A molecular mechanism to regulate lysosome motility for lysosome positioning and tubulation - PubMed (original) (raw)

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

Xinran Li et al. Nat Cell Biol. 2016 Apr.

Abstract

To mediate the degradation of biomacromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca(2+) 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 Ca(2+)-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 Ca(2+) sensor that associates physically with the minus-end-directed dynactin-dynein motor, while PtdIns(3,5)P(2), a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PtdIns(3,5)P(2)-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. Ca(2+) release from lysosomes thus provides an on-demand mechanism regulating lysosome motility, positioning and tubulation.

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Figures

Figure 1

Figure 1. TRPML1 channel activity is required for acute, minus-end-directed retrograde transport of lysosomes

(a, b) Representative HeLa cells (a) and Cos1 cells (b) transfected with the late-endosome and lysosome marker Lamp1-mCherry under 2 h serum starvation with (right) or without (middle) the TRPML1 inhibitor ML-SI3 (25 μM). Insets illustrate the size of lysosomes with and without starvation (same magnification, blue arrows indicate enlarged lysosomes). (c) Representative WT mouse fibroblasts transfected with Lamp1-mCherry in 2 h starvation condition (right). (d) Lysosome (labelled with Lamp1) distribution in WT fibroblasts in the presence of the TRPML1 inhibitor ML-SI1 (25 μM) with (right) or without (left) starvation. (e) Lysosome distribution in WT fibroblasts treated with mTOR inhibitor Torin 1 (1 μM) or together with ML-SI3 (25 μM) for 2 h. (f) Quantitative analyses of lysosome distribution in the experiments shown in (c) and (d). The intracellular distribution of Lamp1-positive vesicles was quantified as described in Materials & Methods. Fibroblasts were chosen for most quantification analyses for their large cell area, regular shape, and ML1 KO availability. (g) Quantification of groups shown in (e). (h-j) FRAP analysis of lysosome movement in WT fibroblasts without any treatment (h), with 15– 30 min starvation (i), or with starvation in the presence of 25 μM ML-SI3 (j). Snap images immediately after (top panels) or 5 min (bottom panels) after photobleaching are shown. Lysosomes that traveled across the midline of the photobleached region (yellow line) towards the nucleus (retrograde) are labeled green; those moving away from the nucleus (anterograde) are labeled red. (k) Quantification of lysosomes in (h–j) undergoing retrograde or anterograde transport. Red lines in images outline cell boundaries and the red “N” marks the nucleus. 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 (for each sample group, no sample was excluded). *p < .05, **p < .01 in ANOVA. Scale bars = 10 μm, and 2 μm for insets.

Figure 2

Figure 2. Activation of TRPML1 is sufficient to promote Ca2+-dependent retrograde migration of lysosomes

HeLa cells (a) Cos1 cells (b) and WT fibroblasts (c) with (bottom) and without (top) 25 μM MLSA1 for 2 h. (d-f) Lysosome distribution in TRPML1-overexpessing WT fibroblasts (d) treated with 10 μM BAPTA-AM for 1 h (e) or 25 μM ML-SI1 for 2 h (f). (g) Quantification of lysosome distribution in the experiments shown in (c) and (d). (h) Quantification of experimental group shown in (e). (i) Quantification of experimental group shown in (f). (j) FRAP analysis of lysosome retrograde transport upon ML-SA1 application. (k) Quantification of lysosomes shown in (j) undergoing retrograde and anterograde transport after photobleaching. Red lines delineate cell boundaries and red “N” mark nuclei. 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 (for each sample group, no sample was excluded). *p < .05, **p < .01 in ANOVA. Scale bars = 10 μm.

Figure 3

Figure 3. Cholesterol accumulation causes perinuclear localization of lysosomes in LSDs

(a) Representative images showing Lamp1-mCherry-transfected ML1 KO fibroblasts in starved cells that were treated with simvastatin and mevalonolactone to deplete cholesterol. (b) Quantification of groups shown in (a). (c) Representative images showing Lamp1-mCherry transfected NPC1 KO fibroblasts (upper left), starved for 3 h (upper right), starved with cholesterol depletion (bottom left), or starved with cholesterol depletion in the presence of 25 μM ML-SI1 (bottom right). (d) Effect of cholesterol depletion on starvation-induced lysosome redistribution in WT fibroblasts. (e) Quantification of groups shown in (c). (f) Quantification of observations shown in (d). (g) Filipin staining of WT fibroblasts (upper left) and WT fibroblasts depleted of cholesterol with simvastatin (upper right), WT fibroblasts treated with 25 μM MLSI3 for 1 h (bottom left) or 24 h (bottom right). (h) ML1 KO fibroblasts with (bottom) or without (upper) cholesterol depletion. (i) NPC1 KO fibroblasts with (bottom) or without (upper) cholesterol depletion. (j) Quantitative comparison of filipin staining in WT fibroblasts treated with different durations of ML-SI3 versus ML1 KO and NPC1 KO fibroblasts. (k) Quantification of filipin stating in WT, ML1 KO, NPC1 KO fibroblasts with or without cholesterol depletion. Red lines outline cell boundaries. 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), (c) and (d), and = 50 μm for (g), (h) and (i).

Figure 4

Figure 4. TRPML1 promotes retrograde migration of lysosomes independent of the Rab7-RILP pathway

(a, b) Representative images showing WT fibroblasts overexpressing Lamp1-mCherry and RILP-GFP (a) or Rab7-Q67L-GFP (b) in the presence or absence of 25 μM ML-SI3 for 2 h. (c) Quantification of groups shown in (a). (d) Quantification of groups shown in (b). (e) Representative images showing WT fibroblasts transfected with Lamp1-mCherry and Rab7-T22N-GFP, then left untreated (upper panels), serum starved for 2 h (middle panels), or incubated with 25 μM ML-SA1 for 2 h (bottom panels). (f) Representative images showing ML1 KO fibroblasts overexpressing Lamp1-mCherry and Rab7-T22N-GFP. (g) Quantification of groups shown in (e). (h) Quantification of the group shown in (f). Red lines outline cell boundaries. 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.

Figure 5

Figure 5. ALG-2 interacts with dynein complexes to mediate TRPML1-dependent minus-end-directed transport of lysosomes

(a) Quantification of the effect of dominant-negative Dynein Intermediate Chain 2 (DynIC2-DN) on the distribution of lysosomes under different conditions (see Supplementary Figure 5j-l). (b) Quantification of the effect of 20 μM Ciliobrevin D (1 h) on lysosome distribution with or without TRPML1 overexpression (see Supplementary Figure 5m). (c) Quantification of the effect of Ciliobrevin D (1 h) on lysosome distribution in the presence or absence of 25 μM MLSA1 (see Supplementary Figure 5n). (d) Lysosome distribution in WT fibroblasts co-transfected with Lamp1-GFP and mCherry-ALG-2. (e) Lysosome distribution in mCherry-ALG-2-transfected cells treated with ML-SI3 (25 μM) for 1 h. (f) Lysosome distribution in WT fibroblasts co-transfected with Lamp1-GFP and mCherry-ALG-2-EEAA (E47A-E114A). (g) Lysosome distribution in ML1 KO fibroblasts transfected with Lamp1-mCherry alone (top), Lamp1-mCherry + GFP-TRPML1 (middle), or Lamp1-mCherry + GFP-TRPML1-R44-A (bottom). (h) Quantification of lysosome distribution in experiments shown in (d-f). (i) Quantification of groups shown in (g). (j) Co-immunoprecipitation of ALG-2 and dynamitin in Cos-1 cells doubly transfected with mCherry-ALG-2 and GFP-Dynamitin, in the absence or presence of 0.5 mM Ca2+ in the lysis buffer. Cell lysates were directly loaded (input), or immunoprecipitated with either anti-GFP or anti-mCherry antibody, and then blotted against GFP. Red lines in images outline cell boundary and the red “N” marks the nucleus. 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.

Figure 6

Figure 6. ALG-2 is required for the TRPML1-promoted acute retrograde migration of lysosomes

(a) DNA sequencing results of the two ALG-2 CRISPR KO HeLa cell lines, the red “ATG” indicates start codon. Mutant #1 had a 2bp deletion on all chromosomes, while mutant #2 had various lengths of out-of-frame deletions. (b) Western blot confirmation of the ALG-2 KO. (c) Representative images showing Lamp1-mCherry distribution of WT (left) and the two mutant lines in complete medium without any treatment. (d, e) WT (d) and ALG-2 KO (e) cells under 2 h of 25 μM ML-SA1 treatment (left), or under 2 h of serum starvation (right). Mutant #2 was chosen for further studies based on their more extended morphology. (f, g) WT (f) and ALG-2 KO (g) cells overexpressing Lamp-mCherry and RILP-GFP in the presence of 25 μM ML-SI3 for 2h. Red asterisks in RILP panels indicate cells not expressing RILP-GFP. (h) Quantification of the groups shown in (c-e). ( i, j) Western blot analysis of endogenous LC3 in WT or ALG-2 KO HeLa cells upon 2 h starvation. LC3-II over LC3-I ratio normalized to WT control cells from 5 independent experiments were quantified in (j). Red lines in images outline cell boundary and the red “N” marks the nucleus. 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.

Figure 7

Figure 7. The PI(3,5)P2–TRPML1-Ca2+ pathway is required for lysosome tubulation

(a) Lamp1-mCherry-transfected fibroblasts were starved for 24 h. A high level of lysosome tubulation was seen in WT fibroblasts, but not in ML1 KO fibroblasts or in WT fibroblasts treated with ML-SI1 (25 μM) during the last hour of starvation. (b) Quantification of lysosome tubules in the groups shown in (a). (c) Lysosome tubulation in macrophages loaded with tetramethylrhodamine-dextran (1 h loading, 2 h chase) and activated with lysopolysaccharides (LPS) for 3 h. Lysosome tubulation was prominent in WT macrophages (left), but not in ML1 KO macrophages (middle) or WT macrophages treated with ML-SI1 (25 μM, 30 min; right). (d) Quantification of the groups shown in (c). (e) Effects of ML-SI1 (25 μM, 1h) or BAPTA-AM (10 μM, 1 h) on spontaneous lysosome tubulation in Lamp1-GFP-expressing CV1 cells. (f) Quantification of groups shown in (e). (g) Representative WT fibroblasts starved for 24 h and treated with 1 μM YM 201636 for 30 min (left), Vac14 KO fibroblasts starved for 24 h (middle), and starved Vac14 KO fibroblasts treated with 10 μM ML-SA1 for 30 min (right). (h) Quantification of the groups shown in (g). (i) Lysosome tubulation after 24 h starvation of ML1 KO fibroblasts transfected with Lamp1-mCherry alone (left), Lamp1-mCherry together with GFP-TRPML1-7Q with (right) or without (middle) a low dose (1 μM) of ML-SA1 for 1 h. (j) Quantification of the groups shown in (i). 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.

Figure 8

Figure 8. TRPML1 regulates switch between the plus- and minus-end directed lysosome motility

(a, b) Effect of ML-SA1 (20 μM, 1 h) on lysosome tubulation in mouse fibroblasts. (c-e) Effects of 1 μM (d) or 25 μM (e) ML-SI1 on lysosome tubulation in TRPML1-overexpressing WT fibroblasts. ML-SIs were applied during the last 2 h of starvation. (f) Quantifications of the groups shown in (c–e). (g) Model illustrating the proposed role of TRPML1 in the regulation of lysosome motibility and tubulation. Under normal growth conditions (I), lysosomes are mostly peripherally distributed. During acute starvation (II), the TRPML1-ALG-2 pathway is activated to increase minus-end-directed motility of lysosomes, resulting in rapid redistribution of lysosomes to the juxtanuclear region, thereby facilitating autophagosome-lysosome fusion. After prolonged starvation (III), reactivation of mTOR turns on the machinery for lysosome tubulation and reformation. While the TRPML1-ALG-2 pathway remains active, the plus-end motility of lysosomes is increased. Subsequently, the “balanced” driving forces on both directions result in the generation of tubular lysosomes. 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. *p < .05, **p < .01 in ANOVA. Scale bars = 10 μm.

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