Filopodia are exploratory finger-like projections composed of multiple long, straight, parallel-bundled actin filaments that protrude from the leading edge of migrating cells. Drosophila melanogaster Enabled (Ena) is a member of the Ena/vasodilatorstimulated phosphoprotein protein family, which facilitates the assembly of filopodial actin filaments that are bundled by Fascin. However, the mechanism by which Ena and Fascin promote the assembly of uniformly thick F-actin bundles that are capable of producing coordinated protrusive forces without buckling is not well understood. We used multicolor evanescent wave fluorescence microscopy imaging to follow individual Ena molecules on both single and Fascinbundled F-actin in vitro. Individual Ena tetramers increase the elongation rate approximately two-to threefold and inhibit capping protein by remaining processively associated with the barbed end for an average of ∼10 s in solution, for ∼60 s when immobilized on a surface, and for ∼110 s when multiple Ena tetramers are clustered on a surface. Ena also can gather and simultaneously elongate multiple barbed ends. Collectively, these properties could facilitate the recruitment of Fascin and initiate filopodia formation. Remarkably, we found that Ena's actin-assembly properties are tunable on Fascinbundled filaments, facilitating the formation of filopodia-like F-actin networks without tapered barbed ends. Ena-associated trailing barbed ends in Fascin-bundled actin filaments have approximately twofold more frequent and approximately fivefold longer processive runs, allowing them to catch up with leading barbed ends efficiently. Therefore, Fascin and Ena cooperate to extend and maintain robust filopodia of uniform thickness with aligned barbed ends by a unique mechanistic cycle.profilin | formin | TIRF microscopy | self organization | single molecule T he actin cytoskeleton facilitates fundamental cellular processes including division, polarization, and motility. The organization and dynamics of particular F-actin networks are determined by the coordinated action of specific subsets of actinbinding proteins with complementary biochemical properties such as sequestering, nucleating, elongating, bundling/crosslinking, and severing (1-3).Cell motility is driven primarily by lamellipodia, protrusive structures at the cell's leading edge composed of a dendritic network of short-branched filaments produced by the rapid capping of filaments nucleated by the actin-related proteins 2 and 3 (Arp2/3) complex (4). Filopodia are exploratory finger-like projections composed of uniformly long, straight, parallel-bundled filaments that extend from lamellipodia. One current model for filopodia assembly is convergent elongation (5). Formin and/or Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins gather and rapidly elongate subpopulations of lamellipodial actin-barbed ends, antagonizing inhibition by capping protein (CP), and the parallel actin-crosslinking protein Fascin aligns and bundles elongating filopodial filaments (...
The importance of Abl kinase activity, the F-actin–binding site, and scaffolding ability in Abl’s many cell biological roles during Drosophila morphogenesis is examined. Abl is a robust multidomain scaffold with different protein motifs and activities contributing differentially to diverse cellular behaviors.
The open source MATLAB application CellGeo is a user-friendly computational platform that allows simultaneous, automated tracking and analysis of dynamic changes in cell shape, including protrusions ranging from filopodia to lamellipodia to growth cones.
De novo germline mutations in GNB1 have been associated with a neurodevelopmental phenotype. To date, 28 patients with variants classified as pathogenic have been reported. We add 18 patients with de novo mutations to this cohort, including a patient with mosaicism for a GNB1 mutation who presented with a milder phenotype. Consistent with previous reports, developmental delay in these patients was moderate to severe, and more than half of the patients were non-ambulatory and nonverbal. The most observed substitution affects the p.Ile80 residue encoded in exon 6, with 28% of patients carrying a variant at this residue. Dystonia and growth delay were observed more frequently in patients carrying variants in this residue, suggesting a potential genotype-phenotype correlation. In the new cohort of 18 patients, 50% of males had genitourinary anomalies and 61% of patients had gastrointestinal anomalies, suggesting a possible association of these findings with variants in GNB1. In addition, cutaneous mastocytosis, reported once before in a patient with a GNB1 variant, was observed in three additional patients, providing further evidence for an association to GNB1. We will review clinical and molecular data of these new cases and all previously reported cases to further define the phenotype and establish possible genotype-phenotype correlations.
Summary Actin regulators facilitate cell migration by controlling cell protrusion architecture and dynamics. As the behavior of individual actin regulators becomes clear, we must address why cells require multiple regulators with similar functions and how they cooperate to create diverse protrusions. We characterized Diaphanous and Enabled as a model, using complementary approaches: cell culture, biophysical analysis, and Drosophila morphogenesis. We found Dia and Ena have distinct biochemical properties that contribute to the different protrusion morphologies each induces. Dia is a more processive, faster elongator, paralleling the long, stable filopodia it induces in vivo, while Ena promotes filopodia with more dynamic changes in number, length and lifetime. Acting together, Ena and Dia induce protrusions distinct from those induced by either alone, with Ena reducing Dia-driven protrusion length and number. Consistent with this, EnaEVH1 binds Dia directly and inhibits DiaFH1FH2-mediated nucleation in vitro. Finally, Ena rescues hemocyte migration defects caused by activated Dia.
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