Additional information regarding α-tubulin PTM contacts with LYTL tubules. (A) U2OS cells were transfected with 3xflag-LRRK2, mNeonGreen-JIP4, and TUBB5-HaloTag. Cells were treated with LLOME (2 h) and observed under a confocal microscope. Time-lapse shows LYTL tubules associated with microtubules (arrowheads) and even a tubule budding alongside a microtubule (outlined arrowhead). (B) U2OS cells were fixed and stained for different α-tubulin PTMs: tyrosinated, acetylated, and detyrosinated. (C) Mouse primary astrocytes were transfected with 3xflag-LRRK2 and pDEST53-JIP4 for 48 h. Cells were treated with LLOME (6 h) and fixed. After fixation, cells were stained for GFP and tyrosinated α-tubulin. Yellow arrowheads indicate contact between LYTL tubules and tyrosinated microtubules. (D) Mouse primary astrocytes were transfected with HaloTag-LRRK2, mNeonGreen-JIP4, and TagRFP-T-A1aY1 (tyrosinated microtubules) for 48 h. Cells were treated with LLOME (6 h) and imaged live. Arrowheads indicate contact between LYTL tubules and tyrosinated microtubules. (E) U2OS cells were transfected with 3xflag-LRRK2, HaloTag-JIP4, and EMTB-mNeonGreen. Cells were treated with LLOME (2 h) and analyzed under a confocal microscope. Time-lapse shows a sorted tubule moving along microtubules on a single z plane. Volume view with depth code shows the tubule attached to a lysosome (t = 66.5 s) and the moment of fission (t = 72.5 s). (F) U2OS cells were transfected with 3xflag-LRRK2, HaloTag-JIP4, and EMTB-mNeonGreen. Cells were treated with LLOME (2 h) and with DMSO or nocodazole (10 µM, 30 min) and analyzed under a confocal microscope. Time-lapse shows the effect of nocodazole on the microtubule network and the dynamics of the LYTL-sorted materials. Scale bar (A and C–E) = 2 µm; (B and F) = 10 µm.
Figure S4.

Additional information regarding α-tubulin PTM contacts with LYTL tubules. (A) U2OS cells were transfected with 3xflag-LRRK2, mNeonGreen-JIP4, and TUBB5-HaloTag. Cells were treated with LLOME (2 h) and observed under a confocal microscope. Time-lapse shows LYTL tubules associated with microtubules (arrowheads) and even a tubule budding alongside a microtubule (outlined arrowhead). (B) U2OS cells were fixed and stained for different α-tubulin PTMs: tyrosinated, acetylated, and detyrosinated. (C) Mouse primary astrocytes were transfected with 3xflag-LRRK2 and pDEST53-JIP4 for 48 h. Cells were treated with LLOME (6 h) and fixed. After fixation, cells were stained for GFP and tyrosinated α-tubulin. Yellow arrowheads indicate contact between LYTL tubules and tyrosinated microtubules. (D) Mouse primary astrocytes were transfected with HaloTag-LRRK2, mNeonGreen-JIP4, and TagRFP-T-A1aY1 (tyrosinated microtubules) for 48 h. Cells were treated with LLOME (6 h) and imaged live. Arrowheads indicate contact between LYTL tubules and tyrosinated microtubules. (E) U2OS cells were transfected with 3xflag-LRRK2, HaloTag-JIP4, and EMTB-mNeonGreen. Cells were treated with LLOME (2 h) and analyzed under a confocal microscope. Time-lapse shows a sorted tubule moving along microtubules on a single z plane. Volume view with depth code shows the tubule attached to a lysosome (t = 66.5 s) and the moment of fission (t = 72.5 s). (F) U2OS cells were transfected with 3xflag-LRRK2, HaloTag-JIP4, and EMTB-mNeonGreen. Cells were treated with LLOME (2 h) and with DMSO or nocodazole (10 µM, 30 min) and analyzed under a confocal microscope. Time-lapse shows the effect of nocodazole on the microtubule network and the dynamics of the LYTL-sorted materials. Scale bar (A and C–E) = 2 µm; (B and F) = 10 µm.

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