Fig. 1. Spatial distribution of actin populations during induction of cell-cell contacts. (A) Keratinocytes grown in low calcium medium (Low Ca²⁺), which does not induce cell-cell contacts, were induced to form cadherin-dependent cell-cell adhesion (standard calcium medium, Std. Ca²⁺). After junction formation, two actin populations can be distinguished on the basis of their location: one is present as a wavy, punctate line at junctions (junctional actin, arrowheads); the second population is a tighter array of thin bundles in the cytoplasm, flanking the junctional-actin population (arrow). By 60 minutes, the two populations are less readily distinguished (mature junctional actin). Bar, 50 µm. (B) Percentage of keratinocytes classified into four different categories according to the spatial organization of thin bundles following junction formation. Categories observed: (1) a wide, loose band of actin filaments, and no F-actin at cell-cell borders; (2) F-actin at junctions is visible (junctional actin) and the band of filaments localizes closer to cell-cell contacts; (3) junctional actin is more intense; filaments are more tightly bundled in a narrower region proximal to junctions or indistinguishable from F-actin at cell-cell contacts; (4) other phenotypes (less than 5-10% of total). Results are the average of two independent experiments, in which 200 cells were scored per time point in each experiment. (C) Following induction of cell-cell contacts in sub-confluent keratinocytes, cytoskeletal changes show a delayed formation of junctional actin and reorganization of thin bundles. For example, thin bundles are not coincident with junctional actin after 120 minutes of junction formation. Additional actin structures are also seen before junctional actin is properly stabilized (i.e. kissing structures, inset 30 minutes). Presumably these structures participate in the migration of two neighbouring cells towards each other as they are not readily observed in confluent cultures.