the presence (bottom) or absence (middle) of 25 μM ML-SA1 for 1 h. (e) Quantification of groups shown in (a). (f) Quantification of groups shown in (b). (g) Quantification of groups shown in (c). (h) Quantification of groups shown in (d), compared to cells expressing Lamp1 alone. (i) Lysosome (labeled with Lamp1-EGFP) distribution in WT fibroblasts transfected with mCherry-tagged dominant-negative Kif5B (Kif5B-DN). (jl) Lysosome (labeled with Lamp1-mCherry) distribution in WT fibroblasts transfected with GFP-tagged dominant-negative cytoplasmic dynein intermediate chain 2 (DynIC2-DN), then left untreated (j), starved for 2 h (k), or treated with 25 μM ML-SA1 for 2 h (l). (m) Effect of dynein inhibitor ciliobrevin D (20 μM, 2 h) on lysosome distribution in TRPML1-expressing fibroblasts. (n) WT fibroblasts treated with ML-SA1 (25 μM) or together with ciliobrevin D (20 μM) for 2 h. (o) Quantification of lysosome distribution in experimental groups shown in (i) and (j). Red lines outline cell boundaries; “N” marks nuclei. (p) Whole-lysosome TRPML1 currents were not activated by Torin-1 in TRPML1-expressing Cos1 cells. ML-SA1 readily activated whole-lysosome TRPML1 currents. (q) Application of ML-SA1 (1, 5, 10 μM) in HEK293T cells for 3 h did not lead to a significant change in the level of phophorylated S6K, a major mROTC1 target. Graphed data are presented as means ± SEM, the numbers of cells (n) used for quantification were pooled across at least three independent experiments and are shown in the parentheses). p < .05, p < .01 in ANOVA. Scale bars = 10 μm. Uncropped western blot images are shown in Supplementary Figure 9.

Supplementary Figure 6 Effects of Syt VII, ALG-2, and ORP1L overexpression on lysosome distribution.

(a,b) WT fibroblasts overexpressing Lamp1-mCherry with (a) or without (b) Syt VII co-expression. (c) Quantification of groups shown in (a,b). (df) WT fibroblasts co-transfected with ALG-2-GFP and Lamp1-mCherry, then left without treatment (d), treated with 25 μM ML-SI3 for 2 h (e), or treated with 25 μM ML-SA1 for 2 h (f). Some perinuclear bright dots of ALG-2 not co-localized with Lamp1 were seen in all treatment conditions. Treatment of ML-SI3 and ML-SA1 resulted in less (ML-SI3) or more (ML-SA1) co-localization with Lamp1 compared to non-treated control cells, respectively. (g) ORP1L overexpression induced lysosome clustering as well as enlargement in WT fibroblasts. Red lines outline cell boundaries and nuclei are marked with red “N”. Graphed data are presented as means ± SEM, the number of cells (n) used for quantification were pooled across at least three independent experiments and are shown in the parentheses. p < .05, p < .01 in ANOVA. Scale bars = 10 μm.

Supplementary Figure 7 ALG-2 mediates the interaction of TRPML1 and dynactin.

(ac) Representative whole-lysosome currents in cells overexpressing WT TRPML1 (a), TRPML1-R44-A (b), or TRPML1-R44LK-AAA (c). Whole-lysosome currents were elicited by the endogenous agonist PI(3,5)P2 or the synthetic agonist ML-SA1. (d,e) Filipin staining of ML1 KO fibroblasts overexpressing TRPML1 (d) or TRPML1-R44-A (e). Purple arrows indicate cells with overexpression. (f) Overexpression of TRPML1 in ML1 KO fibroblasts causes perinuclear accumulation of lysosomes, but contrary to ML1 KO fibroblasts without TRPML1 expression, this was reversible through application of 25 μM ML-SI3 for 2 h. (g) Quantification of groups shown in (f). (h) Cells expressing mCherry or mCherry-ALG-2 were subject to Co-IP with either anti-mCherry or anti-Dynamitin, then blotted against mCherry (top) or Dynamitin (bottom). Asterisk indicates a non-specific band seen with HEK293 pull-down. (i) Cos1 cells expressing either mCherry or mCherry-ALG-2 were subject to Co-IP with either anti-mCherry or anti-GFP antibodies, then blotted against mCherry. (j) Cos1 cells co-expressing GFP-dynamitin and mCherry-ALG-2 were subject to Co-IP with either anti-mCherry or anti-GFP antibodies, then blotted against GFP. Red lines outline cell boundaries and nuclei are marked with a red “N”. Graphed data are presented as means ± SEM, the numbers of cells (n) used for quantification were pooled across at least three independent experiments and are shown in the parentheses. p < .05, p < .01 in ANOVA. Scale bars = 10 μm for (f), and 50 μm for (d,e). Uncropped western blot images are shown in Supplementary Figure 9.

Supplementary Figure 8 Modulation of lysosome tubulation by TRPML1, motor proteins, and ALG-2.

(a) Quantification of lysosome tubulation in CV-1 cells. Dominant-negative constructs of Kif5B and DynIC2 eliminated spontaneous tubulation in Lamp1-GFP-expressing CV1 cells almost completely. (b) Representative images of Lamp1-mCherry-transfected NRK cells starved for 16 h (left), or starved for 16 h with the last 2 h in the presence of 25 μM ML-SI3 (middle) or ML-SI1 (right). (c) Quantification of groups shown in (b). (d) Quantification of lysosome tubulation in WT fibroblasts transfected with Lamp1-GFP, or Lamp1-GFP plus ALG-2-mCherry, with or without starvation for 24 h. ALG-2 expression inhibited lysosome tubulation strongly in starved cells. (e,f) Same region of a Lamp1-GFP-transfected fibroblast starved for 24 h under conventional (e) or STED super-resolution (f) confocal imaging. The super-resolution image was taken 6 s after the conventional confocal image. Yellow arrows point to several small lysosomes that are lined-up to have a tubular appearance under the conventional confocal images; red arrows point to a genuine tubule. Graphed data are presented as means ± SEM, the number of cells (n) used for quantification were pooled across at least three independent experiments and are shown in the parentheses p < .05, p < .01. Scale bars = 10 μm for (b), and = 2 μm for (e,f).

Supplementary Figure 9 Unprocessed scans of original western blots used in the main and supplementary figures.

For each image, black boxes indicate roughly the positions of each original blotting membranes, and the red dotted boxes indicate the regions used in the figures. Each image is labeled on top with the panel in the figure they appeared.

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Video (1 frame/s) of a Lamp1-mCherry-transfected WT fibroblast. Photobleaching was conducted at the 5th frame (T = 0 s). Scale bar = 10 μm. (AVI 2410 kb)

FRAP analysis of directional movement of lysosomes under normal conditions.

Video (1 frame/s) of a Lamp1-mCherry-transfected WT fibroblast. Photobleaching was conducted at the 5th frame (T = 0 s). Scale bar = 10 μm. (AVI 2410 kb)

41556_2016_BFncb3324_MOESM19_ESM.avi

A Lamp1-mCherry-transfected WT fibroblast imaged 15 30 min after starvation. Scale bar = 10 μm. (AVI 2946 kb)

FRAP analysis of directional movement of lysosomes under acute starvation.

A Lamp1-mCherry-transfected WT fibroblast imaged 15 30 min after starvation. Scale bar = 10 μm. (AVI 2946 kb)

41556_2016_BFncb3324_MOESM20_ESM.avi

Imaging of a Lamp1-mCherry-transfected WT fibroblast after it was incubated with 10 μM BAPTA-AM for 1 h. Scale bar = 10 μm. (AVI 1621 kb)

FRAP analysis of directional movement of lysosomes in the presence of the Ca2+ chelator BAPTA-AM.

Imaging of a Lamp1-mCherry-transfected WT fibroblast after it was incubated with 10 μM BAPTA-AM for 1 h. Scale bar = 10 μm. (AVI 1621 kb)

41556_2016_BFncb3324_MOESM21_ESM.avi

A Lamp1-mCherry-transfected WT fibroblast imaged 15 30 min after starvation in the presence of ML-SI3 (25 μM). Scale bar = 10 μm. (AVI 1954 kb)

FRAP analysis of directional movement of lysosomes under starvation condition in the presence of ML-SI3.

A Lamp1-mCherry-transfected WT fibroblast imaged 15 30 min after starvation in the presence of ML-SI3 (25 μM). Scale bar = 10 μm. (AVI 1954 kb)

41556_2016_BFncb3324_MOESM22_ESM.avi

A Lamp1-mCherry-transfected WT fibroblast imaged 15 30 min after application of ML-SA1 (25 μM). Scale bar = 10 μm. (AVI 2554 kb)

FRAP analysis of directional movement of lysosomes in the presence of ML-SA1.

A Lamp1-mCherry-transfected WT fibroblast imaged 15 30 min after application of ML-SA1 (25 μM). Scale bar = 10 μm. (AVI 2554 kb)

41556_2016_BFncb3324_MOESM23_ESM.avi

A Lamp1-mCherry-transfected WT fibroblast was imaged 1 frame/10 s for 250 frames. The video plays 300× real-time speed. Scale bar = 10 μm. (AVI 840 kb)

Time-lapse imaging of lysosome migration under normal conditions.

A Lamp1-mCherry-transfected WT fibroblast was imaged 1 frame/10 s for 250 frames. The video plays 300× real-time speed. Scale bar = 10 μm. (AVI 840 kb)

41556_2016_BFncb3324_MOESM24_ESM.avi

A Lamp1-mCherry-transfected WT fibroblast was imaged at 1 frame/10 s for 250 frames in the presence of 25 μM ML-SA1, which was applied 3 min after the start of the imaging (i.e. frame 18). The video plays 300× real-time speed. Scale bar = 10 μm. (AVI 753 kb)

Time-lapse imaging of lysosome migration upon acute application of ML-SA1.

A Lamp1-mCherry-transfected WT fibroblast was imaged at 1 frame/10 s for 250 frames in the presence of 25 μM ML-SA1, which was applied 3 min after the start of the imaging (i.e. frame 18). The video plays 300× real-time speed. Scale bar = 10 μm. (AVI 753 kb)

41556_2016_BFncb3324_MOESM25_ESM.avi

TRPML1-GFP-expressing WT fibroblast was imaged at 1 frame/10 s for 250 frames in the presence of 25 μM ML-SI3, which was applied 3 min after the start of the imaging (i.e. frame 18). The video plays 300× real-time speed. Scale bar = 10 μm. (AVI 925 kb)

Time-lapse imaging of lysosome migration in TRPML1-expressing cells upon acute application of ML-SI3.

TRPML1-GFP-expressing WT fibroblast was imaged at 1 frame/10 s for 250 frames in the presence of 25 μM ML-SI3, which was applied 3 min after the start of the imaging (i.e. frame 18). The video plays 300× real-time speed. Scale bar = 10 μm. (AVI 925 kb)

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Li, X., Rydzewski, N., Hider, A. et al. A molecular mechanism to regulate lysosome motility for lysosome positioning and tubulation. Nat Cell Biol 18, 404–417 (2016). https://doi.org/10.1038/ncb3324

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