Focal adhesions are large multi-protein assemblies that form at the basal surface of cells on planar dishes, which mediate cell signaling, force transduction, and adhesion to the substratum. While much is known about focal adhesion components in 2-D systems, their role in migrating cells within a more physiological three-dimensional (3-D) matrix is largely unknown. Live-cell microscopy shows that for cells fully embedded in a 3-D matrix, focal adhesion proteins, including vinculin, paxillin, talin, α-actinin, zyxin, VASP, FAK, and p130Cas, do not form aggregates but are diffusively distributed throughout the cytoplasm. Despite the absence of detectable focal adhesions, focal adhesion proteins still modulate cell motility but in a manner distinct from cells on planar substrates. Rather, focal adhesion proteins in matrix-embedded cells regulate cell speed and persistence by affecting protrusion activity and matrix deformation, two processes that play no direct role in controlling 2-D cell speed. This study shows that membrane protrusions constitute a critical motility/matrix-traction module that drives cell motility in a 3-D matrix.

Mesenchymal cell migration through a three-dimensional (3D) matrix typically involves major matrix remodeling. The direction of matrix deformation occurs locally in all three dimensions, which cannot be measured by current techniques. To probe the local, 3D, real-time deformation of a collagen matrix during tumor cell migration, we developed an assay whereby matrix-embedded beads are tracked simultaneously in all three directions with high resolution. To establish a proof of principle, we investigated patterns of collagen I matrix deformation near fibrosarcoma cells in the absence and presence of inhibitors of matrix metalloproteinases and acto-myosin contractility. Our results indicate that migrating cells show patterns of local matrix deformation toward the cell that are symmetric in magnitude with respect to the axis of cell movement. In contrast, patterns of matrix release from the cell are asymmetric: the matrix is typically relaxed first at the back of the cell, allowing forward motion, and then at the cell's leading edge. Matrix deformation in regions of the matrix near the cell's leading edge is elastic and mostly reversible, but induces irreversible matrix rupture events near the trailing edge. Our results also indicate that matrix remodeling spatially correlates with protrusive activity. This correlation is mediated by myosin II and Rac1, and eliminated after inhibition of pericellular proteolysis or ROCK. We have developed an assay based on high-resolution 3D multiple-particle tracking that allows us to probe local matrix remodeling during mesenchymal cell migration through a 3D matrix and simultaneously monitor protrusion dynamics.

Cells often migrate in vivo in an extracellular matrix that is intrinsically three-dimensional (3D) and the role of actin filament architecture in 3D cell migration is less well understood. Here we show that, while recently identified linkers of nucleoskeleton to cytoskeleton (LINC) complexes play a minimal role in conventional 2D migration, they play a critical role in regulating the organization of a subset of actin filament bundles – the perinuclear actin cap - connected to the nucleus through Nesprin2giant and Nesprin3 in cells in 3D collagen I matrix. Actin cap fibers prolong the nucleus and mediate the formation of pseudopodial protrusions, which drive matrix traction and 3D cell migration. Disruption of LINC complexes disorganizes the actin cap, which impairs 3D cell migration. A simple mechanical model explains why LINC complexes and the perinuclear actin cap are essential in 3D migration by providing mechanical support to the formation of pseudopodial protrusions.