The ER is present at Lamp1 tubule fission sites marked by Arf1 positive vesicles and PI4KIIIβ positive vesicles are found at endolysosomal and phagolysosomal tubule fission sites. (A) Time-lapse imaging of a COS7 cell expressing the ER marker GFP-KDEL, Arf1-BFP, and Lamp1-mCherry incubated for 8 h in HBSS media that shows the concomitant presence of the ER and of an Arf1 positive vesicle to a Lamp1 positive tubule fission event. Quantification of such event is shown in the table. n = 38 events from 19 cells. Yellow arrow indicates fission. Scale bar: 2 µm. (B) Representative time-lapse images showing the absence of ub-GFP-SKL (Peroxisome) at a Lamp1 positive tubule fission site in MEFs starved (HBSS) for 8 h and quantification of percentage of fission events marked by ub-GFP-SKL. Negative control analysis was performed with the Lamp1-mCherry signal rotated by 90°. n = 49 events from 22 cells. Yellow arrows indicate fission. Scale bar: 1 µm. (C and D) Representative time-lapse imaging showing a GFP-PI4KIIIβ positive vesicle marking fission site of a tubule from an (C) endolysosome in a MEF cell. Endolysosomes were formed by 24 h incubation with sucrose and their tubulation and fission was observed after 1 h of treatment with invertase (0.5 mg/ml). Endolysosomes were defined as positive for Lamp1 and overnight chased fluorescent 10 kD Dextran; (D) Phagolysosome in RAW264.7 cells phagocytosing SRBCs. Yellow arrows indicate fission. Scale bar: 1 µm.