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Volume 7 issue 4 April 2006
Rab6A and Rab6A' GTPases play non-overlapping roles in membrane trafficking
Elaine Del Nery, Stéphanie Miserey-Lenkei, Thomas Falguières, Clément Nizak, Ludger Johannes, Franck Perez and Bruno Goud
Online Supplemental Material: Legends to Figures
Figure S1: (A): Endogenous and overexpressed Rab6•GTP dynamics in vivo. HeLa cells expressing AA2-YFP (recombinant antibody AA2 specifically interacts with Rab6•GTP) and CFP-Rab6A' were time-lapse imaged on a confocal microscope. CFP-Rab6A' is present on the Golgi and the endoplasmic reticulum, and on 5-10 µm long membrane tubules moving from the Golgi towards the periphery at ER entry points and sometimes back to the Golgi. Rab6 is detected in its GTP-bound form by AA2-YFP on the Golgi and all motile structures, but not on the endoplasmic reticulum where CFP-Rab6A' is therefore likely in its GDP-bound form. Endogenous Rab6•GTP shows similar dynamics and localization but endogenous transport intermediates are 1-2 µm long tubulo-vesicular structures, and thus overexpression of Rab6 may modulate the geometry of transport intermediates by tuning the traffic rate. Similar phenotype has been previously described for Rab6A transport intermediates (29).
(B) GFP-Rab6A depletion was achieved by transfecting cells with Rab6AsiRNA or Rab6A/A' siRNA, both targeting the Rab6A mRNA. Silent mutations impairing Rab6A' siRNA recognition prevent silencing of GFP-Rab6A in Rab6A' siRNA treated cells. The level of β-tubulin is not affected after siRNAs treatment.
Figure S1 (.eps)
Online Supplemental Material: Legends to videos
Video 1: CFP-Rab6A' vs. AA2-YFP.
Movie corresponding to supplementary Fig. S1A. HeLa cells were co-transfected with CFP- Rab6A' and the anti- Rab6•GTP fluorescent intrabody AA2-YFP. The dynamics of overexpressed CFP- Rab6A' are very similar to those reported for overexpressed fluorescent Rab6 chimeras (15, 29) : 5-20 µm long tubules emanate from the Golgi and move towards the periphery. AA2 detects Rab6A' in its GTP-bound form on the Golgi and all along moving structures (but not on the endoplasmic reticulum, where Rab6A' is present). This suggests that the level of Rab6A' expression modulates the geometry of transport intermediates in a very similar manner to the one we previously described for Rab6A'.
Figure S1 Video1 (.mov)
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