RAC1 is a widely studied Rho GTPase, a class of molecules that modulate numerous cellular functions essential for normal development. RAC1 is highly conserved across species and is under strict mutational constraint. We report seven individuals with distinct de novo missense RAC1 mutations and varying degrees of developmental delay, brain malformations, and additional phenotypes. Four individuals, each harboring one of c.53G>A (p.Cys18Tyr), c.116A>G (p.Asn39Ser), c.218C>T (p.Pro73Leu), and c.470G>A (p.Cys157Tyr) variants, were microcephalic, with head circumferences between À2.5 to À5 SD. In contrast, two individuals with c.151G>A (p.Val51Met) and c.151G>C (p.Val51Leu) alleles were macrocephalic with head circumferences of þ4.16 and þ4.5 SD. One individual harboring a c.190T>G (p.Tyr64Asp) allele had head circumference in the normal range. Collectively, we observed an extraordinary spread of $10 SD of head circumferences orchestrated by distinct mutations in the same gene. In silico modeling, mouse fibroblasts spreading assays, and in vivo overexpression assays using zebrafish as a surrogate model demonstrated that the p.Cys18Tyr and p.Asn39Ser RAC1 variants function as dominant-negative alleles and result in microcephaly, reduced neuronal proliferation, and cerebellar abnormalities in vivo. Conversely, the p.Tyr64Asp substitution is constitutively active. The remaining mutations are probably weakly dominant negative or their effects are context dependent. These findings highlight the importance of RAC1 in neuronal development. Along with TRIO and HACE1, a sub-category of rare developmental disorders is emerging with RAC1 as the central player. We show that ultra-rare disorders caused by private, non-recurrent missense mutations that result in varying phenotypes are challenging to dissect, but can be delineated through focused international collaboration.Developmental disorders (DDs) are etiologically extremely heterogeneous and affect 2%-5% of individuals.

A microtubule arm regulates cell–cell repulsion, pointing hemocytes in opposite directions when they contact each other in Drosophila embryos.

Mutation of a critical residue of fascin eliminates the protein’s actin-bundling activity but maintains its positive role in filopodia formation

The group I family of pleckstrin homology (PH) domains are characterized by their inherent ability to specifically bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and its corresponding inositol head-group inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). In vivo this interaction results in the regulated plasma membrane recruitment of cytosolic group I PH domain-containing proteins following agonist-stimulated PtdIns(3,4,5)P(3) production. Among group I PH domain-containing proteins, the Ras GTPase-activating protein GAP1(IP4BP) is unique in being constitutively associated with the plasma membrane. Here we show that, although the GAP1(IP4BP) PH domain interacts with PtdIns(3,4, 5)P(3), it also binds, with a comparable affinity, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) (K(d) values of 0.5 +/- 0.2 and 0.8 +/- 0.5 microm, respectively). Intriguingly, whereas this binding site overlaps with that for Ins(1,3,4,5)P(4), consistent with the constitutive plasma membrane association of GAP1(IP4BP) resulting from its PH domain-binding PtdIns(4,5)P(2), we show that in vivo depletion of PtdIns(4,5)P(2), but not PtdIns(3,4,5)P(3), results in dissociation of GAP1(IP4BP) from this membrane. Thus, the Ins(1,3,4,5)P(4)-binding PH domain from GAP1(IP4BP) defines a novel class of group I PH domains that constitutively targets the protein to the plasma membrane and may allow GAP1(IP4BP) to be regulated in vivo by Ins(1,3,4,5)P(4) rather than PtdIns(3,4,5)P(3).

The p38 MAPK pathway regulates multiple neutrophil functional responses via activation of the serine-threonine kinase MAPK-activated protein kinase 2 (MAP-KAPK2). To identify substrates of MAPKAPK2 that mediate these responses, a proteomic approach was used in which in vitro phosphorylation of neutrophil lysates by exogenously added active recombinant MAPKAPK2 was followed by protein separation using two-dimensional electrophoresis. Peptide mass fingerprinting of peptides defined by MALDI-MS was then utilized to identify phosphorylated proteins detected by autoradiography. Six candidate substrates were identified, including the p16 subunit of the seven-member Arp2/3 complex (p16-Arc). In vitro studies confirmed that MAPKAPK2 interacts with and phosphorylates the A isoform, but not the B isoform, of p16-Arc with a stoichiometry of 0.6 to 0.7. MAPKAPK2 also phosphorylated p16-Arc in intact Arp2/3 complexes precipitated from neutrophil lysates. Mutation of serine-77 to alanine on the A isoform prevented phosphorylation by MAPKAPK2. The ability of MAPKAPK2 to phosphorylate one isoform of p16-Arc suggests a possible mechanism by which the p38 MAPK cascade regulates remodeling of the actin cytoskeleton.

SUMMARYEffective wound closure mechanisms are essential for maintenance of epithelial structure and function. The repair of wounded epithelia is primarily driven by the cells bordering the wound, which become motile after wounding, forming dynamic actin protrusions along the wound edge. The molecular mechanisms that trigger wound edge cells to become motile following tissue damage are not well understood. Using wound healing and dorsal closure in Drosophila, we identify a direct molecular link between changes in cell-cell adhesion at epithelial edges and induction of actin protrusion formation. We find that the scaffolding protein Par3/Bazooka and the lipid phosphatase Pten are specifically lost from cell-cell junctions at epithelial edges. This results in a localized accumulation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), which promotes the formation of actin protrusions along the epithelial edge. Depleting PIP3 results in defective epithelial closure during both dorsal closure and wound healing. These data reveal a novel mechanism that directly couples loss of epithelial integrity to activation of epithelial closure.

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Live imaging of epidermal wound closure in Drosophila embryos reveals that assembly of motile F-actin at wound edges requires the endocytic remodeling of adherens junctions and is mediated by Diaphanous, SCAR, and WASp.