Abp1 utilizes the Arp2/3 complex activator Scar/WAVE in bristle development

Abstract

SummaryMany developmental processes rely on cortical actin dynamics; however, the mechanisms of its fine control at the cell cortex are still largely unknown. Our analyses demonstrate that the lipid-and F-actin-binding protein Abp1 is crucial for actin-driven bristle development in Drosophila melanogaster. Combined genetic, cell biological and biochemical analyses reveal that Abp1 triggers cortical Arp2/3-mediated actin nucleation by complex formation with Scar in bristle development. The role of the plasma-membrane-associated Abp1 subpool was highlighted by constitutively membrane-anchored Abp1. Such gain-of-function experiments led to a severe splitbristle phenotype, which was negatively correlated with bristle length. This phenotype was dependent on Scar but not on WASP and required the Scar-interacting SH3 domain of Abp1. Strikingly, knockout of abp1 led to defects in both microchaete and macrochaete bristle integrity. Importantly, Arp2-and Scar-deficient flies displayed similar bristle phenotypes. Microchaetes of flies deficient for Abp1, Arp2 and Scar functions had kinks, whereas those of wasp heterozygous flies did not. Electron microscopy analyses revealed that abp1 knockout, Arp2 RNAi and Scar RNAi all led to distorted macrochaetes with an excessive number of ridges. Interestingly, despite the physical association of Abp1 with Scar and its ability to use the Arp2/3 complex activator as an effector, abp1 knockout did not affect Scar stability. This is in contrast to classical Scar complex components, such as Kette or Sra-1. Our work reveals that Abp1 is an important, Scar-interacting factor controlling cortical Arp2/3-mediated actin nucleation and unravels a novel layer of complexity in the scrupulous control of cortical actin nucleation during sensory organ formation.

A spastic paraplegia mouse model reveals REEP1-dependent ER shaping

“…Subcellular fractions were obtained by differential centrifugation at 4°C (10 minutes at 1,000 g to pellet nuclei, 15 minutes at 10,000 g to pellet mitochondria, 1 hour at 100,000 g to pellet the membrane fraction). The postmitochondrial fraction was further fractionated by a discontinuous iodixanol gradient, and individual fractions were diluted, pelleted, resolved in SDS sample buffer (42), and quantitatively evaluated by fluorescence-based immunoblotting using a LI-COR Odyssey system.…”

Cooperative functions of the two F-BAR proteins Cip4 and Nostrin in regulating E-cadherin in epithelial morphogenesis

“…Members of the Fes-CIP4 homology Bin-amphiphysin-Rvs161/ 167 (F-BAR) protein family form crescent-shaped dimers that are able to shape membranes into vesicles and tubules (McMahon and Gallop, 2005;Fütterer and Machesky, 2007;Heath and Insall, 2008;Koch et al, 2012b;Qualmann et al, 2011;Suetsugu, 2010;Suetsugu and Gautreau, 2012). F-BAR proteins have been grouped into six subfamilies, the Cdc42-interacting protein 4 (Cip4) subfamily, the Fes subfamily of non-receptor tyrosine kinases, the protein kinase C and casein kinase substrate in neurons protein (pacsin) subfamily, the Slit-Robo RhoGTPase-activating proteins (SrGAPs), the FCH-domain-only (FCHO) and the proline-serinethreonine phosphatase-interacting protein (PSTPIP) subfamilies Heath and Insall, 2008).…”

Terminal Axonal Arborization and Synaptic Bouton Formation Critically Rely on Abp1 and the Arp2/3 Complex

“…In flies, Abp1 interacts with the Arp2/3 complex activator Scar and abp1 knock-out flies (for genomic scheme see Fig. 1A) show defects in the development of micro- and macrochaete bristles reflecting impairments in the otherwise highly regular F-actin organization of these sensory organs at the thorax of flies [15].…”

“…Abp1 RNAi [15] and Abp1-GFP were cloned into pUAST. Abp1 SH3 P523A (Abp1 SH3*) [15] as well as the SH3 domain of Abp1 were cloned into pGEX-5X-1.…”

“…Affinity-purified anti-Abp1 antibodies described previously were used at 1∶100 for immunostainings [15]. Other primary antibodies used include rabbit anti-GFP (1∶1000; Invitrogen and Abcam, respectively), guinea pig anti-WASP (1∶250) and anti-Scar (1∶250) [20].…”