Mammalian diaphanous-related formin Dia1 controls the organization of E-cadherin-mediated cell-cell junctions

Abstract

The formin-homology protein Dia1 is a target of RhoA and a potent activator of nucleation and elongation of actin filaments. Here, we demonstrate that short-hairpin (sh) RNA-mediated downregulation of Dia1 in human MCF7 epithelial cells disrupts adherens junctions, as manifested by the significantly decreased localization of E-cadherin and associated proteins to cell-cell contacts. Expression of mouse Dia1, which is insensitive to the human Dia1-specific shRNA, rescued the junctional integrity. Coexpression of GFP-tagged Dia1 and a constitutively active RhoA mutant, RhoA-V14, resulted in localization of the exogenous GFP-Dia1 to the cell-cell junctions. This localization was accompanied by a strong increase in the width of the adhesion zone and augmentation of the actin, E-cadherin and β-catenin content of the junctions. A constitutively active Dia1 mutant lacking the N-terminal portion was unable to localize to cell-cell junctions and did not show any junction-strengthening effect. The adherens junction enhancement induced by Dia1 and active RhoA did not require microtubules, but depended on the activity of myosin II. Inhibition of myosin II activity abolished the Dia1-mediated reinforcement of cell-cell junctions and instead induced the formation of numerous actin-rich filopodia at the contact zone. Thus, Dia1 localizes to and controls cadherin-mediated junctions in a RhoA-dependent manner.

Changes in E-cadherin rigidity sensing regulate cell adhesion

“…In particular, formin activity, which polymerizes long, parallel actin bundles found in filopodia (46, 47), appeared to be essential for cell adhesion and dynamic filopodialike protrusions on a 30-kPa Ecad-Fc PA gel, but not on a 60-kPa Ecad-Fc PA gel. These results are consistent with previous studies that demonstrated a role for the diaphanous formin mDia1 (11,65,66) and formin-like proteins FMNL2 and FMNL3 (12,13) in the recruitment of actin and E-cadherin and the formation of filopodia at initial cell-cell contacts. At present, we do not know which formin(s) is (are) regulated by the stiffness of E-cadherin interactions.…”

“…Adhesion is initiated when filopodia from opposing cells come into contact with one another (7,8), and this process is regulated by Cdc42 activity (9, 10) and formin-dependent actin polymerization (11)(12)(13)(14). Intermediate stages of cell-cell contact formation involve lateral expansion of the contact by Rac1-induced and Arp2/3-dependent lamellipodial activity (15, 16).…”

Phosphorylation-mediated 14-3-3 Protein Binding Regulates the Function of the Rho-specific Guanine Nucleotide Exchange Factor (RhoGEF) Syx*

“…Dia1, a RhoA effector that is proposed to regulate cell junction stability (27,28), belongs to the formin family and can remodel the actin cytoskeleton (29). We previously identified Dia1 as the key downstream effector of Syx in regulating junction integrity (5).…”

Rho GEFs in endothelial junctions

“…16,29 In epithelial cells, expression of Dia1 reinforces the adhesion zone, whereas Dia1 knockdown results in decreased junctional E-cadherin. 30 Formin-1, a Dia1 related protein, is required at the intercellular junctions to direct actin cable formation and to support cell-cell contact in mouse keratinocytes in vivo. 31 Furthermore, Dia1 knockout mice exhibit severe defects in neuroepithelial junction formation and polarization, supporting its indispensable role in regulating cell-cell adhesion and monolayer integrity.…”

“…Dia1 is proposed to have a housekeeping function in stabilizing cell junctions. 30 Even though the mechanism of Dia1 recruitment to cell-cell contacts is unclear, its basal activity is likely maintained by upstream RhoA signaling at the junctions. ROCK binds several junctional proteins such as p120, Shroom 3, and p114RhoGEF, 43,50,51 suggesting that its recruitment to the cell membrane is highly regulated and that its activation is dependent on its immediate associated protein complex.…”