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

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 FMNL3-containing 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.

3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.

Cell invasion is a multi-step process, initiated by the acquisition of a migratory phenotype and the ability to move through complex 3D extracellular environments. We determine the composition of cell-matrix adhesion complexes of invasive breast cancer cells in 3D matrices and identify an interaction complex required for invasive migration. ßPix and myosin18A (Myo18A) drive polarized recruitment of non-muscle myosin 2A (NM2A) to adhesion complexes at the tips of protrusions. Actomyosin force engagement then displaces the Git1-ßPix complex from paxillin, establishing a feedback loop for adhesion maturation. We observe active force transmission to the nucleus during invasive migration that is needed to pull the nucleus forward. The recruitment of NM2A to adhesions creates a non-muscle myosin isoform gradient, which extends from the protrusion to the nucleus. We postulate that this gradient facilitates coupling of cell-matrix interactions at the protrusive cell front with nuclear movement, enabling effective invasive migration and front-rear cell polarity.

Focal Adhesions Undergo Longitudinal Splitting into Fixed-Width Units.

Focal adhesions (FAs) and stress fibers (SFs) act in concert during cell motility and in response to the extracellular environment. Although the structures of mature FAs and SFs are well studied, less is known about how they assemble and mature de novo during initial cell spreading. In this study using live-cell Airyscan microscopy, we find that FAs undergo "splitting" during their assembly, in which the FA divides along its longitudinal axis. Before splitting, FAs initially appear as assemblies of multiple linear units (FAUs) of 0.3-µm width. Splitting occurs between FAUs, resulting in mature FAs of either a single FAU or of a small number of FAUs that remain attached at their distal tips. Variations in splitting occur based on cell type and extracellular matrix. Depletion of adenomatous polyposis coli (APC) or vasodilator-stimulated phosphoprotein (VASP) results in reduced splitting. FA-associated tension increases progressively during splitting. Early in cell spreading, ventral SFs are detected first, with other SF sub-types (transverse arcs and dorsal SFs) being detected later. Our findings suggest that the fundamental unit of FAs is the fixed-width FAU, and that dynamic interactions between FAUs control adhesion morphology.

Cell type-dependent mechanisms for formin-mediated assembly of filopodia.

Filopodia are finger-like protrusions from the plasma membrane and are of fundamental importance to cellular physiology, but the mechanisms governing their assembly are still in question. One model, called convergent elongation, proposes that filopodia arise from Arp2/3 complex-nucleated dendritic actin networks, with factors such as formins elongating these filaments into filopodia. We test this model using constitutively active constructs of two formins, FMNL3 and mDia2. Surprisingly, filopodial assembly requirements differ between suspension and adherent cells. In suspension cells, Arp2/3 complex is required for filopodial assembly through either formin. In contrast, a subset of filopodia remains after Arp2/3 complex inhibition in adherent cells. In adherent cells only, mDia1 and VASP also contribute to filopodial assembly, and filopodia are disproportionately associated with focal adhesions. We propose an extension of the existing models for filopodial assembly in which any cluster of actin filament barbed ends in proximity to the plasma membrane, either Arp2/3 complex dependent or independent, can initiate filopodial assembly by specific formins.

The formin FMNL3 assembles plasma membrane protrusions that participate in cell-cell adhesion.

FMNL3 is a vertebrate-specific formin protein previously shown to play a role in angiogenesis and cell migration. Here we define the cellular localization of endogenous FMNL3, the dynamics of GFP-tagged FMNL3 during cell migration, and the effects of FMNL3 suppression in mammalian culture cells. The majority of FMNL3 localizes in a punctate pattern, with >95% of these puncta being indistinguishable from the plasma membrane by fluorescence microscopy. A small number of dynamic cytoplasmic FMNL3 patches also exist, which enrich near cell-cell contact sites and fuse with the plasma membrane at these sites. These cytoplasmic puncta appear to be part of larger membranes of endocytic origin. On the plasma membrane, FMNL3 enriches particularly in filopodia and membrane ruffles and at nascent cell-cell adhesions. FMNL3-containing filopodia occur both at the cell-substratum interface and at cell-cell contacts, with the latter being 10-fold more stable. FMNL3 suppression by siRNA has two major effects: decrease in filopodia and compromised cell-cell adhesion in cells migrating as a sheet. Overall our results suggest that FMNL3 functions in assembly of actin-based protrusions that are specialized for cell-cell adhesion.