Hayley L. Cox

Myosin II isoforms identify distinct functional modules that support integrity of the epithelial zonula adherens

Classic cadherin receptors cooperate with regulators of the actin cytoskeleton to control tissue organization in health and disease. At the apical junctions of epithelial cells, the cadherin ring of the zonula adherens (ZA) couples with a contiguous ring of actin filaments to support morphogenetic processes such as tissue integration and cellular morphology. However, the molecular mechanisms that coordinate adhesion and cytoskeleton at these junctions are poorly understood. Previously we identified non-muscle myosin II as a target of Rho signalling that supports cadherin junctions in mammalian epithelial cells. Myosin II has various cellular functions, which are increasingly attributable to the specific biophysical properties and regulation of its different isoforms. Here we report that myosin II isoforms have distinct and necessary roles at cadherin junctions. Although two of the three mammalian myosin II isoforms are found at the ZA, their localization is regulated by different upstream signalling pathways. Junctional localization of myosin IIA required E-cadherin adhesion, Rho/ROCK and myosin light-chain kinase, whereas junctional myosin IIB depended on Rap1. Further, these myosin II isoforms support E-cadherin junction integrity by different mechanisms. Myosin IIA RNA-mediated interference (RNAi) selectively perturbed the accumulation of E-cadherin in the apical ZA, decreased cadherin homophilic adhesion and disrupted cadherin clustering. In contrast, myosin IIB RNAi decreased filament content, altered dynamics, and increased the lateral movement of the perijunctional actin ring. Myosin IIA and IIB therefore identify two distinct functional modules, with different upstream signals that control junctional localization, and distinct functional effects. We propose that these two isoform-based modules cooperate to coordinate adhesion receptor and F-actin organization to form apical cadherin junctions.

Neogenin recruitment of the WAVE regulatory complex maintains adherens junction stability and tension

To maintain tissue integrity during epithelial morphogenesis, adherens junctions (AJs) must resist the mechanical stresses exerted by dynamic tissue movements. Junctional stability is dependent on actomyosin contractility within the actin ring. Here we describe a novel function for the axon guidance receptor, Neogenin, as a key component of the actin nucleation machinery governing junctional stability. Loss of Neogenin perturbs AJs and attenuates junctional tension. Neogenin promotes actin nucleation at AJs by recruiting the Wave regulatory complex (WRC) and Arp2/3. A direct interaction between the Neogenin WIRS domain and the WRC is crucial for the spatially restricted recruitment of the WRC to the junction. Thus, we provide the first example of a functional WIRS–WRC interaction in epithelia. We further show that Neogenin regulates cadherin recycling at the AJ. In summary, we identify Neogenin as a pivotal component of the AJ, where it influences both cadherin dynamics and junctional tension.

Supplementary Figure 1, (d) of the paper Feedback regulation through myosin II confers robustness on RhoA signalling at E-cadherin junctions

Spinning disk confocal images were taken on MCF-7 cells expressing the F-actin marker RFP-UtrCH and GFP-tagged AHPH, AHPH+C3-T (C3 Transferase ,1 mg/ml; 3 hours), D AH, AHPHC2mut, AHPHA740D, AHPHE758K and AHPHA740D E758K. Images were acquired as Z-stacks and the plane corresponding to the apical zonula adherens is shown in the figure.

Supplementary Figure 1, (f) of the paper Feedback regulation through myosin II confers robustness on RhoA signalling at E-cadherin junctions

Spinning disk confocal images (Z-stacks) of wild type and Ect-2 knockdown MCF-7 cells expressing the GTP-RhoA biosensor GFP-AHPH.

Supplementary Figure 1, (h) of the paper Feedback regulation through myosin II confers robustness on RhoA signalling at E-cadherin junctions.

Images showing western blot analysis of E-cadherin and beta-tubulin expression in wild type (ctrl) and E-cadherin knockdown (E-cad KD) MCF-7 cells.

Supplementary Figure 1, (l) of the paper Feedback regulation through myosin II confers robustness on RhoA signalling at E-cadherin junctions.

Images show immunoblot analysis of NMIIA and beta-tubulin expression in cell lysates from Control (Ctrl) and NMIIA knockdown MCF-7 cells.

Supplementary Figure 2, (m) of the paper Feedback regulation through myosin II confers robustness on RhoA signalling at E-cadherin junctions.

Confocal images showing the localization of p190B RhoGAP at the apical adherens junctions of MCF-7 cells treated with DMSO or treated with blebbistatin (BLB) (100mM, 2 hours) alone or in combination with Src inhibitors SU6656 (10 mM) or PP2 (10 mM)) for 4 hours. Cells were fixed with methanol and stained for p190B GAP.

Supplementary Figure 1, (j) of the paper Feedback regulation through myosin II confers robustness on RhoA signalling at E-cadherin junctions.

Confocal images (Z-stacks) showing NMIIA and E-cadherin localization in MCF-7 cells treated with vehicle (DMSO) or blebbistatin (BLB, 100mM, 2 hours). The images shown correspond to the plane where the apical zonula adherens is located.