Abstract
Actin regulation is required for membrane activities that drive cell adhesion and migration. The Rho GTPase family plays critical roles in actin and membrane dynamics; however, the roles of the Rho GTPase family are not limited to cell adhesion and migration. Using micron-sized obstacles to induce the formation of self-contacts in epithelial cells, we previously showed that self-adhesion is distinct from cell-to-cell adhesion in that self-contacts are eliminated by membrane fusion. In the current study, we identified Rho GTPases, RhoA, Rac1, and Cdc42, as potential upstream regulators of membrane fusion. The RhoA downstream effector myosin II is required for fusion as the expression of mutant myosin light chain reduced membrane fusion. Furthermore, an inhibitor of the Arp2/3 complex, a downstream effector of Rac1 and Cdc42, also reduced self-contact-induced membrane fusion. At self-contacts, while the concentration of E-cadherin diminished, the intensity of GFP-tagged Arp3 rapidly fluctuated then decreased and stabilized after membrane fusion. Taken together, these data suggest that the Arp2/3 complex-mediated actin polymerization brings two opposing membranes into close apposition by possibly excluding E-cadherin from contact sites, thus promoting membrane fusion at self-contacts.
Highlights
Normal epithelial cells remove self-junctions through membrane fusion
A Micro-fabricated Pillar Array Promotes Self-contact-induced Membrane Fusion—To promote the formation of cell self-contacts, MDCK epithelial cells were plated on a microfabricated pillar array with individual pillars measuring 20 m high and 5 m in diameter and arranged in a series of hexagons with an 18-m pitch along each hexagon side (Fig. 1A) [4]
We previously showed that ROCK inhibitors reduce self-contact-induced membrane fusion, presumably by reducing myosin II activity [4]
Summary
Results: Self-contact induced membrane fusion is regulated by Rho GTPases via myosin II and the Arp2/3 complex. Conclusion: Self-contact-induced membrane fusion requires Arp2/3 activity to bring membranes into close apposition to fuse. Actin regulation is required for membrane activities that drive cell adhesion and migration. An inhibitor of the Arp2/3 complex, a downstream effector of Rac and Cdc, reduced self-contact-induced membrane fusion. In Caenorhabditis elegans, a fusion process, termed auto-fusion, fuses two opposing plasma membrane regions of a single cell to remove self-contacts [1,2,3]. Some progress has been made in understanding cell-to-cell fusion, the molecular components and regulation of self-contact-induced membrane fusion remain unclear. Actin polymerization is required for cell adhesion and cell migration, very little is known about actin dynamics at self-contact-induced membrane fusion. Our results demonstrate a unique role of the Arp2/3 complex-induced actin assembly in the organization of E-cadherin at self-contacts
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