A study of desmosome formation in kidney epithelial cells

Measures, Helen Rachel (1988) A study of desmosome formation in kidney epithelial cells. University of Southampton, Doctoral Thesis.

Abstract

Desmosomes are intercellular junctions mediating cell-cell adhesion. When epithelial cells are cultured in medium of low calcium concentration (< 0.05mM, LCM) desmosomes do not form but do so when the external calcium concentration is raised above 0.1 mM (SM). It has been shown previously in Madin Darby bovine kidney (MDBK) cells that the onset of desmosome formation after the calcium switch is slow (up to 8 hours). Since desmosomal proteins 1 and 2 (dp1/2) and desmosomal glycoprotein 1 (dg1) were not detected, by immunofluorescence, in MDBK cells cultured in LCM it was suggested that dp1/2 and dg1 had to be newly synthesized in these cells, after the calcium switch, before they could form desmosomes. In this study monoclonal and polyclonal antibodies specific for the individual desmosomal components were raised in order to investigate calcium-induced desmosome formation in these cells. Immunofluorescence studies using actinomycin D and cycloheximide, inhibitors of RNA and protein synthesis respectively, indicated that both protein and RNA synthesis were required after the switch for desmosome formation. However, metabolic labelling and immunoprecipitation showed that dp1/2 and dg1, as well as the desmosomal glycoproteins 2 and 3 (dg2/3) were synthesized in cells cultured in LCM and in SM. The rate of synthesis of the components was shown to increase at least 5-6 fold by 8 hours after the switch, compared to that in LCM. Biochemical extraction of cells revealed that the desmosomal components became insoluble, i.e. associated with the cytoskeleton, under both culture conditions. The rate of turnover of these components was faster in cells cultured in LCM than in those switched into SM, suggesting that the insoluble components were becoming stabilized by assembly into desmosomes. As previously shown for dg2/3, dg1 was exposed at the cell surface under both culture conditions, and accumulated in the cytoskeleton following the calcium switch, reaching a plateau after 48 hours. A model for the mechanism of desmosome formation in MDBK cells is proposed whereby the cell surface glycoproteins interact with similar molecules on apposing cells when the calcium concentration is raised. This interaction induces dp1/2, in association with keratin filaments, to move to the cell periphery and interact at these regions of intercellular contact, which in turn stabilizes the desmosomal components within the complex. An increase in the rate of synthesis of the components aids the continued recruitment of components and the formation of morphologically recognizable desmosomes. (DX86706)

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