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. 2023 Jun 8;222(9):e202205128. doi: 10.1083/jcb.202205128

Figure 2.

Figure 2.

Arf1 positive vesicles recruited to Lamp1 positive organelles tubule fission sites are positive for PI4KIIIβ and TGN46 but negative for endosome markers. (A and B) Time-lapse images and quantification of MEFs incubated for 8 h in HBSS media showing GFP-PI4KIIIβ (A) or TGN46-mEmerald (B) positive vesicles at the sites of Lamp1 positive tubule fission. Yellow arrows indicate fission. Scale bars: 2 µm. (C) Representative time-lapse images of a MEF as in A but showing a Lamp1-mCherry tubule fission event marked by a vesicle positive for both Arf1-SNAP and GFP-PI4KIIIβ. Yellow arrow indicates fission. Scale bar: 1 µm. (D–F) Time-lapse images of MEFs expressing Lamp1-mCherry and (D) GFP-Rab5, (E) GFP-EEA1, or (F) CFP-Rab11 incubated for 8 h in HBSS media. Yellow arrows indicate fission. Scale bars: 1 µm. (G) Quantification of vesicles positive for proteins in D–F shows them to be absent at Lamp1 positive tubule fission events. Also shown is the quantification of images in D where lamp1 was rotated by 90°. (H) Representative images of MEFs expressing Arf1-SNAP with GFP-Rab5, GFP-EEA1, CFP-Rab11, or GFP-PI4KIIIβ. Scale bar: 5 µm. (I) Pearson’s coefficient measurement of cells is described in H as indicated. The graph shows the mean ± SEM from three independent experiments. One-way ANOVA with Dunnett’s multiple comparison test. The number of events and cells quantified are shown with each quantification data.