Ena/VASP processive elongation is modulated by avidity on actin filaments bundled by the filopodia cross-linker fascin

Abstract

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.

Existing actin filaments orient new filament growth to provide structural memory of filament alignment during cytokinesis

“…In principle, this tethering could be mediated by the simultaneous association of CYK-1 dimers with a growing filament's barbed-end and adjacent filaments ( Figure 6F, left), as recently reported for another elongation factor Ena/VASP (Harker et al, 2019). However, Ena/VASP forms tetramers that can associate with multiple filaments while promoting processive filament elongation (Harker et al, 2019). In contrast, Formin dimers use both FH2 domains to associate with the barbed-end of the elongating actin filament to maintain rapid processive elongation.…”

“…One possible scenario is that one or more factors tether a growing filament's barbed end to the side of an existing filament. In principle, this tethering could be mediated by the simultaneous association of CYK-1 dimers with a growing filament's barbed-end and adjacent filaments ( Figure 6F, left), as recently reported for another elongation factor Ena/VASP (Harker et al, 2019). However, Ena/VASP forms tetramers that can associate with multiple filaments while promoting processive filament elongation (Harker et al, 2019).…”

Coefficient of variation as an image-intensity metric for cytoskeleton bundling

“…Bundling of the cytoskeleton, which consists of actin filaments and microtubules, is a critical step in the formation of the high-order structures that are tightly related to cellular activities including cell division [1][2][3][4] , cell morphogenesis 5,6 , and cell motility [7][8][9] . Microscopic assessment of cytoskeletal bundling is a fundamental experimental method in the field of cell biology.…”

Pharmaco-Genetic Screen To Uncover Actin Regulators Targeted by Prostaglandins DuringDrosophilaOogenesis

“…One intriguing means by which PGs may regulate Ena activity is through controlling Fascin, a known downstream effector of PGs (Groen et al 2012; Groen et al 2015). Indeed, Fascin has be shown to promote Ena processivity, increasing filament elongation (Winkelman et al 2014; Harker et al 2019).…”