Analysis of actin filament bundle dynamics during contact formation in live epithelial cells

The actin cytoskeleton is an integral component of the cell-cell adherens junction complex. We used fluorescence labeling of actin filaments and time-lapse laser scanning confocal microscopy to investigate the functional relationship between the organization of the actin cytoskeleton and formation of adherens junctions in live epithelial cells. Rhodamine-phalloidin was loaded into cultured cells by wounding epithelial monolayers in the presence of fluorescent analog. Rhodamine-phalloidin was incorporated into the actin filaments in stress fibers, circumferential bundles, and marginal bundles. Cells containing labeled actin filaments appeared physiologically normal since the rates of migration, rates of pseudopodial protrusion/retraction, ability to form contacts, and sensitivity to cytochalasin B were equivalent to non-loaded, control epithelial cells. Marginal actin bundles initially formed as bow-shaped bundles that were observed to straighten as the bundles flowed rearward and away from the free cell edge. When lamellae from adjacent cells made contact, rearward flow of marginal bundles ceased and the bundles started to disassemble with higher frequency. Next, we observed the formation of arc-like bundles at the edges of contacting cells, a position suggestive of a role in lateral expansion of the contact. During later stages of contact formation, new actin bundles assembled along the length of the expanding cell-cell boundary. These newly formed bundles are likely to participate in the establishment of the initial cadherin/actin cytoskeleton linkage and eventually form the circumferential bundles at the cell-cell adherens junction. Additionally, indirect immunolocalization studies characterized the location of myosin-II. A model is presented describing the function of the spatial and temporal dynamics of actin filament bundles and myosin-II activity in the formation of adherens junctions.