Feedback interactions between cell-cell adherens junctions and cytoskeletal dynamics in Newt lung epithelial cells

To test how cell-cell contacts regulate microtubule (MT) and actin cytoskel etal dynamics, we examined dynamics in cells that were contacted on all sid es with neighboring cells in an epithelial eel sheet that was undergoing mi gration as a wound-healing response. Dynamics were recorded using time-laps e digital fluorescence microscopy of microinjected, labeled tubulin and act in. 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 instabi lity. In the contacted rear and side edges of these migrating cells, a majo rity of MTs were also quiescent, indicating that cell-cell contacts may loc ally regulate MT dynamics. Using photoactivation of fluorescence techniques to mark MTs, we found that MTs in fully contacted cells did not undergo re trograde flow toward the cell center, such as occurs at the leading edge of motile cells. Time-lapse fluorescent speckle microscopy of fluorescently l abeled actin in fully contacted cells revealed that actin did not flow rear ward 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 mu M or 1 00 nM nocodazole, breakage of cell-cell contacts occurred, indicating that MT growth is required for maintenance of cell-cell contacts. Analysis of fi xed cells indicated that during nocodazole treatment, actin became reduced in adherens junctions, and junction proteins alpha- and beta-catenin were l ost from adherens junctions as cell-cell contacts were broken. These result s indicate that a MT plus end capping protein is regulated by cell-cell con tact, and in turn, that MT growth regulates the maintenance of adherens jun ctions contacts in epithelia.