Helical buckling of actin inside filopodia generates traction

Abstract

Cells can interact with their surroundings via filopodia, which are membrane protrusions that extend beyond the cell body. Filopodia are essential during dynamic cellular processes like motility, invasion, and cell-cell communication. Filopodia contain crosslinked actin filaments, attached to the surrounding cell membrane via protein linkers such as integrins. These actin filaments are thought to play a pivotal role in force transduction, bending, and rotation. We investigated whether, and how, actin within filopodia is responsible for filopodia dynamics by conducting simultaneous force spectroscopy and confocal imaging of F-actin in membrane protrusions. The actin shaft was observed to periodically undergo helical coiling and rotational motion, which occurred simultaneously with retrograde movement of actin inside the filopodium. The cells were found to retract beads attached to the filopodial tip, and retraction was found to correlate with rotation and coiling of the actin shaft. These results suggest a previously unidentified mechanism by which a cell can use rotation of the filopodial actin shaft to induce coiling and hence axial shortening of the filopodial actin bundle.membrane-cytoskeleton interactions | membrane nanotubes | filopodial retrograde flow | helical buckling | filopodia rotation T ubular membrane remodeling driven by the actin cytoskeleton plays a major role in both pathogenesis and in a healthy immune response. For instance, invadopodia, podosomes, and filopodia are crucial for invasion and migration of cells (1). Filopodia are thin (100 to 300 nm), tube-like, actin-rich structures that function as "antennae" or "tentacles" that cells use to probe and interact with their microenvironment (2-4). Such structures have been studied in vitro using model systems where the point-like 3D contacts with the extracellular matrix (ECM) have been mimicked by using optically trapped dielectric particles, functionalized with relevant ligands. These model systems allow for mechanical and visual control over filopodial dynamics and have significantly advanced the understanding of cellular mechanosensing (5).Extraction of membrane tubes, using optical trapping, has been used to investigate mechanical properties of the membrane-cytoskeleton system (6-10), or membrane cholesterol content (11), and has revealed important insight into the mechanism that peripheral proteins use to shape membranes (12). Motivated by the pivotal role of F-actin in the mechanical behavior of filopodia and other cellular protrusions, special focus has been on revealing the presence of F-actin within extracted membrane tubes (1, 13-16). However, apart from a single study (9), fluorescent visualization of the F-actin was achieved by staining and fixation of the cells, and literature contains conflicting results regarding the presence or absence of actin in membrane tubes pulled from living cells (8,11,17).Filopodia in living cells have the ability to rotate and bend by a so-far unknown mechanism (13,(18)(19)(20). For instance, filopodia ...

Automated analysis of filopodial length and spatially resolved protein concentration via adaptive shape tracking

“…For instance, filopodia in C2C12 cells often appeared as lamellipodia retreated, whereas filopodia in COS cells actively extended from the cell body (Supplemental Figure S2, A and B). Furthermore, we also found helical buckling inside filopodia (Figure 6D), which was recently described as being used by cells to generate traction (Leijnse et al , 2015). Together these results suggest that various subforms of filopodial protrusions may exist.…”

Formin 2 regulates the stabilization of filopodial tip adhesions in growth cones and affects neuronal outgrowth and pathfinding in vivo

“…Forces originating from F-actin retrograde flow are transduced onto the substrate by dynamic molecular clutches that link the actin to the extracellular substrate (Bornschlögl et al, 2013;Chan and Odde, 2008;Mallavarapu and Mitchison, 1999). Additionally, helical buckling-induced shortening of the filopodial actin shaft can also contribute to filopodial traction (Leijnse et al, 2015). We propose that Fmn2 knockdown results in disorganized actin bundles in filopodia, impairing its ability to transduce forces to the tip adhesions (Fig.…”

“…In addition, unbalanced actin polymerization at the tip increases membrane protrusion. Recently, a second mechanism of filopodial force generation has been described involving helical buckling of filopodial actin bundles pulling on the substrate engaged to the filopodial tip (Leijnse et al, 2015). These studies highlight the importance of filopodial tip adhesions, although less is known about the mechanisms underlying adhesion dynamics and force transduction through the filopodial shaft.…”

Coordinating Neuronal Actin–Microtubule Dynamics

“…Membrane tension also opposes actin-mediated protrusion (6) [15]. Further means of force generation to enhance forward movement of the growth cone may include the helical buckling of F-actin bundles (7) [170]. Colour scheme as for Figures 1 and 3: brown area, growth cone C-domain; peach area, growth cone T-domain; pale yellow area, growth cone P-domain.…”