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. 2004 Aug 24;2(9):e261. doi: 10.1371/journal.pbio.0020261

Figure 3. Rabankyrin-5 Associates with Two Types of Rab5-Containing Vesicles in A431 Cells, Early Endosomes, and Macropinosomes.

Figure 3

(A) A431 cells stably transfected for GFP-Rab5wt were immunostained for endogenous Rabankyrin-5 and EEA1. While there is perinuclear overlap between Rab5, Rabankyrin-5, and EEA1 in nontransfected cells (arrowheads), some smaller peripheral structures are devoid of EEA1 (arrows).

(B) Overexpression of Rabankyrin-5 in A431 by using a recombinant adenovirus construct of Rabankyrin-5 causes an accumulation of peripheral, enlarged Rab5-positive structures, costained mainly by Rabankyrin-5 (arrows) but not detectable for EEA1.

(C) Rabankyrin-5 localises on EGF-induced and -enriched macropinosomes. Serum-starved A431 cells (16 h) were incubated for 7 min with 100 ng/ml rhodamine-conjugated EGF to induce macropinocytosis and 1 μg/ml Cy5-labelled transferrin. Endogenous Rabankyrin-5 localises to enlarged EGF-containing macropinosomes, indicated by the lack of transferrin labelling (arrows), but also to EGF- and transferrin-containing endosomes (arrowheads).

(D) Rabankyrin-5 structures contain tyrosine-phosphorylated proteins. A431 cells, stably transfected for GFP-Rab5, were stimulated with 50 ng/ml EGF for 7 min and immediately processed for immunofluorescence. Costaining of Rabankyrin-5 and tyrosine-phosphorylated proteins (α-4G10) reveal the localisation of Rabankyrin-5 to plasma membrane ruffles. Scale bars represent 10 μm.