A Rac1-FMNL2 signaling module affects cell-cell contact formation independent of Cdc42 and membrane protrusions.

Hanna Grobe,Robert Grosse,Andrea Wüstenhagen,Katharina Grikscheit,Christian Baarlink,Yulia Komarova

doi:10.1371/journal.pone.0194716

Abstract

De novo formation of epithelial cell-cell contacts relies on actin-based protrusions as well as tightly controlled turnover of junctional actin once cells encounter each other and adhesion complexes assemble. The specific contributions of individual actin regulators on either protrusion formation or junctional actin turnover remain largely unexplored. Based on our previous findings of Formin-like 2 (FMNL2)-mediated control of junctional actin dynamics, we investigated its potential role in membrane protrusions and impact on newly forming epithelial contacts. CRISPR/Cas9-mediated loss of FMNL2 in human MCF10A cells combined with optogenetic control of Rac1 activity confirmed its critical function in the establishment of intercellular contacts. While lamellipodial protrusion rates remained unaffected, FMNL2 knockout cells were characterized by impaired filopodia formation similar to depletion of the Rho GTPase Cdc42. Silencing of Cdc42, however, failed to affect FMNL2-mediated contact formation. Hence, we propose a cell-cell contact-specific and Rac1-mediated function of FMNL2 entirely independent of Cdc42. Consistent with this, direct visualizations of native epithelial junction formation revealed a striking and specifically Rac1- and not Cdc42-dependent recruitment of FMNL2 to newly forming junctions as well as established cell-cell contacts within epithelial sheets.

Highlights

Protrusive membrane structures such as filopodia or lamellipodia are important mediators of cellular motility and are critically involved in cell migration, tumor cell invasion or epithelial differentiation [1, 2]
Using human breast epithelial MCF10A cells, we recently identified a critical role of Formin-like 2 (FMNL2) in the assembly of junctional actin at newly forming cell-cell contacts in a 3D matrix [21]
Our results indicate that functions of FMNL2 in the establishment and maintenance of cell-cell contacts cannot be attributed to defects in the formation of actin-based membrane protrusions, which were recently described for FMNL2 in migrating B16-F1 melanoma cells [23]

Summary

Introduction

Protrusive membrane structures such as filopodia or lamellipodia are important mediators of cellular motility and are critically involved in cell migration, tumor cell invasion or epithelial differentiation [1, 2]. Previous studies using MDCK cells revealed the extension of Rac1-dependent lamellipodia in cells facing each other, which upon encounter initiate novel cell-cell contact sites characterized by subsequent lateral expansion and accumulation of the adhesion receptor E-Cadherin [5, 6] This reorganization of cell-cell adhesions was shown to coincide with a substantial rearrangement of the actin cytoskeleton at newly forming junctions [6]. Other studies highlighted the importance of filopodia in the establishment of cell-cell contacts showing that primary mouse keratinocytes extend filopodial structures enriched for E-cadherin at their tips to contact neighboring cells These filopodia generate so-called “adhesion zippers” which eventually develop further into mature intercellular adhesions [7]. Both lamellipodia and filopodia could be observed at the leading edge during dorsal closure in Drosophila [8] allowing to speculate on a potential interplay of these distinct cellular protrusions during the process of epithelialization

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