Diaphanous regulates myosin and adherens junctions to control cell contractility and protrusive behavior during morphogenesis

Catarina C. F. Homem Mark Peifer ) Formins are key regulators of actin nucleation and elongation. Diaphanous-related formins, the best-known subclass, are activated by Rho and play essential roles in cytokinesis. In cultured cells, Diaphanous-related formins also regulate cell adhesion, polarity and microtubules, suggesting that they may be key regulators of cell shape change and migration during development. However, their essential roles in cytokinesis hamper our ability to test this hypothesis. We used loss- and gain-of-function approaches to examine the role of Diaphanous in Drosophila morphogenesis. We found that Diaphanous has a dynamic expression pattern consistent with a role in regulating cell shape change. We used constitutively active Diaphanous to examine its roles in morphogenesis and its mechanisms of action. This revealed an unexpected role in regulating myosin levels and activity at adherens junctions during cell shape change, suggesting that Diaphanous helps coordinate adhesion and contractility of the underlying actomyosin ring. We tested this hypothesis by reducing Diaphanous function, revealing striking roles in stabilizing adherens junctions and inhibiting cell protrusiveness. These effects also are mediated through coordinated effects on myosin activity and adhesion, suggesting a common mechanism for Diaphanous action during morphogenesis. INTRODUCTION Drosophila embryogenesis provides a superb model of how cell migration and shape changes reshape the body plan during morphogenesis. Small changes in individual cell shapes collectively generate large-scale tissue reorganization. For example, mesoderm invagination requires coordinated apical constriction, germband extension requires cell intercalation, while in dorsal closure epidermal cells elongate in a polarized fashion. One current challenge is to determine how cell fate is translated into cell shape change. Cell-cell adhesion and the cytoskeleton must be tightly coordinated during morphogenesis. Cell shape changes require remodeling of adherens junctions (AJs) and the actomyosin cytoskeleton. AJs mediate cell-cell adhesion via transmembrane cadherins, with -catenin (Drosophila Armadillo; Arm) and catenin bound to their cytoplasmic tails (Halbleib and Nelson, 2006). -Catenin also interacts with actin. AJs and actin are intimately interrelated, as disrupting one disrupts the other (e.g. Cox et al., 1996; Quinlan and Hyatt, 1999), but mechanisms regulating this coordination are not well understood. During morphogenesis, many epithelial cells polarize in the plane of the epithelium, orthogonal to the apicobasal axis. This planar cell polarity involves asymmetric distribution of AJ and cytoskeletal proteins around the apical circumference (Zallen, 2007). For example, during Drosophila germband extension, F-actin enrichment at anterior/posterior (A/P) cell borders is the first break in symmetry. Nonmuscle Myosin 2 (hereafter Myosin) then becomes enriched at A/P cell borders, while Bazooka/PAR-3 and AJ proteins accumulate at reciprocal dorsal/ventral (D/V) cell borders (Bertet et al., 2004; Blankenship et al., 2006; Zallen and Wieschaus, 2004). Planar polarization of AJs, actin and Myosin also occur during dorsal closure, when epidermal cells elongate along the D/V axis and leading-edge cells construct an actomyosin cable along their dorsal borders (Kaltschmidt et al., 2002; Kiehart et al., 2000). Actin and Myosin regulators, like Rho family GTPases, direct cell movement, shape changes and planar cell polarity. Rho regulates the cytoskeleton and AJs, and Drosophila Rho1 mutants have dorsal closure defects (Magie et al., 1999). Two major Rho effectors are Rho kinases (ROCKs), which are Myosin regulators, and Diaphanousrelated formins (DRFs), which are actin regulators. Embryos zygotically lacking Drosophila ROCK are normal, owing to maternal contribution, while removing maternal ROCK blocks oogenesis (Verdier et al., 2006b; Winter et al., 2001). Myosin heavy chain [MHC; encoded by zipper (zip)] is required for proper dorsal closure (Franke et al., 2005). However, zip zygotic mutants retain maternal MHC and thus do not reveal the full spectrum of functions of Myosin. DRFs, a second class of Rho effectors, nucleate actin filaments and promote filament elongation (Kovar, 2006). DRFs normally are autoinhibited via intramolecular interactions between the N-terminal GTPase-binding domain (GBD) and C-terminal DAD domain. Rho binds the GBD, activating DRFs. DRFs are essential regulators of cytokinesis in yeast, nematodes and flies (Castrillon and Wasserman, 1994; Severson et al., 2002; Swan et al., 1998). Although DRFs exact mechanism of action is not clear, they are crucial in assembling/stabilizing the contractile actomyosin ring. In Drosophila Diaphanous (Dia) is the sole DRF. Dia is essential in conventional cytokinesis and in more specialized events of early embryogenesis, when Dia localizes to tips of syncytial and cellularization furrows, coordinating actin assembly as cells form (Afshar et al., 2000; Grosshans et al., 2005). Formins also have roles outside of cytokinesis. DRFs can affect transcription by activating MAL/SRF through G-actin depletion, and can stabilize polarized microtubules (Faix and Grosse, 2006). In cultured mammalian cells, both Dia1 and Formin 1 promote AJ stability; AJ stabilization by Formin 1 requires its actin polymerization function but is independent of microtubule binding (Kobielak et al., 2004; Sahai and Marshall, 2002; Carramusa et al., 2007). DRF roles in morphogenesis remain largely unknown. Two challenges impede progress. First, mammals have three DRFs that are at least partially redundant. Mouse Dia1 mutant mice have hematopoiesis defects, but are otherwise normal (Eisenmann et al., 2007; Peng et al., 2007). Human DIA1 mutations result in deafness (Lynch et al., 1997), but these mutations may be gain of function. Uncovering the full function of mammalian DRFs will require multiple knockouts. The second, more difficult challenge is their essential role in cytokinesis. Although flies and nematodes have only a single DRF, its inactivation disrupts cytokinesis from the onset of development (Afshar et al., 2000; Swan et al., 1998), preventing analysis of morphogenesis. To address this challenge, we used loss and gain-of-function genetic tools available in Drosophila to study the function of Dia during morphogenesis, and to address its mechanisms of action. These studies suggest that Dia plays an important and unexpected role in coordinating actomyosin contractility and adhesion at AJs, and provide evidence that it acts upstream of Myosin as well as actin. MATERIALS AND METHODS Genetics Mutations and Balancer chromosomes are described at FlyBase (flybase.bio.indiana.edu). Fly stocks and sources are in Table 1. Wild-type was y w. Females carrying UAS-transgenes were crossed to males with GAL4-drivers their expression patterns are described in Table 1. To generat (...truncated)