Research ArticleCELL BIOLOGY

LRRK2 mediates tubulation and vesicle sorting from lysosomes

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Science Advances  11 Nov 2020:
Vol. 6, no. 46, eabb2454
DOI: 10.1126/sciadv.abb2454
  • Fig. 1 LRRK2 and JIP4 are localized at the membrane of a subset of lysosomes in primary astrocytes.

    (A) Representative confocal images of 3xflag-LRRK2 and LAMP1 expression in mouse primary astrocytes. (B) Representative confocal images of astrocytes expressing 3xflag-LRRK2 costained with LAMP2, RAB7, EEA1, and CTSB. (C) Outline of the APEX2 proteomic approach to detect LRRK2-membrane interactors. (D) Venn diagrams showing the number of common proteins detected in both replicates (left) and the number of proteins selected as candidates due to having a twofold enrichment in LRRK2 versus negative control in both replicates (right). (E) Scatter plot depicting the 64 LRRK2-interacting candidates from the APEX2 screening. LRRK2 is marked in red, and proteins involved in vesicle-mediated transport are marked in green. (F) Gene Ontology (GO) search of the top 5 enriched terms for biological process of the 64 LRRK2 potential interacting partners, with P values adjusted using a Bonferroni correction indicated on the horizontal axis. (G) Representative confocal image of an astrocyte expressing 3xflag-LRRK2 and GFP-JIP4 and stained for LAMP1. White arrowheads show colocalization, and yellow arrowheads show structures without localization. Scale bar, 20 μm.

  • Fig. 2 Lysosomal membrane permeabilization enhances LRRK2 recruitment to the lysosomal membrane.

    (A) Confocal images of astrocytes untreated or treated with LLOME expressing 3xflag-LRRK2 and LAMP1. The histogram shows the number of LRRK2-positive lysosomes per cell. Data are mean ± SEM (n = 20 cells per N, N = 5). One-way ANOVA with Dunnett’s. (B and C) Live-cell confocal images of astrocytes expressing Emerald-GFP-LRRK2 exposed to LysoTracker Red DND-99 (B) or Magic Red CTSB (C) and treated with LLOME. White arrowheads show absence of colocalization between LRRK2 and the two dyes. (D to F) Confocal images of astrocytes expressing 3xflag-LRRK2, EGFP-Gal3, and LAMP1, untreated or treated with LLOME (D). Yellow arrowheads indicate absence of colocalization, while white arrowheads show colocalization. (E) Histogram shows the number of Gal3-positive lysosomes per cell in cotransfected cells. Data are means ± SEM. One-way ANOVA with Dunnett’s (n = 10 to 20 cells per N, N = 4). (F) Colocalization analysis using n = 20 cells from a single experiment. The percentage of LRRK2-positive/Gal-positive lysosomes normalized by the total number of LRRK2-positive lysosomes was measured in each cell. Box plot shows the median, and the whiskers show the 10th to 90th percentile. One-way ANOVA with Dunnett’s. (G) Confocal images of astrocytes expressing EGFP-Gal3 and LAMP1, pretreated with DMSO or MLi-2 and incubated with LLOME (4 hours). Data are means ± SD. Unpaired t test (n = 20 to 39 cells, N = 2). (H) Western blot of astrocytes pretreated with MLi-2 before adding LLOME. Histogram shows normalized LC3-II levels using two-way ANOVA with Tukey’s. Data are means ± SEM from n = 3. AU, arbitrary units. (I) Working model suggesting that the function of LRRK2 at ruptured lysosomes is independent of lysophagy. Scale bar, 20 μm. *P < 0.05; **P < 0.01, ***P < 0.001.

  • Fig. 3 LRRK2 recruits JIP4 through its kinase activity.

    (A) Representative confocal images of astrocytes expressing 3xflag-LRRK2, GFP-JIP4, and LAMP1 untreated or treated with LLOME. Histogram depicts the number of JIP4-positive lysosomes per cell after LLOME (n = 20 cells per N, N = 3). Data are means ± SEM. One-way ANOVA with Dunnett’s post hoc test. (B) Representative confocal images of astrocytes expressing 3xflag-LRRK2, GFP-JIP4, and LAMP1. Cells were pretreated with DMSO or MLi-2 and incubated with LLOME (6 hours). (C) Astrocytes expressing GFP-JIP4 and LAMP1 were pretreated with DMSO or MLi-2 before adding LLOME (10 hours). Histogram shows the number of JIP4-positive lysosomes per cell (n = 20 to 30 cells per N, from N = 3). Data are means ± SEM using unpaired t test with Welch’s correction. (D) Western blot confirming lack of LRRK2 expression in KO astrocytes compared to WT. (E) Images of Lrrk2-WT or Lrrk2-KO astrocytes transfected with GFP-JIP4 and treated with LLOME (10 hours). Statistical analysis used an unpaired t test with Welch’s correction (n = 13 cells per condition in a single experiment). (F) Astrocytes expressing GFP-JIP4 were cotransfected with 3xflag-LRRK2-WT, 3xflag-LRRK2-G2019S, or 3xflag-LRRK2-R1441C, stained for LAMP1, and treated with LLOME (6 hours). Histogram shows the number of JIP4-positive lysosomes per cell. Data are means ± SEM (n = 20 cells per N, from N = 3). One-way ANOVA with Dunnett’s. Yellow arrowheads indicate LRRK2-positive/JIP4-negative lysosomes. White arrowheads show colocalization. Scale bar, 20 μm. *P < 0.05; **P < 0.01,****P < 0.0001.

  • Fig. 4 LRRK2 phosphorylates RAB35 and RAB10 in the membrane of ruptured lysosomes.

    (A) Cartoon of the APEX2 screening in HEK293FT cells in the presence or absence of LLOME. (B) Volcano plot showing the subset of proteins with fold change > 2 and false discovery rate (FDR)–corrected P < 0.08 (n = 3) in proximity with LRRK2 in LLOME-treated cells. (C and D) Astrocytes expressing 3xflag-LRRK2, RAB35 (C), GFP-RAB10 (D), and LAMP1 treated or not with LLOME. The number of RAB35-positive (C) (n = 20 cells per N, N = 3) or RAB10-positive (D) (n = 20 cells in a single experiment) lysosomes per cell was quantified. Data are means ± SEM. One-way ANOVA with Dunnett’s. (E) Western blot images of HEK293FT cells transfected with 3xflag-LRRK2 (WT or K1906M) and pretreated with DMSO or MLi-2 before LLOME was added. Data are means ± SEM (n = 4). Two-way ANOVA with Tukey’s. (F) Phos-tag gel image of HEK293FT cells expressing 3xflag-LRRK2 and GFP-RAB35, pretreated with DMSO or MLi-2 and incubated with LLOME. Normalized phospho-RAB35 levels were measured (n = 4). Data are means ± SEM. Two-way ANOVA with Tukey’s. (G) Astrocyte expressing 3xflag-LRRK2, 2xmyc-RAB10, and RAB10-T73. (H and I) Astrocytes expressing 3xflag-LRRK2, RAB35 (H), GFP-RAB10 (I), and LAMP1 pretreated with DMSO or MLi-2 and incubated with LLOME. (J and K) Astrocytes were transfected with GFP-JIP4 and 2xmyc-RAB35-(WT/T72A) (J) or 2xmyc-RAB10-(WT/T73A) (K) and treated with LLOME. JIP4-positive lysosomes per cell were quantified (n = 20 cells per N, N = 3). Unpaired t test with Welch’s. Data are means ± SEM. (L) HEK293FT cells transfected with 3xflag-LRRK2 along with 2xmyc-RAB10-(WT/T73A) or 2xmyc-RAB35-(WT/T72A) were treated with LLOME, and lysates were subjected to immunoprecipitation with anti-myc antibodies. White arrowheads indicate colocalization. Scale bar, 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 5 JIP4 promotes the formation of LAMP1-negative lysosomal tubular structures.

    (A) Representative Airyscan image of a LLOME-treated astrocyte (6 hours) expressing GFP-JIP4, 3xflag-LRRK2, and LAMP1. (B) Three-dimensional (3D) surface reconstruction was done in astrocytes treated with LLOME (6 hours) and previously transfected with 3xflag-LRRK2, mNeonGreen-JIP4, and LAMP1-HaloTag. (C) Super-resolution images comparing the number of JIP4-positives tubules in cells expressing LRRK2-WT and G2019S after LLOME treatment (6 hours). (D) FIB-SEM image of a tubule stemming from a lysosome in a 3xflag-LRRK2-G2019S–transfected astrocyte. Top panel shows the Airyscan image of two LAMP1-HaloTag/mNeonGreen-JIP4–labeled lysosomes. Bottom panel shows the correlated EM image, and right panel shows a lysosome with a tubule in different z planes. (E) 3D surface reconstruction of (D) showing a microtubule and the endoplasmic reticulum (ER) in contact with the lysosome. (F) Super-resolution image of an astrocyte expressing 3xflag-LRRK2-G2019S, GFP-JIP4, and α-tubulin. (G) Representative super-resolution images of astrocytes expressing 3xflag-LRRK2-G2019S, GFP-JIP4, and LAMP1 and treated with LLOME (6 hours). Cells were treated with DMSO or nocodazole (Noc). JIP4 tubulation index was measured using an unpaired t test with Welch’s correction (n = 36 to 38 cells pooled from three experiments). Box plot shows the median, and the whiskers show the 10th to 90th percentile. (H) Confocal super-resolution images of astrocytes expressing 3xflag-LRRK2-G2019S, RAB10, and LAMP1 and incubated with LLOME (10 hours). RAB10 tubulation index was measured using an unpaired t test with Welch’s correction (n = 41 cells pooled from two experiments). Box plot shows the median, and the whiskers show the 10th to 90th percentile. White and red (FIB-SEM picture) arrowheads indicate lysosomal tubular structures, and blue arrowheads indicate microtubules. Scale bar, 5 μm or 2 μm (B and D). *P < 0.05, **P < 0.01, ****P < 0.0001.

  • Fig. 6 JIP4-positive tubules are resolved in vesicular structures that contact with other lysosomes.

    (A) Time-lapse fast Airyscan confocal images of a LLOME-treated (6 hours) astrocyte expressing 3xflag-LRRK2-G2019S and mNeonGreen-JIP4 showing a group of JIP4-positive lysosomes for over 2 min, where the different tubular dynamics were under display (15 slices per frame were taken at 1 frame per second). (B and C) Astrocytes expressing 3xflag-LRRK2-G2019S, mNeonGreen-JIP4, and LAMP1-HaloTag and treated with LLOME (6 hours) were analyzed with a SoRa spinning disk super-resolution microscope. (B) A single frame of an astrocyte, where several JIP4-positive vesicles (white arrowhead) are in close proximity to other lysosomes (blue arrowhead). (C) Time-lapse confocal images of a resolved tubule that, after ejection to the cytosol, contacts other lysosomes (15 slices per frame, 1 frame per second). (D) FIB-SEM image of a JIP4-positive vesicle associated with a lysosome in a 3xflag-LRRK2-G2019S–transfected astrocyte after LLOME treatment (6 hours). Top panel shows the Airyscan image of a group of lysosomes (LAMP1) and a vesicle labeled with JIP4 (white arrow). Bottom panel shows the correlated EM image, with a red arrowhead pointing to the vesicle. (E) LLOME-treated astrocytes (6 hours) expressing 3xflag-LRRK2-G2019S and mNeonGreen-JIP4 and stained with Magic Red CTSB were analyzed with a fast Airyscan confocal microscope for almost 4 min, at 6.05 s per frame. (F) Schematic representation of our working model. White arrowhead marks a JIP4-positive vesicle associated with an active lysosome (red). White arrowheads indicate JIP4-positive lysosomal tubules, and yellow arrowheads show resolved tubules (vesicular structures and scissioned tubules) (A to C). Scale bars, 2 μm (D), 2.5 μm (A, C, and E), and 5 μm (B).

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