LYTL tubules elongate through tyrosinated microtubules. (A) U2OS cells were transfected with 3xflag-LRRK2 and GFP-JIP4. Cells were treated with LLOME (2 h) and fixed. After fixation, cells were stained for GFP, tyrosinated α-tubulin, and acetylated α-tubulin. (B) Box plot showing the LYTL tubule contact fraction with the different α-tubulin PTMs. Unpaired t test with Welch’s correction was applied (P < 0.0001, n = 49 tubules). Arrowheads show lysosomal tubules. Scale bar = 2 µm. (C) Mouse primary astrocytes were transfected with 3xflag-LRRK2 and GFP-JIP4. Cells were treated with LLOME (6 h) and fixed. After fixation, cells were stained for GFP, tyrosinated α-tubulin, and acetylated α-tubulin. (D and E) Time-lapse experiment of U2OS cells transfected with 3xflag-LRRK2, mNeonGreen-JIP4, and TagRFP-T-A1aY1 (tyrosinated microtubules) and treated with LLOME for 2 h before imaging. (F) The enzyme TTL is responsible for adding the tyrosine residue into the C-terminal tail of α-tubulin. (G) U2OS cells were incubated with a nontargeting control (NTC) or TTL siRNA and transfected 24 h later with 3xflag-LRRK2 and mNeonGreen-JIP4. Cells were treated with LLOME (2 h) and imaged live. The #JIP4+ tubules/JIP4+lysosomes ratio was quantified. (H) Graph depicts the ratio in the NTC and siTTL groups. Unpaired t test with Welch’s correction was applied. Data are mean ± SEM (P < 0.0001, n = 27–29 cells). Magenta arrowheads show colocalization between LYTL tubules and only tyrosinated microtubules (filled) and tyrosinated + acetylated microtubules (empty). White arrowheads in A show the length of a LYTL tubule; in D, show a budding tubule elongating on a tyrosinated microtubule; and in E, a fissioned tubule traveling to a tyrosinated microtubule. Yellow arrowheads indicate JIP4-positive tubules. (I) U2OS cells were treated with NTC, siJIP4, and siRILPL1 siRNA for 72 h and lysed. Lysates were blotted against anti-tyrosinated α-tubulin, total α-tubulin, JIP4, and RILPL1 antibodies. (J) Histograms showing tyrosinated α-tubulin, JIP4, and RILPL1 protein levels, normalized to total α-tubulin. Data are mean ± SEM from three independent replicates. One-way ANOVA with Dunnett’s post hoc test. Scale bar (C) = 10 µm; (A and D–G) = 2 µm. Insets = 2 µm. Source data are available for this figure: SourceData F8.
Figure 8.

LYTL tubules elongate through tyrosinated microtubules. (A) U2OS cells were transfected with 3xflag-LRRK2 and GFP-JIP4. Cells were treated with LLOME (2 h) and fixed. After fixation, cells were stained for GFP, tyrosinated α-tubulin, and acetylated α-tubulin. (B) Box plot showing the LYTL tubule contact fraction with the different α-tubulin PTMs. Unpaired t test with Welch’s correction was applied (P < 0.0001, n = 49 tubules). Arrowheads show lysosomal tubules. Scale bar = 2 µm. (C) Mouse primary astrocytes were transfected with 3xflag-LRRK2 and GFP-JIP4. Cells were treated with LLOME (6 h) and fixed. After fixation, cells were stained for GFP, tyrosinated α-tubulin, and acetylated α-tubulin. (D and E) Time-lapse experiment of U2OS cells transfected with 3xflag-LRRK2, mNeonGreen-JIP4, and TagRFP-T-A1aY1 (tyrosinated microtubules) and treated with LLOME for 2 h before imaging. (F) The enzyme TTL is responsible for adding the tyrosine residue into the C-terminal tail of α-tubulin. (G) U2OS cells were incubated with a nontargeting control (NTC) or TTL siRNA and transfected 24 h later with 3xflag-LRRK2 and mNeonGreen-JIP4. Cells were treated with LLOME (2 h) and imaged live. The #JIP4+ tubules/JIP4+lysosomes ratio was quantified. (H) Graph depicts the ratio in the NTC and siTTL groups. Unpaired t test with Welch’s correction was applied. Data are mean ± SEM (P < 0.0001, n = 27–29 cells). Magenta arrowheads show colocalization between LYTL tubules and only tyrosinated microtubules (filled) and tyrosinated + acetylated microtubules (empty). White arrowheads in A show the length of a LYTL tubule; in D, show a budding tubule elongating on a tyrosinated microtubule; and in E, a fissioned tubule traveling to a tyrosinated microtubule. Yellow arrowheads indicate JIP4-positive tubules. (I) U2OS cells were treated with NTC, siJIP4, and siRILPL1 siRNA for 72 h and lysed. Lysates were blotted against anti-tyrosinated α-tubulin, total α-tubulin, JIP4, and RILPL1 antibodies. (J) Histograms showing tyrosinated α-tubulin, JIP4, and RILPL1 protein levels, normalized to total α-tubulin. Data are mean ± SEM from three independent replicates. One-way ANOVA with Dunnett’s post hoc test. Scale bar (C) = 10 µm; (A and D–G) = 2 µm. Insets = 2 µm. Source data are available for this figure: SourceData F8.

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