RhoGEF2 and the formin Dia control the formation of the furrow canal by directed actin assembly during Drosophila cellularisation

Jrg Grohans 2 Christian Wenzl 2 Hans-Martin Herz 2 Slawomir Bartoszewski 2 Frank Schnorrer 1 Nina Vogt 1 Heinz Schwarz 1 H.-Arno Mller 0 0 Institut fur Genetik, Heinrich-Heine-Universitat Dusseldorf , Universitatsstrasse 1 Geb. 26.02., 40225 Dusseldorf , Germany 1 Max-Planck-Institut fur Entwicklungsbiologie , Spemannstrae 35, 72076 Tubingen , Germany 2 ZMBH, Universitat Heidelberg , Im Neuenheimer Feld 282, 69120 Heidelberg , Germany - The physical interaction of the plasma membrane with the associated cortical cytoskeleton is important in many morphogenetic processes during development. At the end of the syncytial blastoderm of Drosophila the plasma membrane begins to fold in and forms the furrow canals in a regular hexagonal pattern. Every furrow canal leads the invagination of membrane between adjacent nuclei. tn Concomitantly with furrow canal formation, actin e filaments are assembled at the furrow canal. It is not known m how the regular pattern of membrane invagination and the p lo morphology of the furrow canal is determined and whether ev actin filaments are important for furrow canal formation. eD We show that both the guanyl-nucleotide exchange factor RhoGEF2 and the formin Diaphanous (Dia) are required for furrow canal formation. In embryos from RhoGEF2 or dia germline clones, furrow canals do not form at all or are considerably enlarged and contain cytoplasmic blebs. Both Many morphogenetic processes during development involve precise changes in the shape of the plasma membrane. Migrating cells form extensions at their leading edge, while during cytokinesis the plasma membrane constricts between the future daughter cells (Glotzer, 2001). The curvature of the plasma membrane is determined to a large degree by the cortical cytoskeleton with actin filaments being an integral part of it (Revenu et al., 2004). Shape changes and internalisation events require a reorganisation of the cell cortex and the actin cytoskeleton, which is controlled by GTPases of the Rho family (Etienne-Manneville and Hall, 2002; Lu and Settleman, 1999a). The enclosure of the nuclei into cells during the cellular blastoderm of Drosophila embryos is achieved by a specialised process of membrane invagination and reorganisation of the actin cytoskeleton (Foe et al., 1993; Schejter and Wieschaus, 1993; Mazumdar and Mazumdar, 2002). Following the exit from the last mitosis of the cleavage stage, the plasma membrane folds in between adjacent nuclei to form the furrow canals that are visible by light microscopy as the cellularisation front after about 10 to 20 minutes when the shape of the nuclei Dia and RhoGEF2 proteins are localised at the invagination site prior to formation of the furrow canal. Whereas they localise independently of F-actin, Dia localisation requires RhoGEF2. The amount of F-actin at the furrow canal is reduced in dia and RhoGEF2 mutants, suggesting that RhoGEF2 and Dia are necessary for the correct assembly of actin filaments at the forming furrow canal. Biochemical analysis shows that Rho1 interacts with both RhoGEF2 and Dia, and that Dia nucleates actin filaments. Our results support a model in which RhoGEF2 and dia control position, shape and stability of the forming furrow canal by spatially restricted assembly of actin filaments required for the proper infolding of the plasma membrane. is already ellipsoid. The furrow canals have a diameter of about 0.2 m, are coated with actin filaments and remain connected with the surface plasma membrane. Apical to the furrow canal the so-called basal junction tethers the two adjacent membranes and thus stabilises the furrow (Hunter and Wieschaus, 2000). The mechanism of the spatially restricted assembly of F-actin at the furrow canal, as well as the factors that nucleate F-actin at this site, have not yet been identified. Furthermore, it is unclear whether actin filaments have an instructive function for the initial formation and shape of the furrow canal. A few genes are known to be involved in furrow canal formation. Embryos mutant for nullo, sry-, nuf, Rab11, Abl, dah or dia lack furrow canals between adjacent nuclei to a variable extent, which leads to the formation of multinuclear cells (Schweisguth et al., 1990; Postner and Wieschaus, 1994; Zhang et al., 1996; Rothwell et al., 1998; Afshar et al., 2000; Riggs et al., 2003; Grevengoed et al., 2003). Among this group of genes, nullo and sry- are particularly interesting because they are early markers for the furrow canal and affect the formation of the basal junction (Hunter and Wieschaus, 2000). Another early marker for the furrow canal is the novel protein Slam, which is required for timed invagination of the furrow. Localised to the furrow canal and the basal junction, Slam recruits MyoII to the furrow canal and affects the accumulation of Arm at the basal junction (Lecuit et al., 2001; Stein et al., 2002). However, a direct link to furrow canal formation and Factin polymerisation has not been established for any of the genes in this group. To identify additional components required for proper cell morphology in the blastoderm we have screened a large collection of female-sterile mutants derived from germline clones (Luschnig et al., 2004) (our unpublished data). We found two allelic mutations that affect the cellularisation front. Mapping and complementation analysis identified RhoGEF2 as the mutated gene. RhoGEF2 is required during gastrulation for apical constriction of the cells undergoing mesoderm invagination (Barrett et al., 1997; Hcker and Perrimon, 1998). Although there is evidence that RhoGEF2 genetically interacts with Rho1 and is controlled by Folded gastrulation during Drosophila gastrulation, the mechanism of how RhoGEF2 controls cell shape at this stage and whether this involves spatially restricted control of F-actin is not understood. Potential effectors of RhoGEF2 and Rho1 are Rho kinase/sqh/myoII (Royou et al., 2004), citron kinase (Shandala et al., 2004; Naim et al., 2004; DAvino et al., 2004), protein kinase N (Lu and Settleman, 1999b) and Diaphanous (Dia). As t there are no indications that Rho kinase/sqh/myoII and citron en kinase would have a similar function for furrow canal m formation as RhoGEF2, we concentrated in our analysis on dia, op which is a member of the protein family with formin-homology le domains (FH) that control formation of actin filaments (Wallar e and Alberts, 2003). Biochemical and structural studies of the v D yeast (BNI1) and mouse (mDia; also known as Diap1 Mouse Genome Informatics) homologues have shown how actin filaments are nucleated (Pruyne et al., 2002; Sagot et al., 2002; Li and Higgs, 2003; Xu et al., 2004; Shimada et al., 2004; Higashida et al., 2004; Romero et al., 2004). mDia1 is assumed to be activated by binding of Rho1 that releases an inhibitory intramolecular interaction of the C-and N-terminal domains of mDia1 (Alberts, 2001; Watanabe et al., 1997; Watanabe et al., 1999). However, this activation (...truncated)