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. RFP, LRRK2, and pT73-RAB10 were blotted. (E) RILP-RHD2 alignment of JIP3, JIP4, RILPL1, and RILPL2 using Clustal (Clustal Omega, EMBL). Arrowheads show the conserved Arg. residues responsible 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. Quantification of RILPL1-positive lysosomes per cell in the different groups. Data are mean ± SEM (P = 0.0009 and P = 0.001, n = 16 cells). Brown–Forsythe and Welch ANOVA with Dunnett’s post hoc test. (H) U2OS cells were transfected with 3xflag-LRRK2, along with 2xmyc-RAB10 (WT) or 2xmyc-RAB10 (T73A). Cells were then treated with LLOME for 2 h, fixed, and stained with anti-flag, anti-myc, and anti-RILPL1 antibodies. Graph shows the quantification of RILPL1 puncta per cell in the different conditions. Data are mean ± SEM (P= 0.0006, n = 28–32 cells). Unpaired t test. (I) HEK239T cells were transfected with HaloTag-LRRK2 and 2xmyc-RILPL1, and one group was also transfected with Ubc-3xHA-JIP4 for 24 h. Cells were then treated or not with LLOME (2 h) and subjected to anti-myc immunoprecipitation. Graph shows the amount of RILPL1:T73-RAB10 interaction in the different groups. Data are mean ± SEM (P < 0.0001, P= 0.0293; N = 5 independent replicates). One-way ANOVA with Tukey’s post hoc test. Scale bar (A, G, and H) = 10 µm; (C) = 20 µm. Insets (A and H) = 2 µm; (C) = 4 µm. Source data are available for this figure: SourceData F3.
Figure 3.

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. RFP, LRRK2, and pT73-RAB10 were blotted. (E) RILP-RHD2 alignment of JIP3, JIP4, RILPL1, and RILPL2 using Clustal (Clustal Omega, EMBL). Arrowheads show the conserved Arg. residues responsible 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. Quantification of RILPL1-positive lysosomes per cell in the different groups. Data are mean ± SEM (P = 0.0009 and P = 0.001, n = 16 cells). Brown–Forsythe and Welch ANOVA with Dunnett’s post hoc test. (H) U2OS cells were transfected with 3xflag-LRRK2, along with 2xmyc-RAB10 (WT) or 2xmyc-RAB10 (T73A). Cells were then treated with LLOME for 2 h, fixed, and stained with anti-flag, anti-myc, and anti-RILPL1 antibodies. Graph shows the quantification of RILPL1 puncta per cell in the different conditions. Data are mean ± SEM (P= 0.0006, n = 28–32 cells). Unpaired t test. (I) HEK239T cells were transfected with HaloTag-LRRK2 and 2xmyc-RILPL1, and one group was also transfected with Ubc-3xHA-JIP4 for 24 h. Cells were then treated or not with LLOME (2 h) and subjected to anti-myc immunoprecipitation. Graph shows the amount of RILPL1:T73-RAB10 interaction in the different groups. Data are mean ± SEM (P < 0.0001, P= 0.0293; N = 5 independent replicates). One-way ANOVA with Tukey’s post hoc test. Scale bar (A, G, and H) = 10 µm; (C) = 20 µm. Insets (A and H) = 2 µm; (C) = 4 µm. Source data are available for this figure: SourceData F3.

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