Roles for Ena/VASP proteins in FMNL3-mediated filopodial assembly

Abstract

Filopodia are actin-dependent finger-like structures that protrude from the plasma membrane. Actin filament barbed-end-binding proteins localized to filopodial tips are key to filopodial assembly. Two classes of barbed-end-binding proteins are formins and Ena/VASP proteins, and both classes have been localized to filopodial tips in specific cellular contexts. Here, we examine the filopodial roles of the FMNL formins and Ena/VASP proteins in U2OS cells. FMNL3 suppression reduces filopodial assembly by 90%, and FMNL3 is enriched at >95% of filopodial tips. Suppression of VASP or Mena (also known as ENAH) reduces filopodial assembly by >75%. However, VASP and Mena do not display consistent filopodial tip localization, but are enriched in focal adhesions (FAs). Interestingly, >85% of FMNL3containing filopodia are associated with FAs. Two situations increase Ena/VASP filopodial localization: (1) expression of myosin-X, and (2) actively spreading cells. In spreading cells, filopodia often mark sites of nascent adhesions. Interestingly, VASP suppression in spreading cells causes a significant increase in adhesion assembly at filopodial tips. This work demonstrates that, in U2OS cells, Ena/VASP proteins play roles in filopodia beyond those at filopodial tips. This article has an associated First Person interview with the first author of the paper.

A direct role for SNX9 in the biogenesis of filopodia

“…Ena/VASP proteins are important in filopodia formation in cortical neurons and retinal ganglion cells, where their roles in elongating F-actin are thought to be direct (Dent et al, 2007; Dwivedy et al, 2007). However, in osteosarcoma cells, although reduced levels of Ena/VASP proteins inhibit filopodia, they localize to focal adhesions in the cell body rather than at filopodia tips, which suggests an indirect role (Young et al, 2018).…”

Loss of Ena/VASP interferes with lamellipodium architecture, motility and integrin-dependent adhesion

“…Indeed, FMNL2/3 removal caused defects partly reminiscent of what’s seen here, at least concerning impact on lamellipodial protrusion, actin filament densities or microspike formation (see below), although phenotypes seen upon Ena/VASP deficiency were much more severe. Thus, the extent of potential redundancy between all these factors will only be revealed by systematic, combinatorial loss of function studies and side-by-side, phenotypic comparison (Young et al, 2018). Derived conclusions will also have to consider the exciting, recently discovered ‘distributive polymerase’ activity ascribed to WAVE (Bieling et al, 2018).…”

“…Formins are dimeric multi domain proteins that remain tightly associated with growing filament barbed ends while accelerating their growth (Kovar et al, 2006). Several formins such as mDia2 (Block et al, 2008; Yang et al, 2007) and the formin-like family members 2 (FMNL2) and 3 (Block et al, 2012; Harris et al, 2010; Kage et al, 2017; Young et al, 2018) localize at lamellipodia and filopodia tips, and have been implicated in driving these protrusions and cell migration.…”

Leading-edge VASP clusters assemble at sites containing lamellipodin and exhibit size-dependent instability

“…Filopodia are thin, finger-like protrusions of the plasma membrane that participate in fundamental cellular processes, including directed migration, substrate adhesion, and cell-cell communication (Blanchoin et al 2014;Mattila and Lappalainen 2008) . Filopodia are defined by morphology rather than function or composition, and growing evidence suggests that different types of filopodia assemble via different mechanisms and employ different sets of actin regulators (Yang and Svitkina 2011;Young et al 2018;Barzik et al 2014;Young et al 2015) .…”