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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2005.09.021 Byline: Frauke Drees (1), Sabine Pokutta (1)(2), Soichiro Yamada (1), W. James Nelson (1), William I. Weis (1)(2) Abstract: Epithelial cell-cell junctions, organized by adhesion proteins and the underlying actin cytoskeleton, are considered to be stable structures maintaining the structural integrity of tissues. Contrary to the idea that [alpha]-catenin links the adhesion protein E-cadherin through [beta]-catenin to the actin cytoskeleton, in the accompanying paper we report that [alpha]-catenin does not bind simultaneously to both E-cadherin-[beta]-catenin and actin filaments. Here we demonstrate that [alpha]-catenin exists as a monomer or a homodimer with different binding properties. Monomeric [alpha]-catenin binds more strongly to E-cadherin-[beta]-catenin, whereas the dimer preferentially binds actin filaments. Different molecular conformations are associated with these different binding states, indicating that [alpha]-catenin is an allosteric protein. Significantly, [alpha]-catenin directly regulates actin-filament organization by suppressing Arp2/3-mediated actin polymerization, likely by competing with the Arp2/3 complex for binding to actin filaments. These results indicate a new role for [alpha]-catenin in local regulation of actin assembly and organization at sites of cadherin-mediated cell-cell adhesion. Author Affiliation: (1) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA (2) Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA Article History: Received 31 March 2005; Revised 28 July 2005; Accepted 12 September 2005 Article Note: (miscellaneous) Published: December 1, 2005

The proto-oncogenic kinase Abelson (Abl) regulates actin in response to cell signaling. Drosophila Abl is required in the nervous system, and also in epithelia[ cells, where it regulates adherens junction stability and actin organization. Abl acts at least in part via the actin regulator Enabled (Ena), but the mechanism by which Abl regulates Ena is unknown. We describe a novel role for Abl in early Drosophila development, where it regulates the site and type of actin structures produced. In Abl's absence, excess actin is polymerized in apical microvilli, whereas too little actin is assembled into pseudocleavage and cellularization furrows. These effects involve Ena misregulation. In abl mutants, Ena accumulates ectopically at the apical cortex where excess actin is observed, suggesting that Abl regulates Ena's subcellular localization. We also examined other actin regulators. Loss of Abl leads to changes in the localization of the Arp2/3 complex and the formin Diaphanous, and mutations in diaphanous or capping protein [beta] enhance abl phenotypes.

Research has been conducted on the profilin and profilin-actin complex interactions with actin filament barbed end. The role of profilin in the nonmuscle beta,gamma actin elongation has been investigated and the details are reported.

All members of the Wiskott-Aldrich syndrome protein (WASp) family contain a carboxyl-terminal verprolin homology, cofilin homology, and acidic region (VCA) domain that binds and activates the Arp2/3 complex, thereby linking these proteins to the induction of actin polymerization. Although the VCA domain imbues WASp and other WASp family members with the capacity to modulate cytoskeletal organization, little is known about the impact of this domain activity on lymphoid cell function. Here we demonstrate that T cell-restricted expression of VCA domain-deleted WASp (WASp [DELTA] VCA) in [WAS.sup.-/-] mice engenders a severe early block in T lymphopoiesis associated with impaired T cell antigen receptor [alpha][beta] expression and a consequent failure to generate single-positive [CD4.sup.+] and [CD8.sup.+] T cells. These latter defects, which are not observed in [WAS.sup.-/-] mice, are associated with impaired induction of cellular actin polymerization and a failure in the terminal differentiation of double-negative thymocytes. These findings indicate that WASp family proteins play an essential role in modulating the signaling events required for early thymocyte development and reveal their capacity to subserve this role to depend on VCA domain-mediated actin polymerization.

The Arp2/3 complex and its activators, Scar/WAVE and Wiskott-Aldrich Syndrome protein (WASp), promote actin polymerization in vitro and have been proposed to influence cell shape and motility in vivo. We demonstrate that the Drosophila Scar homologue, SCAR, localizes to actin-rich structures and is required for normal cell morphology in multiple cell types throughout development. In particular, SCAR function is essential for cytoplasmic organization in the blastoderm, axon development in the central nervous system, egg chamber structure during oogenesis, and adult eye morphology. Highly similar developmental requirements are found for subunits of the Arp2/3 complex. In the blastoderm, SCAR and Arp2/3 mutations result in a reduction in the amount of cortical filamentous actin and the disruption of dynamically regulated actin structures. Remarkably, the single Drosophila WASp homologue, Wasp, is largely dispensable for these numerous Arp2/3-dependent functions, whereas SCAR does not contribute to cell fate decisions in which Wasp and Arp2/3 play an essential role. These results identify SCAR as a major component of Arp2/3-dependent cell morphology during Drosophila development and demonstrate that the Arp2/3 complex can govern distinct cell biological events in response to SCAR and Wasp regulation.

The Arp2/3 complex has been shown to dramatically increase the slow spontaneous rate of actin filament nucleation in vitro, and it is known to be important for remodeling the actin cytoskeleton in vivo. We isolated and characterized loss of function mutations in genes encoding two subunits of the Drosophila Arp2/3 complex: Arpc1, which encodes the homologue of the p40 subunit, and Arp3, encoding one of the two actin-related proteins. We used these mutations to study how the Arp2/3 complex contributes to well-characterized actin structures in the ovary and the pupal epithelium. We found that the Atp2/3 complex is required for ring canal expansion during oogenesis but not for the formation of parallel actin bundles in nurse cell cytoplasm and bristle shaft cells. The requirement for Atp2/3 in ring canals indicates that the polymerization of actin filaments at the ring canal plasma membrane is important for driving ring canal growth.

Results demonstrate that hydrolysis of magnesium bound ATP actin is three times faster as shown by mechanical quenched-flow technique. Hydrolysis rates of the point end and the barbed end are similar with no effect on the latter by saturating concentration of profilin.

The authors review the publications on the structure and function of the Arp2/3 complex. The topics of interest include the crystal structure, the cryo-EM structure and the reconstruction of the complex from the subunits.

The thymosin beta-4 polypeptides are potential actin regulators which may act by regulating actin polymerization. The beta-4 receptors were overexpressed in epithelial and fibroblast cells by microinjection techniques and the actin responses were monitored by measuring the amount of fluorescent phalloidin binding. Actin depolymerization was observed to occur in a dose-dependent manner. The results clearly demonstrated that beta-4 can induce extensive depolymerization of actin in living cells.

The AF-6 protein is a multidomain protein that contains two potential Ras-binding domains within its N terminus. Because of this feature, AF-6 has been isolated in both two-hybrid and biochemical approaches and is postulated to be a potential Ras-effector protein. Herein, we show that it is specifically the first Ras-binding domain of AF-6 that mediates this interaction and that the Ras-related RaplA protein can associate with this motif even more efficiently than the oncogenic Ha-, K-, and N-Ras GTPases. We further demonstrate that both Ras and Rap1 interact with full-length AF-6 in vivo in mammalian cells and that a fraction of Rap1 colocalizes with AF-6 at the membrane. Dominant active Rap1A, in contrast to Ras, when introduced into epithelial MDCK and MCF-7 cells, does not perturb AF-6-specific residency in cell-cell adhesion complexes. In a pursuit to gain further understanding of the role of AF-6 in junctions, we identified profilin as an AF-6-binding protein. Profilin activates monomeric actin units for subsequent polymerization steps at barbed ends of actin filaments and has been shown to participate in cortical actin assembly. To our knowledge, AF-6 is the only integral component in cell-cell junctions discovered thus far that interacts with profilin and thus could modulate actin modeling proximal to adhesion complexes.