JCS02384 Supplementary Material

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• Supplemental Figure 1 (Adobe PDF) -

  Fig. S1. Activated forms of RhoB and RhoA induce opposite changes in cell morphology. Cells were microinjected with myc-RhoB^G14V or myc-RhoA^G14V cDNAs (injected cells are marked with an asterisk). Myc and F-actin were subsequently immunodetected. Cells expressing RhoB^G14V showed and elongated shape and contained thin stress fibres running between the two cell poles (a). RhoA^G14V induced stress fibre formation but induced cell contraction (b). Bars: 10 mm.

• Supplemental Figure 2 (Adobe PDF) -

  Fig. S2. Myosin IIA co-localises with RhoB^G14V-positive vesicles. Cells were microinjected with myc-RhoB^G14V cDNA. Myc (a) and myosin IIA (b) were subsequently immunodetected. In control non-injected cells, mysosin IIA distributes evenly along stress fibres (b). However, myosin IIA concentrates at sites where RhoB-positive vesicles are located in RhoB^G14V expressing cells (b). Bars: 10 mm.

• Supplemental Figure 3 (Adobe PDF) -

  Fig. S3. Intracellular distribution of RhoBwt and RhoB^T19N. Cells were microinjected with myc-RhoBwt or myc-RhoB^T19N (GDP-bound mutant) cDNAs and subsequently labelled for myc. RhoBwt mainly localised to juxtanuclear vesicles but it was also present at the plasma membrane (a). RhoB^T19N was largely found in the cytoplasm and a small fraction localised to the plasma membrane (b). Bars: 10  mm.

• Supplemental Figure 4 (Adobe PDF) -

  Fig. S4. EGF-receptor degradation in HeLa cells. HeLa cells were transfected with RhoB^G14V, Dia1-N1 or Dia1-DN3 alone or with RhoB^G14V together with Dia1-N1. Lysates of the transfected cells were analysed by western blot with an antibody specific for the EGF-receptor. Cells transfected with RhoB^G14V or Dia1-DN3 accumulated non-degraded receptor. Co-transfection of Dia1-N1 and RhoB^G14V reversed the accumulation of non-degraded receptor. RhoGDI was detected to control for equal protein loading.

• Movie 1 -

  Movie 1. Activated RhoB impairs endosome motility. Cells stably expressing GFP-FYVE were microinjected with myc-RhoBG14V cDNA and dynamics of endosomes were followed by time-lapse fluorescence microscopy. Images were recorded every 15 seconds for a period of time of approximately 9 minutes. Non-injected control cells show moving vesicles, particularly at the cell periphery. In contrast, vesicles in the RhoB^G14V expressing cell (marked in Fig. 4) do not show directed movement.