Essential role for Abi1 in embryonic survival and WAVE2 complex integrity

Abl interactor 1 (Abi1) plays a critical function in actin cytoskeleton dynamics through participation in the WAVE2 complex. To gain a better understanding of the specific role of Abi1, we generated a conditional Abi1-KO mouse model and MEFs lacking Abi1 expression. Abi1-KO cells displayed defective regulation of the actin cytoskeleton, and this dysregulation was ascribed to altered activity of the WAVE2 complex. Changes in motility of Abi1-KO cells were manifested by a decreased migration rate and distance but increased directional persistence. Although these phenotypes did not correlate with peripheral ruffling, which was unaffected, Abi1-KO cells exhibited decreased dorsal ruffling. Western blotting analysis of Abi1-KO cell lysates indicated reduced levels of the WAVE complex components WAVE1 and WAVE2, Nap1, and Sra-1/PIR121. Although relative Abi2 levels were more than doubled in Abi1-KO cells, the absolute Abi2 expression in these cells amounted only to a fifth of Abi1 levels in the control cell line. This finding suggests that the presence of Abi1 is critical for the integrity and stability of WAVE complex and that Abi2 levels are not sufficiently increased to compensate fully for the loss of Abi1 in KO cells and to restore the integrity and function of the WAVE complex. The essential function of Abi1 in WAVE complexes and their regulation might explain the observed embryonic lethality of Abi1-deficient embryos, which survived until approximately embryonic day 11.5 and displayed malformations in the developing heart and brain. Cells lacking Abi1 and the conditional Abi1-KO mouse will serve as critical models for defining Abi1 function.

[1]  Kenji Nakamura,et al.  Rac1 is required for the formation of three germ layers during gastrulation , 1998, Oncogene.

[2]  K. Rottner,et al.  Abi1 regulates the activity of N-WASP and WAVE in distinct actin-based processes , 2005, Nature Cell Biology.

[3]  A. Pendergast,et al.  Regulation of Cell-Cell Adhesion by Abi/Diaphanous Complexes , 2009, Molecular and Cellular Biology.

[4]  John C. Dawson,et al.  N-WASP involvement in dorsal ruffle formation in mouse embryonic fibroblasts. , 2006, Molecular biology of the cell.

[5]  William C. Wetsel,et al.  Abi2-Deficient Mice Exhibit Defective Cell Migration, Aberrant Dendritic Spine Morphogenesis, and Deficits in Learning and Memory , 2004, Molecular and Cellular Biology.

[6]  F. Alt,et al.  WAVE2 deficiency reveals distinct roles in embryogenesis and Rac‐mediated actin‐based motility , 2003, The EMBO journal.

[7]  G. Scita,et al.  Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1 , 2003, The Journal of cell biology.

[8]  R. Vale,et al.  Molecular requirements for actin-based lamella formation in Drosophila S2 cells , 2003, The Journal of cell biology.

[9]  T. Stradal,et al.  Protein complexes regulating Arp2/3-mediated actin assembly. , 2006, Current opinion in cell biology.

[10]  L. Machesky,et al.  Inclusion of Scar/WAVE3 in a similar complex to Scar/WAVE1 and 2 , 2005, BMC Cell Biology.

[11]  K. Rottner,et al.  Sra‐1 and Nap1 link Rac to actin assembly driving lamellipodia formation , 2004, The EMBO journal.

[12]  B. Mayer,et al.  Abi1/Hssh3bp1 pY213 links Abl kinase signaling to p85 regulatory subunit of PI‐3 kinase in regulation of macropinocytosis in LNCaP cells , 2010, FEBS letters.

[13]  R. Insall,et al.  Abi Mutants in Dictyostelium Reveal Specific Roles for the SCAR/WAVE Complex in Cytokinesis , 2008, Current Biology.

[14]  A. Debnath,et al.  Allosteric inhibition of the nonMyristoylated c-Abl tyrosine kinase by phosphopeptides derived from Abi1/Hssh3bp1. , 2008, Biochimica et biophysica acta.

[15]  Hong Gu,et al.  Identification of a Candidate Human Spectrin Src Homology 3 Domain-binding Protein Suggests a General Mechanism of Association of Tyrosine Kinases with the Spectrin-based Membrane Skeleton* , 1998, The Journal of Biological Chemistry.

[16]  C. Piccoli,et al.  Isolation and characterization of e3B1, an eps8 binding protein that regulates cell growth , 1997, Oncogene.

[17]  A. Pendergast,et al.  Abl Interactor 1 (Abi-1) Wave-Binding and SNARE Domains Regulate Its Nucleocytoplasmic Shuttling, Lamellipodium Localization, and Wave-1 Levels , 2004, Molecular and Cellular Biology.

[18]  K. Siminovitch,et al.  Abelson-interactor-1 promotes WAVE2 membrane translocation and Abelson-mediated tyrosine phosphorylation required for WAVE2 activation. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[19]  C. Betsholtz,et al.  EPS8 and E3B1 transduce signals from Ras to Rac , 1999, Nature.

[20]  S. Gygi,et al.  Purification and architecture of the ubiquitous Wave complex. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[21]  D. Yamazaki,et al.  PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation of lamellipodia , 2004, Nature Cell Biology.

[22]  Anne J. Ridley,et al.  The small GTP-binding protein rac regulates growth factor-induced membrane ruffling , 1992, Cell.

[23]  F. Alt,et al.  Cdc42 is required for PIP2-induced actin polymerization and early development but not for cell viability , 2000, Current Biology.

[24]  T. Pollard,et al.  Cellular Motility Driven by Assembly and Disassembly of Actin Filaments , 2003, Cell.

[25]  M. Kirschner,et al.  Activation of the WAVE complex by coincident signals controls actin assembly. , 2009, Molecular cell.

[26]  A. Ismail,et al.  The WAVE Regulatory Complex is Inhibited , 2009, Nature Structural &Molecular Biology.

[27]  P. Chavrier,et al.  Regulation of phagocytosis by Rho GTPases. , 2005, Current Topics in Microbiology and Immunology.

[28]  E. Derivery,et al.  The Wave complex is intrinsically inactive. , 2009, Cell motility and the cytoskeleton.

[29]  Andrea Disanza,et al.  Abi1 is essential for the formation and activation of a WAVE2 signalling complex , 2004, Nature Cell Biology.

[30]  Jacob R. Haling,et al.  Abl-interactor-1 (Abi1) has a role in cardiovascular and placental development and is a binding partner of the α4 integrin , 2010, Proceedings of the National Academy of Sciences.

[31]  A. VanDongen,et al.  Localization and Phosphorylation of Abl-Interactor Proteins, Abi-1 and Abi-2, in the Developing Nervous System , 2000, Molecular and Cellular Neuroscience.

[32]  Zbyszek Otwinowski,et al.  Structure and Control of the Actin Regulatory WAVE Complex , 2010, Nature.

[33]  K. Rottner,et al.  Actin pedestal formation by enteropathogenic Escherichia coli and intracellular motility of Shigella flexneri are abolished in N‐WASP‐defective cells , 2001, EMBO reports.

[34]  Sheila M. Thomas,et al.  N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility , 2001, Nature Cell Biology.

[35]  K. Anderson,et al.  Rac1-Dependent Collective Cell Migration Is Required for Specification of the Anterior-Posterior Body Axis of the Mouse , 2010, PLoS biology.

[36]  K. Rottner,et al.  Bacteria-host-cell interactions at the plasma membrane: stories on actin cytoskeleton subversion. , 2005, Developmental cell.

[37]  T. Takenawa,et al.  Identification of two human WAVE/SCAR homologues as general actin regulatory molecules which associate with the Arp2/3 complex. , 1999, Biochemical and biophysical research communications.

[38]  J. Small,et al.  Scar/WAVE is localised at the tips of protruding lamellipodia in living cells , 2001, FEBS letters.

[39]  S. Heck,et al.  Differential Regulation of Macropinocytosis by Abi1/Hssh3bp1 Isoforms , 2010, PloS one.

[40]  B. Baum,et al.  Abi, Sra1, and Kette Control the Stability and Localization of SCAR/WAVE to Regulate the Formation of Actin-Based Protrusions , 2003, Current Biology.

[41]  K. Rottner,et al.  The Abl interactor proteins localize to sites of actin polymerization at the tips of lamellipodia and filopodia , 2001, Current Biology.

[42]  Klemens Rottner,et al.  Filopodia formation in the absence of functional WAVE- and Arp2/3-complexes. , 2006, Molecular biology of the cell.

[43]  D. Yamazaki,et al.  Differential roles of WAVE1 and WAVE2 in dorsal and peripheral ruffle formation for fibroblast cell migration. , 2003, Developmental cell.

[44]  R. Grosse,et al.  Staying in shape with formins. , 2006, Developmental cell.

[45]  K. Anderson,et al.  Axis specification and morphogenesis in the mouse embryo require Nap1, a regulator of WAVE-mediated actin branching , 2006, Development.