RILPL1 binds to p150Gluedand favors its recruitment to lysosomes. (A) U2OS cells were transfected with HaloTag-LRRK2 and mNeonGreen-JIP4 for 36 h. Cells were then treated with LLOME (2 h), fixed, and stained with pericentrin. Volume view image of JIP4+ tubules and centrosomes (pericentrin). (B) The orientation of the JIP4+ tubules was manually annotated as “toward the centrosome” (centrosome) or “elsewhere” (other) (n = 28 tubules). (C) Box plot depicting the tubule-to-centrosome angle (n = 72 tubules from 14 cells). (D) HEK293FT cells were transfected with 3xflag-LRRK2 and mCherry-RILPL1 for 24 h. Cells were treated or not with LLOME for 2 h, and lysates were subjected to immunoprecipitation with anti-RFP antibodies. (E) HEK293T cells stably expressing GFP-LRRK2 and TMEM192-3xHA were seeded and treated with DMSO, LLOME, or LLOME + MLi2 24 h later. Lysosomes were purified with anti-HA beads following the LYSO-IP technique. Lysosomes were then lysed, and their content was analyzed via immunoblotting. (F) Quantification of T73-RAB10, RILPL1, and p150Glued protein levels from the lysosomal fraction. Data are mean ± SEM from three independent replicates. P (T73-RAB10) <0.0001; P (RILPL1) = 0.0008; P (p150Glued) = 0.0368. One-way ANOVA with Dunnett’s. (G) HEK293T cells stably expressing GFP-LRRK2 and TMEM192-3xHA were seeded and treated with NTC and siRILPL1 for 48 h later. Then, cells were treated with LLOME for 2 h. Lysosomes were purified with anti-HA beads following the LYSO-IP technique. Lysosomes were then lysed, and their content was analyzed via immunoblotting. (H) Quantification of p150Glued protein levels from the lysosomal fraction. Data are mean ± SEM from four independent replicates. P = 0.0036. Unpaired t test. (I) Schematic representation of our experimental model where the pRAB proteins recruit RILPL1, which in turn binds to p150Glued and recruits it to membrane-damaged lysosomes. (J) Cartoon explaining the FRB–FKBP system to trap RILPL1 to the outer mitochondrial membrane (OMM). (K) U2OS cells were transfected with Mito-YFP-FRB and 3xflag-FKBP-RILPL1 for 24 h. Cells were treated or not with rapamycin or rapamycin+dynarrestin. (L) Box plot showing the perinuclear Mito/Total ratio in all groups. Data are mean ± SEM (P = 0.0198, n = 39–42 cells). Brown–Forsythe and Welch ANOVA with Dunnett’s post hoc test. Scale bar = 10 µm. NTC, nontargeting control. Source data are available for this figure: SourceData F7.
Figure 7.

RILPL1 binds to p150 Glued and favors its recruitment to lysosomes. (A) U2OS cells were transfected with HaloTag-LRRK2 and mNeonGreen-JIP4 for 36 h. Cells were then treated with LLOME (2 h), fixed, and stained with pericentrin. Volume view image of JIP4+ tubules and centrosomes (pericentrin). (B) The orientation of the JIP4+ tubules was manually annotated as “toward the centrosome” (centrosome) or “elsewhere” (other) (n = 28 tubules). (C) Box plot depicting the tubule-to-centrosome angle (n = 72 tubules from 14 cells). (D) HEK293FT cells were transfected with 3xflag-LRRK2 and mCherry-RILPL1 for 24 h. Cells were treated or not with LLOME for 2 h, and lysates were subjected to immunoprecipitation with anti-RFP antibodies. (E) HEK293T cells stably expressing GFP-LRRK2 and TMEM192-3xHA were seeded and treated with DMSO, LLOME, or LLOME + MLi2 24 h later. Lysosomes were purified with anti-HA beads following the LYSO-IP technique. Lysosomes were then lysed, and their content was analyzed via immunoblotting. (F) Quantification of T73-RAB10, RILPL1, and p150Glued protein levels from the lysosomal fraction. Data are mean ± SEM from three independent replicates. P (T73-RAB10) <0.0001; P (RILPL1) = 0.0008; P (p150Glued) = 0.0368. One-way ANOVA with Dunnett’s. (G) HEK293T cells stably expressing GFP-LRRK2 and TMEM192-3xHA were seeded and treated with NTC and siRILPL1 for 48 h later. Then, cells were treated with LLOME for 2 h. Lysosomes were purified with anti-HA beads following the LYSO-IP technique. Lysosomes were then lysed, and their content was analyzed via immunoblotting. (H) Quantification of p150Glued protein levels from the lysosomal fraction. Data are mean ± SEM from four independent replicates. P = 0.0036. Unpaired t test. (I) Schematic representation of our experimental model where the pRAB proteins recruit RILPL1, which in turn binds to p150Glued and recruits it to membrane-damaged lysosomes. (J) Cartoon explaining the FRB–FKBP system to trap RILPL1 to the outer mitochondrial membrane (OMM). (K) U2OS cells were transfected with Mito-YFP-FRB and 3xflag-FKBP-RILPL1 for 24 h. Cells were treated or not with rapamycin or rapamycin+dynarrestin. (L) Box plot showing the perinuclear Mito/Total ratio in all groups. Data are mean ± SEM (P = 0.0198, n = 39–42 cells). Brown–Forsythe and Welch ANOVA with Dunnett’s post hoc test. Scale bar = 10 µm. NTC, nontargeting control. Source data are available for this figure: SourceData F7.

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