CRITICAL ROLE FOR INTRACELLULAR CALCIUM IN TIGHT JUNCTION BIOGENESIS

Using the Madin Darby canine kidney (MDCK) cell ''calcium switch,'' we have previously demonstrated that, as MDCK cells establish contact an d ultimately form tight junctions, there are marked global and localiz ed changes in intracellular calcium at the sites of cell-cell contact (Nigam et al., 1992, Proc. Natl. Acad. Sci. USA, 89:6162-6166). We hav e now examined whether intracellular Ca++ is critical to the biogenesi s of tight junctions by chelating this ion and monitoring the formatio n of junctions by electrical, immunocytochemical, and biochemical crit eria. Intracellular Catt was chelated with the cell-permeant chelators , dimethyl BAPTA-AM and BAPTA-AM. By digital imaging of fura-2 loaded cells, it was demonstrated that both agents efficiently chelated Ca+during the ''switch'' in a dose-dependent manner which paralleled thei r respective in vitro affinities for Ca++. Chelation of Ca++ during th e switch markedly attenuated the development of transepithelial electr ical resistance (TER), a measure of tight junction assembly. Immunoflu orescent staining of the tight junctional protein, zonula occludens-1 (ZO-1), revealed that chelation of intracellular Ca++ retarded the mov ement oi ZO-1 from intracellular sites to the plasma membrane during t he switch. During the development of tight junctions, a fraction of ZO -1 redistributed from the Triton X-100 soluble to the Triton X-100 ins oluble pool; chelation of Ca++ during the induction of cell-fell conta ct prevented this stabilization into the Triton X-100 insoluble fracti on. Taken together, these data indicate an important role for intracel lular Ca++ in tight junction biogenesis and suggest a specific role fo r calcium in the early sorting and possible cytoskeletal association o f tight junction components. (C) 1994 Wiley-Liss, Inc.