BPS2026 – Probing the role of VASP condensates in the assembly of filopodia at the leading edge of motile cells

Filopodia are slender, actin-filled plasma membrane protrusions that play essential roles in sensing the extracellular environment, cell migration, adhesion, and tissue morphogenesis. The actin polymerase, vasodilator-stimulated phosphoprotein (VASP), is a central regulator of filopodia formation, dynamics, and architecture. VASP is recruited to the membrane through interactions with lamellipodin and other cytoskeletal proteins, forming dynamic clusters that accumulate at the tips of nascent filopodia. Within these clusters, VASP promotes processive actin filament elongation, shielding filament barbed ends from capping, while organizing actin filaments into parallel bundles within tubular membrane protrusions. Recently our lab reported that VASP can form protein condensates that facilitate actin polymerization and bundling in vitro. Building on these findings, here we report the role of VASP condensates during assembly of filopodia at the leading edge of motile cells. Specifically, we developed an optogenetic tool, the VASP-CRY2 chimera, in which the VASP tetramerization domain was replaced by CRY2, a domain which forms reversible multimers upon blue-light exposure. We expressed VASP-CRY2 in mouse melanoma cells lacking VASP and its homologs. Without blue light, the protein remained monomeric and biologically inactive, failing to localize filopodia tips or rescue filopodia loss from VASP knockout. However, upon blue light exposure, VASP-Cry2 assembled into flexible clusters that localized to filopodia tips, leading to a dramatic increase in the number of filopodia within minutes. Interestingly, exposing cells to higher blue light formed rigid VASP condensates, inhibiting filopodia assembly. These results suggest that flexible VASP condensates provide an optimal platform for assembly of filopodia. Our ongoing work seeks to determine how condensates balance the recruitment of multiple biochemical factors to the filopodial tip, while remaining sufficiently flexible to promote membrane and cytoskeletal remodeling.