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Fig. 5. Macropinosome-derived SNX5-positive tubules traffic to smaller SNX5-positive early endosomes. (A) HEK-GFP-SNX5 cell monolayers were treated and analysed using the same conditions as in Fig. 1A. The interval between images captured was 1 second for a total period of 3 minutes. Tif stacks were pseudocoloured to highlight variations in fluorescent intensity (black/blue, low intensity; white/yellow, high intensity). Arrows highlight the emergence and movement of a discrete SNX5-positive patch across the cytoplasmic face of the macropinosome. Asterisks indicate point of termination of tubules. Bar, 5 µm. (B) To visualise the kinetics of the structures presented in A, the movie was separated into individual frames using Image J v1.31 and associated Macro programs and stacked in the Z axis using software specifically developed for the task. The frames were arranged with the first frame in the foreground with each successive frame behind the first. Numbers in the image indicate frame numbers captured at 1-second intervals. Presented is a single view from Movie 6 in supplementary material. Red arrows highlight the path of an SNX5-positive patch across the cytoplasmic face of the macropinosome. Green arrows highlight the termination point of the tubules and asterisks indicate the acceptor endosome. (C) Fluorescent compaction was used to monitor the fusion event between the tubule and the acceptor endosome (asterisk). The relative fluorescent intensities of the tubule (
) and the acceptor endosome pre-fusion (ß) and post-fusion (
) were measured using Image Jv1.31.
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