In Vivo Dynamics of Rac-Membrane Interactions -- Moissoglu et al. 17 (6): 2770 Data Supplement - Supplemental Material -- Molecular Biology of the Cell

In Vivo Dynamics of Rac-Membrane Interactions
Mol. Biol. Cell Moissoglu et al. 17: 2770

Supplemental Material

This article contains the following supporting material:

Supplemental Text -
Figure 1 - Sensitivity of the method to parameter changes in the presence of noise. The fitting procedure as described in the text was applied to “data” generated in the presence of noise by Eqs (S5) and (S6) for representative parameter sets. Measurement error mimicked by the superimposed noise is 15%.
Figure 2 - Validation of the compartmental approximation. “Data” are generated from realistic simulations performed on 3D cell geometry taken from experimental images. The cell geometry used in the simulations has an overall surface-to-volume ratio of 0.524 μm^-1 whereas the surface-to-volume ratio of the unbleached region is 0.828 μm^-1, hence r values are greater than the corresponding r₀ ‘s. The diffusion coefficients in the cytosol and in the membrane are 20 μm²/sec and 0.25 μm²/sec, respectively, and the corresponding κ _diff is 0.0352 s^-1.
Figure 3 - Geometry of protrusions. Representative cells expressing the indicated constructs were imaged under the same experimental conditions used for photobleaching. Arrowheads point to typical protrusive areas. Z profiles (lower panel) were acquired across a vertical plane defined by the white lines. The optical slice is 1ìm and the spatial interval is 1 ìm. Scale bar is 20 ìm.
Figure 4 - Effect of Rac regulators on Rac activation. Lysates from NIH3T3 cells transfected with the indicated constructs were incubated with GST-PBD and bound, active GFP-Rac was detected by immunoblotting. Lysates were also probed for total GFP-Rac and RhoGDI. Numbers indicate levels of active Rac normalized to total Rac. Amount of active Rac in RhoGDI RNAi-treated cells was expressed relative to that in control RNAi-treated cells (arbitrarily set to 1). Amount of active Rac in all other conditions was expressed relative to that in cells transfected with GFP-wtRac (also set to 1). Vertical lines indicate that samples were run in the same SDS-PAGE gel. Data are representative of three independent experiments.