RhoGEF2 and the formin Dia control the formation of the furrow canal by directed actin assembly duringDrosophilacellularisation

Abstract

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. Concomitantly with furrow canal formation, actin filaments are assembled at the furrow canal. It is not known how the regular pattern of membrane invagination and the morphology of the furrow canal is determined and whether actin filaments are important for furrow canal formation. 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 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.

Regulation of the Rac GTPase pathway by the multifunctional Rho GEF Pebble is essential for mesoderm migration in theDrosophilagastrula

“…After lysis in 50 mM Tris-HCl (pH 8), 100 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, 1 mM PMSF, the fusion proteins were purified by affinity chromatography (wash buffer, 50 mM Tris-HCl pH 8, 500 mM NaCl, 10 mM MgCl 2 , 1 mM DTT; elution buffer, 50 mM Tris-HCl pH 8, 50 mM NaCl, 20 mM glutathione, 1 mM DTT). The GEF assay was performed as described previously (Grosshans et al, 2005). Briefly, 0.2 μM GSTGTPases were loaded with [8-3 H]GDP (Amersham).…”

The Drosophila MAST kinase Drop out is required to initiate membrane compartmentalisation during cellularisation and regulates dynein-based transport

“…We conclude that dop is required for the initial redistribution of F-actin to the sites of incipient furrows. Rho1, an important regulator of furrow formation and actin polymerisation at the furrows (Grosshans et al, 2005), exhibited a highly abnormal distribution that was similar to the abnormal localisation of F-actin (Fig. 4C).…”

“…Another early zygotic gene, nullo, is required for the proper recruitment of F-actin during furrow canal formation (Sokac and Wieschaus, 2008b). Nullo and the actin regulator RhoGEF2 have been proposed to act in parallel pathways controlling processes that are distinct but both essential for F-actin network formation during the establishment of the furrow canal (Grosshans et al, 2005). Since early F-actin rearrangements are largely normal in nullo and RhoGEF2 single mutants, we propose that Dop is essential for the initial early focussing of F-actin, whereas Nullo and RhoGEF2 are required to elaborate and maintain F-actin levels to stabilise the furrows.…”

“…Specimens were mounted in Mowiol/DABCO, imaged on a Leica SP2 confocal microscope and processed using ImageJ, Photoshop (Adobe) or Volocity (PerkinElmer). For transmission electron microscopy, samples were fixed by highpressure freezing and processed by freeze-substitution as described (Grosshans et al, 2005). For scanning electron microscopy, embryos were staged under halocarbon oil, fixed and processed as described (Müller and Wieschaus, 1996).…”

“…Cortical asymmetry becomes evident by accumulation of proteins including F-actin and Myosin II, which mark the sites where cleavage furrows invaginate (Sokac and Wieschaus, 2008b). These incipient cleavage furrows mature to form furrow canals that are stabilised by actin/myosin, the actin regulators RhoGEF2, Rho1 and Diaphanous (Dia), and additional regulatory proteins including Slow as Molasses (Slam), Septins, Nullo and Patj (Hunter and Wieschaus, 2000;Lecuit et al, 2002;Grosshans et al, 2005;Sokac and Wieschaus, 2008b;Wenzl et al, 2010). Basal adherens junctions (bAJs) form apical to the furrow canal and move inward with the furrows (Müller and Wieschaus, 1996;Hunter and Wieschaus, 2000).…”