An Order of Magnitude Faster AIP1-Associated Actin Disruption than Nucleation by the Arp2/3 Complex in Lamellipodia

The mechanism of lamellipod actin turnover is still under debate. To clarify the intracellular behavior of the recently-identified actin disruption mechanism, we examined kinetics of AIP1 using fluorescent single-molecule speckle microscopy. AIP1 is thought to cap cofilin-generated actin barbed ends. Here we demonstrate a reduction in actin-associated AIP1 in lamellipodia of cells overexpressing LIM-kinase. Moreover, actin-associated AIP1 was rapidly abolished by jasplakinolide, which concurrently blocked the F-actin-cofilin interaction. Jasplakinolide also slowed dissociation of AIP1, which is analogous to the effect of this drug on capping protein. These findings provide in vivo evidence of the association of AIP1 with barbed ends generated by cofilin-catalyzed filament disruption. Single-molecule observation found distribution of F-actin-associated AIP1 throughout lamellipodia, and revealed even faster dissociation of AIP1 than capping protein. The estimated overall AIP1-associated actin disruption rate, 1.8 µM/s, was one order of magnitude faster than Arp2/3 complex-catalyzed actin nucleation in lamellipodia. This rate does not suffice the filament severing rate predicted in our previous high frequency filament severing-annealing hypothesis. Our data together with recent biochemical studies imply barbed end-preferred frequent filament disruption. Frequent generation of AIP1-associated barbed ends and subsequent release of AIP1 may be the mechanism that facilitates previously observed ubiquitous actin polymerization throughout lamellipodia

Molecular and functional analysis of cadherin-based adherens junctions

Adherens junctions are specialized forms of cadherin-based adhesive contacts important for tissue organization in developing and adult organisms. Cadherins form protein complexes with cytoplasmic proteins (catenins) that convert the specific, hemophilic-binding capacity of the extracellular domain into stable cell adhesion. The extracellular domains of cadherins form parallel dimers that possess intrinsic hemophilic-binding activity. Cytoplasmic interactions can influence the function of the ectodomain by a number of potential mechanisms, including redistribution of binding sites into clusters, providing cytoskeletal anchorage, and mediating physiological regulation of cadherin function, Adherens junctions are likely to serve specific, specialized functions beyond the basic adhesive process. These functions include coupling cytoskeletal force generation to strongly adherent sites on the cell surface and the regulation of intracellular signaling events

Proteolytic E-cadherin activation followed by solution NMR and X-ray crystallography

Cellular adhesion by classical cadherins depends critically on the exact proteolytic removal of their N-terminal prosequences. In this combined solution NMR and X-ray crystallographic study, the consequences of propeptide cleavage of an epithelial cadherin construct (domains 1 and 2) were followed at atomic level. At low protein concentration, the N-terminal processing induces docking of the tryptophan-2 side-chain into a binding pocket on the same molecule. At high concentration, cleavage induces dimerization (K(D)=0.72 mM, k(off)=0.7 s(−1)) and concomitant intermolecular exchange of the βA-strands and the tryptophan-2 side-chains. Thus, the cleavage represents the switch from a nonadhesive to the functional form of cadherin

Gab2 and Src co-operate in human mammary epithelial cells to promote growth factor independence and disruption of acinar morphogenesis

The Gab2 docking protein is a target of several oncogenic protein tyrosine kinases and potentiates activation of the Ras/extracellular signal regulated kinase and phosphatidylinositol 3-kinase (PI3-kinase) pathways. Since Gab2 is phosphorylated by c-Src, and both proteins are overexpressed in breast cancers, we have determined the biological consequences of their co-expression in the immortalized human mammary epithelial cell line MCF-10A. While overexpression of c-Src did not affect acinar morphogenesis or growth factor dependence in three-dimensional culture, c-Src co-operated with Gab2 to promote epidermal growth factor (EGF)-independent acinar growth. In contrast, expression of v-Src or the activated mutant c-SrcY527F led to a spectrum of aberrant phenotypes ranging from spheroids with incomplete luminal clearance to highly disrupted, dispersed structures. Gab2 co-expression shifted the phenotypic distribution towards the dispersed phenotype, an effect not observed with a Gab2 mutant unable to bind the p85 subunit of PI3-kinase (Gab2Δp85). In v-Src-expressing cells, Gab2, but not Gab2Δp85, significantly decreased E-cadherin adhesive strength without altering its surface expression. Gab2 associated with E-cadherin in the presence and absence of v-Src, indicating that the ability of Gab2 to weaken the strength of cell–cell contacts may reflect enhanced activation of PI3-kinase at adherens junctions. Gab2 also increased migration and invasion of these cells in transwell assays, but these effects were p85-independent. Overall, these findings demonstrate a novel mechanism whereby Gab2 may promote metastatic spread and indicate that Gab2 may play several roles during breast cancer progression