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Fig. 7. The formation of Rab8-specific tubular structures is actin- and RhoA-dependent. HeLa cells were transfected overnight with plasmids containing either pEGFP-RhoA-T19N (A,B) or pEGFP-RhoA-G14V (C,D) at 37°C. The transfectants were then incubated in 0.1 µM cytochalasin D (CD) for 30 minutes at 37°C. The cells were fixed, processed for immunofluorescence and stained to detect endogenous Rab8. RhoA proteins were detected through GFP fluorescence. Note that expression of RhoA-T19N did not inhibit CD induced formation of Rab8-specific tubules, whereas RhoA-G14V did (arrow). (K) Quantification of cells containing Rab8-specific tubular structures after CD-treatment, in the absence or presence of GFP-RhoA-G14V and GFP-RhoA-T19N. CD indicates untransfected cells that obtained cytochalasin D. C indicates cells in the absence of CD. A total of about 50 cells were counted per experimental condition. Values, given as percentage of cells (n=50) exhibiting Rab8-specific tubules in each scoring category, from three separate experiments are shown as the mean ±s.e.m. Formation of GFP-Rab8b-wt (NIH3T3; E,F) and GFP-Rab8-wt (Hela; G-J) tubular structures (arrows) were seen after cytochalasin D treatment (30 minutes) (supplementary material Movies 4-6). GFP-Rab8 and GFP-Rab8b vesicles formed at the cell periphery (arrows) and fused to a tubular network in the cell center (arrowhead). There is also an anterograde transport of small Rab8-specific vesicles to the outermost region of the cell, and into forming and retracting filopodia (arrowhead) (I and J; supplementary material Movie 6). Bars, 20 µM (A-F), 10 µM (G-J).
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