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Intact epithelial sheets were removed from rabbit corneas using Dispase II, a bacterial neutral protease. The freed sheets were placed on denuded corneal basal laminae and incubated at 35 degrees C for 3, 6, 18, or 24 h. Epithelial-basal lamina preparations were incubated in culture medium that either contained (a) varying concentrations of Ca2+ ions, (b) calmodulin antagonists, (c) exogenous calmodulin following an initial 6-h incubation in the presence of antagonists, or that lacked (d) Mg2+ ions. Tissues were processed for electron microscopy, and micrographs were taken of basal cell membranes. At least four experiments were conducted for each treatment, and for each experiment the total number of hemidesmosomes were counted along the basal membrane-basal lamina surface of eight cells. The number of hemidesmosomes formed was directly proportional to the increasing concentration of Ca2+. The presence of absence of Mg2+ ions did not change the numbers of hemidesmosomes formed. Calmodulin antagonists inhibited hemidesmosome formation, and this inhibition was reversed by the addition of calmodulin. Thus, hemidesmosome formation is Ca2+ dependent and appears to be mediated by a calmodulin-regulated mechanism.

Role of calcium and calmodulin in hemidesmosome formation in vitro.