Functional roles of Rho-GEF PLEKHG4B in regulation of actin remodeling and cell-cell junction formation

概要

Cell-cell junctions regulate various structural and functional characteristics of cells, including morphology, polarity, motility, proliferation and differentiation, and thus play essential roles in maintaining correct tissue architecture, development, and homeostasis. Adherens junctions (AJs) are cadherin-based cell-cell junctions, whose formation requires dynamic actin cytoskeletal reorganization. Rho family small GTPases (Rho GTPases) are key molecules for actin cytoskeletal remodeling and they are activated by Rho guanine nucleotide exchange factors (Rho-GEFs). Although previous studies showed that Rho GTPases and several Rho-GEFs are involved in the formation of epithelial cell-cell adhesions, the complexity of the actin cytoskeletal regulation at junctions has been still elusive.

　Considering that genes of Rho-GEFs are identified more than four times as many as those of Rho GTPases in human genome, Rho-GEF genes appear to have diversified for the precise actin cytoskeletal remodeling in various cellular events by spatiotemporal regulation of Rho GTPases. PLEKHG4B (pleckstrin homology domain-containing family G member 4B) is a Dbl-like Rho-GEF, whose structure is closely related to Solo (also known as ARHGEF40) and PLEKHG4 (also known as puratrophin-1). These three Rho-GEFs share a common domain structure with an N-terminal Solo domain, a CRAL/TRIO domain and spectrin repeats in the medial region, and a C-terminal DH-PH domain, except that PLEKHG4 lacks the N-terminal Solo domain. While the understanding of the cellular roles of Solo and PLEKHG4 have been recently advanced, the function of PLEKHG4B remains to be investigated.

　In this study, I first found that overexpression of PLEKHG4B induced F-actin-rich membrane protrusions in epithelial cells through Cdc42 and Rac1 activation. The protrusions preferentially extended toward adjacent cells, indicating that PLEKHG4B-mediated actin remodeling is cell-cell contact-dependent. To investigate which Rho GTPases are the targets of PLEKHG4B, I conducted pulldown assays and revealed that knockdown of PLEKHG4B significantly decreased Cdc42 activity and tended to increase RhoA activity in the cells. I also examined PLEKHG4B knockdown on actin filaments organization and cell-cell adhesions. Control A549 epithelial monolayer cells showed the 'closed junctions' with closely packed actin bundles along the cell-cell contacts, but PLEKHG4B knockdown suppressed closed junction formation and exhibited the 'open junctions' with split actin bundles located away from the cell-cell boundary (Fig. 1). In calcium- switch assays, PLEKHG4B knockdown delayed the conversion of open junctions to closed junctions and -catenin accumulation at cell-cell junctions, suggesting that PLEKHG4B is involved in cell-cell adhesion maturation. Further, I demonstrated that PLEKHG4B knockdown abrogated the reduction in myosin activity normally seen in the later stage of junction formation. The aberrant myosin activation and impairments in closed junction formation in PLEKHG4B-knockdown cells were reverted by ROCK inhibition or LARG and PDZ-RhoGEF knockdown. These results suggest that PLEKHG4B enables actin remodeling during epithelial cell-cell junction maturation, probably by reducing myosin activity in the later stage of junction formation, through suppressing LARG, PDZ-RhoGEF and their downstream RhoA-ROCK pathway.

　To gain insights into the mechanisms governing the localization and function of PLEKHG4B, proteomics analysis was conducted to search for its binding partners (Ohta, master’s thesis, 2020). Among several proteins identified, I focused on annexin A2 (ANXA2), a phospholipid- and actin- binding protein that mediates the organization of membrane microdomains and regulates actin dynamics at specific membrane sites. I showed that ANXA2 binds to PLEKHG4B and is involved in the localization of PLEKHG4B to cell-cell junctions.

　Collectively, these results highlight a crucial role of PLEKHG4B, the Rho-GEF targeting Cdc42, in the reorganization of the actin cytoskeleton and formation of cell-cell adhesions through regulating other RhoA-targeting GEFs and associating with ANXA2 (Fig. 2). Further studies on the spatiotemporal regulation of Rho-GEFs and their interplay with the components of the actin cytoskeleton, cell adhesion molecules, and cell polarity complexes, will advance our understanding of the mechanisms underlying the formation and remodeling of cell-cell adhesions, and their roles in epithelial development and homeostasis, as well as in cancer invasion and metastasis.

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