Plectin-containing, centrally-localized focal adhesions exert traction forces in primary lung epithelial cells

Abstract

SummaryReceptor clustering upon cell attachment to the substrate induces assembly of cytoplasmic protein complexes termed focal adhesions (FAs), which connect, albeit indirectly, the extracellular matrix to the cytoskeleton. A subset of cultured primary alveolar epithelial cells (AEC) display a unique pattern of vinculin/paxillin/talin-rich FAs in two concentric circles when cultured on glass and micropatterned substrates: one ring of FAs located at the cell periphery (pFAs), and another FA ring located centrally in the cell (cFAs). Unusually, cFAs associate with an aster-like actin array as well as keratin bundles. Moreover, cFAs show rapid paxillin turnover rates following fluorescence recovery after photobleaching and exert traction forces similar to those generated by FAs at the cell periphery. The plakin protein plectin localizes to cFAs and is normally absent from pFAs, whereas tensin, a marker of mature/fibrillar adhesions, is found in both cFAs and pFAs. In primary AEC in which plectin expression is depleted, cFAs are largely absent, with an attendant reorganization of both the keratin and actin cytoskeletons. We suggest that the mechanical environment in the lung gives rise to the assembly of unconventional FAs in AEC. These FAs not only show a distinctive arrangement, but also possess unique compositional and functional properties.

Loss of β-PIX inhibits focal adhesion disassembly and promotes keratinocyte motility via myosin light chain activation

“…The impact of β-PIX KD on FAs, cytoskeleton organization and MLC activation led us to investigate whether these changes result in modulation of the traction forces exerted by cells on their substrate. To do so, we plated HaCaT cells onto a fluorescent bead-embedded gel substrate as described previously (Eisenberg et al, 2013;Hiroyasu et al, 2016). We then compared traction force localization and magnitudes exerted by a control cells, β-PIX KD HaCaT clones and GFP-tagged β-PIX expressing β-PIX KD HaCaT cells.…”

“…Traction force microscopy was performed as described elsewhere (Eisenberg et al, 2013;Hiroyasu et al, 2016). In brief, cells were plated on polyacrylamide gels embedded with fluorescent beads.…”

Intermediate Filaments and the Plasma Membrane

“…Indeed, these same investigators speculated that FAs are the IF equivalent of the microtubule-organizing center (MTOC), a suggestion supported by other studies mentioned above that reveal that newly synthesized vimentin IFs appear to assemble from FAs. Moreover, more recently, keratin IF interaction with an unusually arranged set of FAs has been observed in primary epithelial cells isolated from the alveolar compartment of the lung (Eisenberg et al 2013). In these cells, keratin IFs wrap around a group of FAs located toward the center of the cells (Fig.…”

“…Indeed, in primary rat and mouse lung alveolar cells, DG is a component of a set of unique matrix adhesions arranged in a ring-like array toward the perinuclear zone (Eisenberg et al 2013). These matrix adhesions not only contain typical FA components such as paxillin, vinculin, and talin, but also possess plectin and DG (Fig.…”

“…These matrix adhesions not only contain typical FA components such as paxillin, vinculin, and talin, but also possess plectin and DG (Fig. 6B) (Eisenberg et al 2013). Moreover, they are associated with, and appear to constrain, the organization of the keratin IF cytoskeleton of the cell (Fig.…”