Abstract
Physical interactions between cells and the extracellular matrix (ECM) guide directional migration by spatially controlling where cells form focal adhesions (FAs), which in turn regulate the extension of motile processes. Here we show that physical control of directional migration requires the FA scaffold protein paxillin. Using single-cell sized ECM islands to constrain cell shape, we found that fibroblasts cultured on square islands preferentially activated Rac and extended lamellipodia from corner, rather than side regions after 30 min stimulation with PDGF, but that cells lacking paxillin failed to restrict Rac activity to corners and formed small lamellipodia along their entire peripheries. This spatial preference was preceded by non-spatially constrained formation of both dorsal and lateral membrane ruffles from 5–10 min. Expression of paxillin N-terminal (paxN) or C-terminal (paxC) truncation mutants produced opposite, but complementary, effects on lamellipodia formation. Surprisingly, pax−/− and paxN cells also formed more circular dorsal ruffles (CDRs) than pax+ cells, while paxC cells formed fewer CDRs and extended larger lamellipodia even in the absence of PDGF. In a two-dimensional (2D) wound assay, pax−/− cells migrated at similar speeds to controls but lost directional persistence. Directional motility was rescued by expressing full-length paxillin or the N-terminus alone, but paxN cells migrated more slowly. In contrast, pax−/− and paxN cells exhibited increased migration in a three-dimensional (3D) invasion assay, with paxN cells invading Matrigel even in the absence of PDGF. These studies indicate that paxillin integrates physical and chemical motility signals by spatially constraining where cells will form motile processes, and thereby regulates directional migration both in 2D and 3D. These findings also suggest that CDRs may correspond to invasive protrusions that drive cell migration through 3D extracellular matrices.
Highlights
Directional cell migration is a multi-step process that involves actin-driven protrusion of the plasma membrane, designation of a leading edge, formation of new cell-extracellular matrix (ECM) adhesions, contraction of the cytoskeleton, and disassembly of rearward adhesions [1]
Focal adhesions and lamellipodia are spatially restricted in square cells Microcontact-printed substrates consisting of arrays of square ECM islands (900–2500 mm2) surrounded by non-adhesive regions were prepared by direct stamping of fibronectin (FN) onto activated PDMS-coated coverslips, followed by blocking of unstamped areas with Pluronic F-127 (Fig. S1) [33,34]
Human fibroblasts plated on these islands spread and adopted square shapes, as previously reported for cells cultured on ECM islands formed by stamping self-assembled monolayers of alkanethiols on gold [9]
Summary
Directional cell migration is a multi-step process that involves actin-driven protrusion of the plasma membrane, designation of a leading edge, formation of new cell-extracellular matrix (ECM) adhesions, contraction of the cytoskeleton, and disassembly of rearward adhesions [1]. Many studies have focused on migration directed by gradients of soluble factors, directional motility can be physically controlled by adhesive gradients (haptotaxis [2]), mechanical stiffness (durotaxis [3,4]); alignment of ECM features (contact guidance [5,6,7]), and variations in the geometry of the ECM that affect cell shape (shape-dependent motility control [8,9,10,11]). Cell spreading on adhesive substrates is driven in part by cytoskeletal traction forces that are resisted mechanically by the ECM [12,13]. Mechanical forces are transmitted between the ECM and cytoskeleton through transmembrane receptors, such as integrins, which are coupled to the cytoskeleton via adaptor proteins in multi-protein anchoring complexes called focal adhesions (FAs) [14]. FAs are considered to be mechanosensitive organelles that facilitate the conversion of mechanical and spatial cues from the microenvironment into changes in cytoskeletal architecture and biochemical signaling [12,15,17]
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