Figure 6.

Inefficient cell–cell junction formation in calcium-treated keratinocytes as a consequence of tyrosine kinase inhibition or lack of the Fyn kinase. (A) Confluent keratinocyte cultures under growing conditions were either kept as untreated controls (Control) or pretreated for 2 h with 100 μM Genistein (Gen) or Tyrphostin 23 (Trph). Incubation was then continued for additional 9 h under high calcium conditions. Cells were fixed and processed for electron microscopy as described in Materials and Methods. The protrusions from neighboring cells in the Genistein-treated cultures were consistently observed. (B) Primary keratinocytes derived from fyn−/− mice and wild-type littermates were exposed to high calcium concentrations (2 mM) for 9 h. Cells were fixed and processed for electron microscopy. Note that in the fyn−/− cultures, cell borders were far apart and connected by protrusions of the cell membrane similar to those found with genistein-treated keratinocytes (as shown in A). Note the presence of well formed cell adhesive junctions in control cells and fyn+/+ cells (A and B, white arrows) and the lack of well-formed junctions and of cytoskeleton organization in the tyrosine kinase inhibitor treated and fyn−/− cells (A and B, black arrows). Adherens junctions and desmosomes can be defined as electron dense intercellular adhesive structures connected with the actin and keratin cytoskeleton, respectively. Unlike in vivo, in most of our EM photographs of cultured keratinocytes it is hard to see electron-dense junctional structures in connection with either the actin or keratin cytoskeleton, and therefore to conclusively distinguish between adherens junctions and desmosomes. In all cases, the observed alterations (>70% reduction in mature cell junction formation) were observed throughout the dishes (in each case examining at least 10 different fields), and were confirmed in a minimal of two independent experiments. Bars: (A) 200 nm; (B) 100 nm.