Understanding how a particular cell type expresses the lamellipodial or filopodial form of the actin machinery is essential to understanding a cell's functional interactions. To determine how a cell "chooses" among these alternative modes of "molecular hardware," we tested the role of key proteins that affect actin filament barbed ends. Depletion of capping protein (CP) by short hairpin RNA (shRNA) caused loss of lamellipodia and explosive formation of filopodia. The knockdown phenotype was rescued by a CP mutant refractory to shRNA, but not by another barbed-end capper, gelsolin, demonstrating that the phenotype was specific for CP. In Ena/VASP deficient cells, CP depletion resulted in ruffling instead of filopodia. We propose a model for selection of lamellipodial versus filopodial organization in which CP is a negative regulator of filopodia formation and Ena/VASP has recruiting/activating functions downstream of actin filament elongation in addition to its previously suggested anticapping and antibranching activities.

Filopodia have been implicated in a number of diverse cellular processes including growth-cone path finding, wound healing, and metastasis. The Ena/VASP family of proteins has emerged as key to filopodia formation but the exact mechanism for how they function has yet to be fully elucidated. Using cell spreading as a model system in combination with small interfering RNA depletion of Capping Protein, we determined that Ena/VASP proteins have a role beyond anticapping activity in filopodia formation. Analysis of mutant Ena/VASP proteins demonstrated that the entire EVH2 domain was the minimal domain required for filopodia formation. Fluorescent recovery after photobleaching data indicate that Ena/VASP proteins rapidly exchange at the leading edge of lamellipodia, whereas virtually no exchange occurred at filopodial tips. Mutation of the G-actin-binding motif (GAB) partially compromised stabilization of Ena/ VASP at filopodia tips. These observations led us to propose a model where the EVH2 domain of Ena/VASP induces and maintains clustering of the barbed ends of actin filaments, which putatively corresponds to a transition from lamellipodial to filopodial localization. Furthermore, the EVH1 domain, together with the GAB motif in the EVH2 domain, helps to maintain Ena/VASP at the growing barbed ends. INTRODUCTIONActin-based protrusions known as filopodia, first described by Porter, Claude, and Fullam as early as 1945 (Porter et al., 1945;Albrecht-Buehler, 1976), are composed of tightly bundled parallel filaments 5-50 m long and 0.1-0.5 m thick (Small, 1988;Mitchison and Cramer, 1996). Filopodia have been implicated in a number of cellular processes including neuronal growth cone pathfinding, embryonic development, wound healing, and metastasis (Jacinto et al., 2000(Jacinto et al., , 2001Vasioukhin and Fuchs, 2001;Dent and Gertler, 2003;Hashimoto et al., 2005). Despite the importance of filopodia to so many diverse cellular functions, the exact mechanism governing their initiation and formation has yet to be fully explained. A number of actin-binding proteins have been implicated in the formation of filopodia, most recently myosin X and the formin family of proteins, which catalyze the formation of long unbranched actin filaments Zigmond, 2004;Bohil et al., 2006;Kovar, 2006). However, in several cell types, filopodia and the sheet-like Arp2/3-based lamellipodia, rapidly interchange during protrusion, suggesting commonalities between the two structures . The type of protrusion that dominates in the subcellular compartment critically depends on whether filaments are allowed to elongate persistently or are capped shortly after nucleation. Accordingly, when the heterodimeric Capping Protein (CP), the major barbed end terminator in lamellipodia, is silenced by small interfering RNA (siRNA), the balance between the two structures, is shifted to favor filopodial formation (Mejillano et al., 2004). A similar phenomenon has been observed in vitro where a decrease in CP levels led to a transition from a dendritic-like network...

The dynamics of actin and microtubules are coordinated in many cellular processes, but little is known about molecules mediating cross-talk. We describe intracellular dynamics of Shot in a structure-function analysis of its role as a cross-linker. Shot interacts with microtubules two ways through EB1 and along microtubule lattices by the GAS2 domain.

Ena/VASP tetramers are processive actin elongation factors that localize to diverse F-actin networks composed of filaments bundled by different cross-linking proteins, such as filopodia (fascin), lamellipodia (fimbrin), and stress fibers (α-actinin). Previously, we found that Ena takes approximately threefold longer processive runs on trailing barbed ends of fascin-bundled F-actin. Here, we used single-molecule TIRFM (total internal reflection fluorescence microscopy) and developed a kinetic model to further dissect Ena/VASP’s processive mechanism on bundled filaments. We discovered that Ena’s enhanced processivity on trailing barbed ends is specific to fascin bundles, with no enhancement on fimbrin or α-actinin bundles. Notably, Ena/VASP’s processive run length increases with the number of both fascin-bundled filaments and Ena “arms,” revealing avidity facilitates enhanced processivity. Consistently, Ena tetramers form more filopodia than mutant dimer and trimers in Drosophila culture cells. Moreover, enhanced processivity on trailing barbed ends of fascin-bundled filaments is an evolutionarily conserved property of Ena/VASP homologues, including human VASP and Caenorhabditis elegans UNC-34. These results demonstrate that Ena tetramers are tailored for enhanced processivity on fascin bundles and that avidity of multiple arms associating with multiple filaments is critical for this process. Furthermore, we discovered a novel regulatory process whereby bundle size and bundling protein specificity control activities of a processive assembly factor.

The authors investigated the regulation of the Drosophila actin-microtubule cross-linker Short stop (Shot) and found that Shot undergoes an intramolecular conformational change that regulates its cross-linking activity. This intramolecular interaction depends on Shot's NH2-terminal actin-binding domain and EF-hand-GAS2 domain.