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Fig. 9. GFP-Rab21 T33N expression inhibits lysosomal delivery of Rh-EGF. HeLa cells transfected with GFP-Rab21 plasmids were incubated for 60 minutes with 150 ng/ml Rh-EGF before washing on ice and incubating (chase) for a further 120 minutes in Rh-EGF-free medium. The cells were fixed and analysed by fluorescence microscopy. Shown is the almost complete lack of Rh-EGF fluorescence in cells expressing moderate levels of GFP-Rab21 wt (A,B) and Rab21 Q78L (E,F). By contrast, undegraded EGF was clearly observed in distinct perinuclear structures close to the nucleus in cells expressing very high levels of GFP-Rab21 wt (C,D). Extensive Rh-EGF fluorescence in small diffuse punctate structures in GFP-Rab21 T33N expressing cells is also visible (G,H). Arrows point to colocalising GFP-Rab21 wt and Rh-EGF. HeLa cells expressing GFP-Rab21 T33N were also fixed and stained for Lamp-1 after this chase period (I-L). None of the undegraded Rh-EGF colocalises with Lamp-1 (arrowheads). Bars, 10 µm.

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