Network interactions allowing myosin II to feedback to RhoA: sequencing, predictive modelling, and experimentation

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8 x supplementary figures; 2 x supplementary table; 3 x videos, supplementary note Supplementary Figure 1 Myosin II supports a stable Rho zone at the ZA Supplementary Figure 2 p190B RhoGAP degrades the junctional Rho zone when NMII is inactivated Supplementary Figure 3 Rnd3 recruits p190B to the ZA when NMII is inhibited Supplementary Figure 4 ROCK-1 phosphorylates Rnd3 to support junctional Rho signaling Supplementary Figure 5 Myosin II scaffolds ROCK-1 at the epithelial Zonula Adherens Supplementary Figure 6 Bistable properties of RhoA localization at cell-cell junctions Supplementary Figure 7 Bright field/DIC images corresponding to Figures 1a, 1f, 2h, 3a, 3b, 5h and supplementary figures 1c, 1e, 4e Supplementary Figure 8 Uncropped western blots Supplementary Table 1 Sequences of siRNA and primers used for the study Supplementary Table 2 Statistical Source data Supplementary Video 1 GFP-AHPH localizes at the zonula adherens of epithelial cells. Z-stacks of GFP-AHPH and RFP-UtrCH acquired by spinning disc confocal microscopy. Supplementary Video 2 GFP-AHPH exhibits stability on the time scale of minutes. Time-lapse imaging of GFP-APHPH (transfected in MCF-7 cells) acquired over a span of 30 minutes. Supplementary Video 3 ROCK-1 inhibition causes accumulation of p190B Rho GAP at the cell-cell junctions. MCF-7 cells were transfected with GFP-p190B RhoGAP and time-lapsed imaging was performed briefly before and after addition of Y-27632 (30 μM) (20-minutes post-treatment). Supplementary Notes include: Experimental foundations of the model. Computational model. Supplementary Note Table 1. Hill (K), maximal association rates (b) and decay (a) constants in the stimulation repression model shown in Scheme 1. Implications of the one dimensional model Effect of removing feedback from ROCK1 to RhoA. Supplementary Note Table 2. Kinetic equations for the reduced subsystem shown in Scheme 2. Effect of removing feedback from NMIIA to ROCK1. Effect of adding negative feedback from Rnd3 to ROCK1. Extension of the stimulation/repression model to 3 dimensions Derivation of the explicit form for the equations used in the spatial model. Supplementary Note Table 3. Kinetic equations for the stimulation repression model in 3 dimensions Supplementary Note Table 4. Steady state cortical concentrations of the different species in the 3 dimensional model Supplementary Note Table 5. Variable transformation between the one dimensional stimulation repression model and its extension in 3 dimensions. Numerical simulations for the 3 dimensional model. Supplementary Note Table 6. Dissociation rates, binding affinities and activation constants used for modeling in three dimensions Supplementary Note Table 7. Initial concentrations and diffusion coefficients of species in the 3 - dimensional modelling. Implications of the model for cortical signaling at the ZA. Supplementary Note Table 6. Dissociation rates, binding affinities and activation constants used for modeling in three dimensions Supplementary Note Table 7. Initial concentrations and diffusion coefficients of species in the 3 - dimensional modelling. Implications of the model for cortical signaling at the ZA.