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

Figure 4. Rabankyrin-5 Localises to Macropinosomes in NIH3T3 Cells.

Figure 4

(A) NIH3T3 cells directly fixed and immunostained for endogenous Rabankyrin-5 and EEA1 reveal segregation of Rabankyrin-5 from EEA1-containing structures (arrows) in the cell periphery, while there is colocalisation in the cell centre (arrowheads).

(B) Overexpression of Rabankyrin-5 increases the number of peripheral enlarged structures devoid of EEA1. NIH3T3 cells were infected with recombinant adenovirus for Rabankyrin-5 for 18 h. Dextran (2,5 mg/ml) uptake was performed for 3 min at 37 °C, fixed, and immunostained for the indicated antigens.

(C and D) Formation of enlarged Rabankyrin-5 structures requires PI3-K activity. NIH3T3 cells either (C) DMSO treated or (D) pretreated for 20 min with wortmannin (WM; 100 nM) were incubated for 8 min with 0,5 μg/ml rhodamine-labelled transferrin and 2,5 mg/ml FITC-labelled dextran (MW, 10.000), fixed, processed for immunofluorescence, and analysed by confocal scanning microscopy.

(E) NIH3T3 cells transiently transfected for YFP-Rabankyrin-5 and CFP-actin were imaged using time-lapse video microscopy to visualise the formation of macropinosomes by actin-driven membrane ruffles. Images were taken for the indicated time points. The arrowhead points towards Rabankyrin-5 association to an enlarged vesicle driven by actin dynamics over time. Scale bars represent 10 μm.