Paxillin Mediates Sensing of Physical Cues and Regulates Directional Cell Motility by Controlling Lamellipodia Positioning

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.

Mechanotransduction of fluid stresses governs 3D cell migration

“…S8), but migration speed was unaffected (SI Appendix, Fig. S8), consistent with the results using a scratch assay in fibroblasts (8).…”

“…The vinculin-GFP plasmid has been described and characterized previously (48) and was a gracious gift of Benjamin Geiger (Weizmann Institute of Science, Rehovot, Israel). siRNA against human paxillin (GTGTGGAGCCTTCTTTGGT) was purchased from Sigma Genosys (8). Cells were transfected with SiLentFect according to the manufacturer's protocol (Bio-Rad) (SI Appendix, Supplementary Methods).…”

“…Paxillin is required for stiffness mechanosensing (6), protrusion formation at regions of high stress (8), and durotaxis (9). To evaluate the role of paxillin in upstream FA polarization and rheotaxis, we treated cells with paxillin siRNA, resulting in knockdown of paxillin expression by 80% as confirmed by immunoblotting (Fig.…”

“…The FA protein paxillin colocalizes with vinculin (4) and mediates β1-integrin FA turnover through interaction with FA kinase (FAK) (5). The FAK-paxillin signaling axis recruits vinculin to β1 integrins at regions of high matrix adhesion tension (6), and paxillin-a key mechanosensor (7)-mediates protrusion formation at regions of high stress on 2D substrates (8), and FAK-paxillin-vinculin signaling is required for mechanosensing and durotaxis (9).…”

Low-intensity pulsed ultrasound promotes cell motility through vinculin-controlled Rac1 GTPase activity

“…These cells are ideal for actin-related studies as they are highly sensitive to changes in intracellular signalling, and undergo fast actin rearrangements (Ballestrem et al, 2000(Ballestrem et al, , 1998. LIPUS stimulation of these cells promoted the formation of actin-based circular dorsal ruffles (CDRs) which are associated with macropinocytosis (Dharmawardhane et al, 2000;Dowrick et al, 1993) and cell motility (Krueger et al, 2003;Sero et al, 2011;Suetsugu et al, 2003). CDR formation increased almost immediately upon stimulation, with the greatest number seen 10 min after the end of LIPUS stimulation ( Fig.…”