Supplementary Figure 2: Myosin II supports a stable Rho zone at the ZA.
From: Feedback regulation through myosin II confers robustness on RhoA signalling at E-cadherin junctions
(a) RhoA, E-cadherin and ZO-1 localization in TCA-fixed MCF-7 cells. (b) Domain organization of human full-length Anillin (FL-Anillin) and the respective constructs used in this study. The residues are numbered according to the Uniprot Id Q9NQW6. FBD (Formin binding domain); MBD (Myosin binding domain); ABD (Actin binding domain); AHD (Anillin homology domain), which is composed of RBD (Rho binding domain) and C2 domain. The AHPH (location biosensor) is composed of AHD and PH (Pleckstrin homology domain). (c) Representative images and fluorescence line-scan analysis of GFP-AHPH and endogenous RhoA at the ZA of MCF-7 cells. White line (merged image) represents the position where fluorescence intensity was measured. (d) Localization of RFP-UtrCH and GFP-tagged AHPH, AHPH + C3-T (C3 Transferase, 1 μg ml−1; 3 h), Δ AH, AHPHC2mut, AHPHA740D, AHPHE758K and AHPHA740D E758K in MCF-7 cells. (e) Lysates from control or ECT2 knockdown MCF-7 cells immunoblotted for ECT2 and GAPDH (loading control). (f) GTP-RhoA distribution in control or ECT2 knockdown MCF-7 cells. (g) Control (Ctrl) or E-cadherin RNAi (E-cad KD) MCF-7 cells fixed and immunostained for GFP (GFP AHPH), E-cadherin (E-cad) and ZO-1. (h) Lysates from control (Ctrl) or E-cadherin RNAi (E-cad KD) MCF-7 cells immunoblotted for E-cadherin and β-tubulin (loading control). (i) GTP-RhoA pull-down from control (Ctrl) or E-cadherin RNAi (E-cad KD) or NMIIA RNAi (NMIIA KD) MCF-7 cells using RBD domain of Rhotekin and immunoblotted for RhoA and β-tubulin. (j) NMIIA and E-cadherin localization in MCF-7 cells treated with vehicle (DMSO) or blebbistatin (BLB, 100 μM, 2 h). (k) RhoA localization in Caco2 and MDCK treated with DMSO or blebbistatin (100 μM, 2 h). (l,m) Lysates from Control (Ctrl), NMIIA knockdown or NMIIB knockdown MCF-7 cells immunoblotted for NMIIA, NMIIB and β-tubulin (loading control). Scale bars, 10 μm. DIC images corresponding to Supplementary Fig. 1d are shown in Supplementary Fig. 7.
