RILPL1 colocalizes with LYTL tubules and reduces tubulation. (A) Mouse primary astrocytes were transfected with HaloTag-LRRK2, mCherry-RILPL1, and LAMP1-mNeonGreen. 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 3xflag-LRRK2, mBaoJin-RILPL1, and HaloTag-JIP4. 36 h later, cells were treated with LLOME for 2 h and fixed. 3D volume view of a lysosome with three LYTL tubules expressing JIP4 and RILPL1. (C) U2OS cells were transfected with 3xflag-LRRK2 and mBaoJin-RILPL1 for 36 h. Cells were then treated with LLOME and imaged live. Time-lapse shows RILPL1 staining dynamic tubules that undergo fission (3D view). (D) 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) Quantification of the number of JIP4+ tubules/total JIP4+ lysosomes. 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 siJIP4 for 24 h. Then cells were transfected with HaloTag-LRRK2 and 2xmyc-RILPL1 for 36 h. Cells were treated with LLOME for 2 h, fixed, and stained with anti-myc and anti-LAMP1 antibodies. (H) Box plot showing the tubule length in both groups. Unpaired t test with Welch’s correction was applied (P = 0.0037, n = 112–130 total tubules analyzed, respectively). Data are mean ± SEM. (I) Quantification of the number of RILPL1+ tubules/total RILPL1+ lysosomes. Data are mean ± SEM (P = 0.001, n = 31–32 cells). Unpaired t test with Welch’s correction for unequal variance was used. (J) 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. (K) 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 and G) = 10 µm; (A, B, and J) = 2 µm. NTC, nontargeting control.
Figure 6.

RILPL1 colocalizes with LYTL tubules and reduces tubulation. (A) Mouse primary astrocytes were transfected with HaloTag-LRRK2, mCherry-RILPL1, and LAMP1-mNeonGreen. 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 3xflag-LRRK2, mBaoJin-RILPL1, and HaloTag-JIP4. 36 h later, cells were treated with LLOME for 2 h and fixed. 3D volume view of a lysosome with three LYTL tubules expressing JIP4 and RILPL1. (C) U2OS cells were transfected with 3xflag-LRRK2 and mBaoJin-RILPL1 for 36 h. Cells were then treated with LLOME and imaged live. Time-lapse shows RILPL1 staining dynamic tubules that undergo fission (3D view). (D) 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) Quantification of the number of JIP4+ tubules/total JIP4+ lysosomes. 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 siJIP4 for 24 h. Then cells were transfected with HaloTag-LRRK2 and 2xmyc-RILPL1 for 36 h. Cells were treated with LLOME for 2 h, fixed, and stained with anti-myc and anti-LAMP1 antibodies. (H) Box plot showing the tubule length in both groups. Unpaired t test with Welch’s correction was applied (P = 0.0037, n = 112–130 total tubules analyzed, respectively). Data are mean ± SEM. (I) Quantification of the number of RILPL1+ tubules/total RILPL1+ lysosomes. Data are mean ± SEM (P = 0.001, n = 31–32 cells). Unpaired t test with Welch’s correction for unequal variance was used. (J) 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. (K) 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 and G) = 10 µm; (A, B, and J) = 2 µm. NTC, nontargeting control.

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