Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. I. Biochemical analysis.
The functional interaction of cells in the formation of tissues requires the establishment and maintenance of cell-cell contact by the junctional complex. However, little is known biochemically about the mechanism(s) that regulates junctional complex assembly. To address this problem, we have initiated a study of the regulation of assembly of one component of the junctional complex, the desmosome, during induction of cell-cell contact in cultures of Madin-Darby canine kidney epithelial cells. Here we have analyzed two major protein components of the desmosomal plaque, desmoplakins I (Mr of 250,000) and II (Mr of 215,000). Analysis of protein levels of desmoplakins I and II by immunoprecipitation with an antiserum that reacts specifically with an epitope common to both proteins revealed that desmoplakins I and II are synthesized and accumulate at steady state in a ratio of 3-4:1 (in the absence or presence of cell-cell contact). The kinetics of desmoplakins I and II stabilization and assembly were analyzed after partitioning of newly synthesized proteins into a soluble and insoluble protein fraction by extraction of whole cells in a Triton X-100 high salt buffer. In the absence of cell-cell contact, both the soluble and insoluble pools of desmoplakins I and II are unstable and are degraded rapidly (t1/2 approximately 8 h). Upon induction of cell-cell contact, the capacity of the insoluble pool increases approximately three-fold as a proportion of the soluble pool of newly synthesized desmoplakins I and II is titrated into the insoluble pool. The insoluble pool becomes relatively stable (t1/2 greater than 72 h), whereas proteins remaining in the soluble pool (approximately 25-40% of the total) are degraded rapidly (t1/2 approximately 8 h). Furthermore, we show that desmoplakins I and II can be recruited from this unstable soluble pool of protein to the stable insoluble pool upon induction of cell-cell contact 4 h after synthesis; significantly, the stabilization of this population of newly synthesized desmoplakins I and II is blocked by the addition of cycloheximide at the time of cell-cell contact, indicating that the coordinate synthesis of another protein(s) is required for protein stabilization.
Pasdar
M
M
Institute for Cancer Research, Philadelphia, Pennsylvania 19111.
Nelson
W J
WJ
D000818:Animals; D000906:Antibodies / Q000032:analysis; D002450:Cell Communication; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002999:Clone Cells; D003598:Cytoskeletal Proteins; D051180:Desmoplakins; D003896:Desmosomes / Q000032:analysis* / Q000378:metabolism; D004847:Epithelial Cells; D007106:Immune Sera / Q000276:immunology; D007118:Immunoassay; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D003598:Cytoskeletal Proteins; D051180:Desmoplakins; D007106:Immune Sera; D008562:Membrane Glycoproteins