Helical buckling of actin inside filopodia generates traction

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 cross-linked 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.Tubular 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, 18–20). For instance, filopodia have been reported to exhibit sharp kinks in neuronal cells (21–23) and macrophages (6). These filopodial kinks have been observed both for surface-attached filopodia (23) as well as for filopodia that were free to rotate in three dimensions (6, 19).Other filaments such as DNA and bacterial flagella are common examples of structures that shorten and bend in response to torsional twist. Filopodia have previously been shown to have the ability to rotate by a mechanism that exists at their base (18, 19) and could have the ability to twist in presence of a frictional force. Rotation of the actin shaft results in friction with the surrounding filopodial membrane, and hence torsional energy can be transferred to the actin shaft. The myosin-Vb motor has been found to localize at the base of filopodia in neurite cells and to be critical for rotational movement of the filopodia; however, inhibition of myosin-Vc did not affect the rotation (19).Here, we reveal how actin filaments can simultaneously rotate and helically bend within cellular membrane tubes obtained by elongation of preexisting filopodia by an optically trapped bead. Simultaneous force measurements and confocal visualization reveal how the actin transduces a force as it rotates and retracts. After ∼100 s, the force exerted by the filopodium starts to exhibit pulling events reflecting transient contact between the actin and the tip region of the filopodium, which is attached to the optically trapped bead. We show that helical bending and rotation of the actin shaft (defined as the visible part of the actin) occurs simultaneously with movement of actin coils inside filopodia, which can occur concomitantly with a traction force exerted at the tip. The velocity of the coils depends on their location along the tube, thus indicating that retrograde flow is not the only mechanism driving the motion. Our data, and accompanying calculations, show that the rotation of the actin shaft, and the resulting torsional twist energy accumulated in the actin shaft, can contribute to shortening and bending of the actin shaft in conjunction with a retrograde flow.