Fig. 2. The GFP-PIKfyve-positive early endosome is a highly dynamic tubular-vesicular compartment. (A,B) One HeLa cell expressing low levels of GFP-PIKfyve was imaged at one frame per second for a period of 10 minutes (A). Boxed areas indicate regions of interest detailed in C and D. Subsequently, data from a 100-second period were selected and each individual image was analysed (see Materials and Methods) to obtain a map of spatial temporal behaviour of the PIKfyve-labelled early endosome (B). Coloured asterisks are used to indicate the time at which the chosen vesicle first appeared in the image plane. Subsequent movements of >0.4 µm were followed over time. As indicated in the timescale bar, each colour corresponds to a specific 5-second period during the image window. The resulting 2D image gives an overview of both, the speed and directionality of the PIKfyve-positive punctae. The trafficking map revealed a directional trend, with several PIKfyve-positive vesicles tracking towards the perinuclear and/or TGN region. (C) The progress of one such vesicle is highlighted, with the first frame being denominated time point zero. (D,E) Live cell imaging also revealed that (D) PIKfyve-labelled vesicles (arrows) were able to undergo homotypic fusion (arrowhead), and that (E) the PIKfyve-labelled tubules were able to emminate from the vesicular elements of the PIKfyve-labelled compartment.