Feedback Interactions between Cell–Cell Adherens Junctions and Cytoskeletal Dynamics in Newt Lung Epithelial Cells

摘要

To test how cell–cell contacts regulate microtubule (MT) and actin cytoskeletal dynamics, we examined dynamics in cells that were contacted on all sides with neighboring cells in an epithelial cell sheet that was undergoing migration as a wound-healing response. Dynamics were recorded using time-lapse digital fluorescence microscopy of microinjected, labeled tubulin and actin. In fully contacted cells, most MT plus ends were quiescent; exhibiting only brief excursions of growth and shortening and spending 87.4% of their time in pause. This contrasts MTs in the lamella of migrating cells at the noncontacted leading edge of the sheet in which MTs exhibit dynamic instability. In the contacted rear and side edges of these migrating cells, a majority of MTs were also quiescent, indicating that cell–cell contacts may locally regulate MT dynamics. Using photoactivation of fluorescence techniques to mark MTs, we found that MTs in fully contacted cells did not undergo retrograde flow toward the cell center, such as occurs at the leading edge of motile cells. Time-lapse fluorescent speckle microscopy of fluorescently labeled actin in fully contacted cells revealed that actin did not flow rearward as occurs in the leading edge lamella of migrating cells. To determine if MTs were required for the maintenance of cell–cell contacts, cells were treated with nocodazole to inhibit MTs. After 1–2 h in either 10 μM or 100 nM nocodazole, breakage of cell–cell contacts occurred, indicating that MT growth is required for maintenance of cell–cell contacts. Analysis of fixed cells indicated that during nocodazole treatment, actin became reduced in adherens junctions, and junction proteins α- and β-catenin were lost from adherens junctions as cell–cell contacts were broken. These results indicate that a MT plus end capping protein is regulated by cell–cell contact, and in turn, that MT growth regulates the maintenance of adherens junctions contacts in epithelia.