Abstract:During healing of the skin, the cytoskeleton of keratinocytes and their matrix adhesions, including focal adhesions (FAs), undergo reorganization. These changes are coordinated by small GTPases and their regulators, including the guanine nucleotide exchange factor β-PIX (also known as ARHGEF7). In fibroblasts, β-PIX activates small GTPases, thereby enhancing migration. In keratinocytes in vitro, β-PIX localizes to FAs. To study β-PIX functions, we generated β-PIX knockdown keratinocytes. During wound closure o… Show more
“…We used the FLP/FRT system to generate clones of dpix or git mutant tissue in otherwise heterozygous animals, and found that loss of dpix or git causes multilayering of the follicular epithelium (Fig 1H–1I’), even in small clones (S1G Fig), indicating that the requirement for dpix and git is autonomous to the follicle cells. Interestingly, while PIX and GIT proteins are known to influence focal adhesion turnover and maturation in cell culture systems [38–41], we did not see an obvious effect on the accumulation of the focal adhesion component talin in monolayered dpix or git mutant tissue (Fig 1H–1I’), however talin levels were often increased in multilayered tissue (S1E and S1F Fig). We did not see a general disruption of apical-basal polarity in monolayered clones, as indicated by unchanged apical localisation of β-heavy-spectrin in dpix clones (S1H–S1H’ Fig).…”
How biochemical and mechanical information are integrated during tissue development is a central question in morphogenesis. In many biological systems, the PIX-GIT complex localises to focal adhesions and integrates both physical and chemical information. We used
Drosophila melanogaster
egg chamber formation to study the function of PIX and GIT orthologues (dPix and Git, respectively), and discovered a central role for this complex in controlling myosin activity and epithelial monolayering. We found that Git’s focal adhesion targeting domain mediates basal localisation of this complex to filament structures and the leading edge of migrating cells. In the absence of
dpix
and
git
, tissue disruption is driven by contractile forces, as reduction of myosin activators restores egg production and morphology. Further,
dpix
and
git
mutant eggs closely phenocopy defects previously reported in
pak
mutant epithelia. Together, these results indicate that the dPix-Git complex controls egg chamber morphogenesis by controlling myosin contractility and Pak kinase downstream of focal adhesions.
“…We used the FLP/FRT system to generate clones of dpix or git mutant tissue in otherwise heterozygous animals, and found that loss of dpix or git causes multilayering of the follicular epithelium (Fig 1H–1I’), even in small clones (S1G Fig), indicating that the requirement for dpix and git is autonomous to the follicle cells. Interestingly, while PIX and GIT proteins are known to influence focal adhesion turnover and maturation in cell culture systems [38–41], we did not see an obvious effect on the accumulation of the focal adhesion component talin in monolayered dpix or git mutant tissue (Fig 1H–1I’), however talin levels were often increased in multilayered tissue (S1E and S1F Fig). We did not see a general disruption of apical-basal polarity in monolayered clones, as indicated by unchanged apical localisation of β-heavy-spectrin in dpix clones (S1H–S1H’ Fig).…”
How biochemical and mechanical information are integrated during tissue development is a central question in morphogenesis. In many biological systems, the PIX-GIT complex localises to focal adhesions and integrates both physical and chemical information. We used
Drosophila melanogaster
egg chamber formation to study the function of PIX and GIT orthologues (dPix and Git, respectively), and discovered a central role for this complex in controlling myosin activity and epithelial monolayering. We found that Git’s focal adhesion targeting domain mediates basal localisation of this complex to filament structures and the leading edge of migrating cells. In the absence of
dpix
and
git
, tissue disruption is driven by contractile forces, as reduction of myosin activators restores egg production and morphology. Further,
dpix
and
git
mutant eggs closely phenocopy defects previously reported in
pak
mutant epithelia. Together, these results indicate that the dPix-Git complex controls egg chamber morphogenesis by controlling myosin contractility and Pak kinase downstream of focal adhesions.
“…β-PIX also accumulates in assembling nascent adhesions in the absence of NMII contractility, and it dissociates from focal adhesions during NMII-meditated focal adhesion maturation ( Kuo et al , 2011 ). In fibroblasts, astrocytes, and certain epithelial cells, β-PIX is a negative regulator of focal adhesion maturation ( Zhao et al , 2000 ; Kuo et al , 2011 ), and loss of β-PIX inhibits focal adhesion disassembly ( Kuo et al , 2011 ; Hiroyasu et al , 2017 ). Indeed, cells depleted for Scrib exhibit large continuous focal adhesions, and we propose that β-PIX in these cells is inactive, leading to aberrant focal adhesion dynamics because of the inhibition of focal adhesion disassembly.…”
Scribble (Scrib) and Lgl1 are conserved polarity proteins that play important roles in different forms of cell polarity. The roles of Scrib and Lgl1 in apical-basal cell polarity has been studied extensively, but little is known about their roles in the cell polarity of migrating cells. Furthermore, the effect of Scrib and Lgl1 interaction on cell polarity is largely unknown. In this study, we show that Scrib, through its LRR domain, forms a complex in vivo with Lgl1. Scrib also forms a complex with myosin II, and Scrib, Lgl1, and myosin II co-localize at the leading edge of migrating cells. The cellular localization and the cytoskeletal association of Scrib and Lgl1 are interdependent, as depletion of either protein affects its counterpart. In addition, depletion of either Scrib or Lgl1 disrupts the cellular localization of myosin II. We show that depletion of either Scrib or Lgl1 affects cell adhesion through the inhibition of focal adhesion disassembly. Finally, we show that Scrib and Lgl1 are required for proper cell polarity of migrating cells. These results provide new insights into the mechanism regulating the cell polarity of migrating cells by Scrib, Lgl1, and myosin II.
“…A key role for βPIX in the regulation of adhesion dynamics and cell migration has also been shown to support lamellipodial protrusions by negatively regulating adhesion maturation and promoting rapid adhesion disassembly 32 . In keratinocytes, βPIX is localized to adhesions in the leading edge during single cell migration and is important for recruiting PAK to adhesions 33 .…”
Cell migration is an important biological phenomenon involved in many homeostatic and aberrant physiological processes. Phosphorylation of the focal adhesion adaptor protein, paxillin, on serine 273 (S273) has been implicated as a key regulator of cell migration. Here, it is shown that phosphorylation on paxillin S273 leads to highly migratory cells with small dynamic adhesions. Adhesions at protrusive edges of the cell were more dynamic than adhesions at retracting edges. Temporal image correlation microscopy revealed that these dynamic adhesions undergo rapid binding of paxillin, PAK1 and βPIX. We identified membrane proximal adhesion subdomains in protrusive regions of the cell that show rapid protein binding that is dependent on paxillin S273 phosphorylation, PAK1 kinase activity and phosphatases. These dynamic adhesion subdomains corresponded to regions of the adhesion that also show co-binding of paxillin/PAK1 and paxillin/βPIX complexes. It is likely that parts of individual adhesions are more dynamic while others are less dynamic due to their association with the actin cytoskeleton. Variable adhesion and binding dynamics are regulated via differential paxillin S273 phosphorylation across the cell and within adhesions and are required for regulated cell migration. Dysregulation through phosphomutants, PAK1-KD or βPIX mutants resulted in large stable adhesions, long protein binding times and slow cell migration. Dysregulation through phosphomimics or PAK1-CA led to small dynamic adhesions and rapid cell migration reminiscent of highly migratory cancer cells. Thus, phosphorylation of paxillin S273 is a key regulator of cell migration through recruitment of βPIX and PAK1 to sites of adhesion.
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