Multiple rows of cells behind an epithelial wound edge extend cryptic lamellipodia to collectively drive cell-sheet movement

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Abstract
The mechanism by which epithelial, endothelial and other strongly cell-cell adhesive cells migrate collectively as continuous sheets is not clear, even though this process is crucial for embryonic development and tissue repair in virtually all multicellular animals. Wound closure in Madin-Darby canine kidney (MDCK) epithelial cell monolayers involves Rac GTPase-dependent migration of cells both at and behind the wound edge. We report here for the first time that cells behind the margin of wounded MDCK cell monolayers, even hundreds of microns from the edge, extend `cryptic' lamellipodia against the substratum beneath cells in front of them, toward the wound, as determined by confocal, two-photon and transmission electron microscopy. These so-called submarginal cells nevertheless strictly maintain their more apical cell-cell contacts when they migrate as part of a coherent cell sheet, hiding their basal protrusions from conventional microscopy. The submarginal protrusions display the hallmarks of traditional lamellipodia based on morphology and dynamics. Cells behind the margin therefore actively crawl, instead of just moving passively when cells at the margin pull on them. The rate of migration is inversely proportional to the distance from the margin, and cells move co-ordinately, yet still in part autonomously, toward the wound area. We also clarify the ancillary role played by nonprotrusive contractile actin bundles that assemble in a Rho GTPase-dependent manner at the margin after wounding. In addition, some cell proliferation occurs at a delay after wounding but does not contribute to closure. Instead, it apparently serves to replace damaged cells so that intact spread cells can revert to their normal cuboidal morphology and the original cell density of the unbroken sheet can be restored.