Fig 2: RILPL1 colocalizes with LYTL tubules and reduces tubulation.(A) Mouse primary astrocytes were transfected with HaloTag-LRRK2, mCherry-RILPL1 and mNeonGreen-LAMP1. 48 h later, cells were treated with LLOME for 6 h and imaged live. The 3D volume shows different RILPL1 tubules emanating from a lysosome. (B) U2OS cells were transfected with HaloTag-LRRK2, mCherry-RILPL1 and mNeonGreen-JIP4. 36 h later, cells were treated with LLOME for 2 h and fixed. 3D volume view of a lysosome with two LYTL tubules expressing JIP4 and mCherry. (C) U2OS cells were treated with NTC or siRILPL1 for 24 h. Then cells were transfected with HaloTag-LRRK2 and mNeonGreen-JIP4 for 36 h. Cells were treated with LLOME for 2 h, fixed and stained with an anti-LAMP1 antibody. (E) Box plot showing the tubule length in both groups. Unpaired t-test was applied (p=0.0458, n= 119–134 total tubules analyzed respectively). Data are mean ± SEM. (F) Tubular ratio quantification. Data are mean ± SEM (p=0.0002, n = 31–33 cells). Unpaired t-test with Welch’s correction was used. (G) U2OS cells were treated with NTC or siRILPL1 for 24 h. Then cells were transfected with 3xflag-LRRK2 and mNeonGreen-JIP4 for 36 h. Cells were treated with LLOME for 2 h and analyzed live. Picture depicts examples of “steady” and “dynamic” tubular events. Graph showing the ratio of steady and dynamic tubular events per cell in the different conditions. Data are mean ± SEM (p=0.0141, n = 20 cells). Unpaired t-test. Scale bar (D)= 10 µm; (A,C,G)= 2 µm.

Fig 3: RILPL1 promotes LRRK2+ lysosome clustering around the centrosome.(A) U2OS cells were transfected with HaloTag-LRRK2 for 36 h. Cells were then treated with LLOME (2 h), fixed and stained with pericentrin. Volume view image of LRRK2+ lysosomes clustered around the centrosome (pericentrin). (B) U2OS cells were treated with a non-targeting control (NTC) or siRILPL1 RNA. 24 h later, cells were transfected with HaloTag-LRRK2 for 36 h. Then, cells were treated with LLOME (2 h), and were fixed and stained with a pericentrin antibody. (C) The distance between LRRK2+ lysosomes and the centrosome was measured in cells treated with NTC or siRILPL1. Data are mean ± SEM (p<0.0001, n = 221–222 lysosomes, from 7 and 9 cells). Unpaired t-test with Welch’s correction. (D) The distance between LRRK2+ lysosomes and the center of the nucleus was measured in cells treated with NTC or siRILPL1. Data are mean ± SEM (p= 0.0206, n = 221–222 lysosomes, from 7 and 9 cells). Unpaired t-test. (E) Time-lapse of a U2OS cell expressing HaloTag-LRRK2 and treated with LLOME. LRRK2 puncta recruitment and movement is followed in different time points up to 120 min. (F) U2OS cells were treated with NTC or siRILPL1 RNA. 24 h later, cells were transfected with HaloTag-LRRK2 for 36 h. Then, cells were treated with LLOME (2 h), and were fixed and stained with JIP4 and LAMP2 antibodies. (G) LRRK2+ lysosomes per cell were quantified. Data are mean ± SEM (n = 33–35 cells). Unpaired t-test. (H) JIP4+ lysosomes per cell were quantified. Data are mean ± SEM (p= 0.0202, n = 33–35 cells). Unpaired t-test. (I) The ratio of LRRK2+/JIP4+ lysosomes compared to LRRK2+/JIP4- lysosomes in NTC vs siRILPL1 treated cells. Data are mean ± SEM (p= 0.0035, n = 31–33 cells). Unpaired t-test with Welch’s correction for unequal variance. Arrowheads indicate LRRK2+/JIP4- lysosomes. Scale bar= 10 µm.

Fig 4: LRRK2 recruits RILPL1 to damaged lysosomes via pRAB proteins.(A) U2OS cells were transfected with HaloTag-LRRK2 for 36 h. Cells were then treated with DMSO, LLOME or LLOME+MLi2 for 2 h before fixation. Cells were then stained for RILPL1 and LAMP2. (B) Quantification of the RILPL1 lysosomes per cell in the three different conditions. Data are mean ± SEM (p<0.0001, n = 21 cells). One-way ANOVA with Tukey’s post hoc tests. (C) Mouse primary astrocytes were transfected with HaloTag-LRRK2, mCherry-RILPL1 and LAMP1-mNeonGreen for 48 h. Astrocytes were treated with LLOME for 6 h and imaged live. (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) RILP-Homoloy Domain 2 (RHD2) alignment of JIP3, JIP4, RILPL1 and RILPL2 using Clustal (Clustal Omega, EMBL). Arrowheads show the conserved Arg. residues responsibles for the binding with the pRAB substrates. Filled arrowhead marks the Arg. that binds with the phosphorylated residue, and the empty arrowhead indicates the Arg. that stabilizes the interaction. (F) Structural model of the interaction between the RHD2 of RILPL1 and RAB10, showing the interaction between T73-RAB10 and R293-RILPL1. (G) U2OS cells were transfected with HaloTag-LRRK2 and mCherry-RILPL1 (WT or R293A) for 36 h. Cells were then treated with DMSO or LLOME for 2 h, fixed and stained for LAMP1. (H) Quantification of RILPL1-positive lysosomes per cell in the different groups. Data are mean ± SEM (p<0.0001, n = 16 cells). One-way ANOVA with Tukey’s post hoc test. Scale bar (A,G)= 10 µm; (C)= 20 µm.

Fig 5: RILPL1 binds to p150Glued 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 were manually annotated as “towards 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 ratio in all groups. Data are mean ± SEM (p= 0.0135, n = 39–42 cells). One-way ANOVA with Dunnett’s post hoc test. Scale bar= 10 µm.