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Fig. 3. Internalised Alexa⁵⁶⁸-Tf and TxR-EGF are transported through the GFP-PIKfyve-labelled early endosome. (A-D) HeLa cells were transiently transfected with pEGFPC1-PIKfyve for 22 hours and serum-starved for 3 hours prior to live cell imaging. Cells were incubated with either (A,B) 200 ng/ml TxR-EGF or (C,D) 20 µg/ml Alexa⁵⁶⁸-Tf. TxR-EGF reached the GFP-PIKfyve-positive compartment after 15 minutes, and is not transported along GFP-PIKfyve-positive tubules but remains in the endosomal body. Series of frames (B) depicting a GFP-PIKfyve-positive tubule (arrow) exiting an endosome positive for GFP-PIKfyve and TxR-EGF (arrowhead); data are also available in supplementary material as Movie 4. (C,D) Alexa⁵⁶⁸-Tf also enters the GFP-PIKfyve-positive compartment after 5 minutes, and was observed to exit the GFP-PIKfyve-positive early endosome through tubular elements. Series of frames (D) depicting Alexa⁵⁶⁸-transferrin-positive tubules exiting the GFP-PIKfyve early endsome; data are also available in supplementary material as Movie 5. Similar data were obtained in more than ten imaged cells for each case. Boxed areas in A and C indicate regions enlarged in the corresponding insets. Bars, 10 µm.

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