Abstract

Perturbation of potentially regulatable endoplasmic reticulum (ER) calcium stores with the Ca-ATPase inhibitor, thapsigargin (TG), perturbs the formation of desmosomes and tight junctions during polarized epithelial cell biogenesis, despite the development of cell contact. In a Madin-Darby canine kidney cell model for intercellular junction assembly, TG treatment inhibited the development of transepithelial electrical resistance (TER), a measure of tight junction assembly, in a dose-dependent manner. The TG-induced inhibition of tight junction assembly was paralleled by a defect in the sorting of the tight junction protein, ZO-1. An even more dramatic delay in sorting of the desmosomal protein, desmoplakin, was observed in the presence of TG. In addition, while both ZO-1 and desmoplakin-I in control cells were shown to become associated with the Triton X-100 insoluble cytoskeleton during intercellular junction assembly, prior treatment with 100 nM TG diminished this biochemical stabilization into the detergent-insoluble fraction, particularly in the case of ZO-1. Although spectrofluorimetric measurements in fura-2 loaded Madin-Darby canine kidney cells confirmed the occurrence of TG-mediated release of calcium from internal stores, total cytosolic calcium during junction assembly remained similar to untreated cells. Therefore, the presence of cytosolic calcium alone is not sufficient for normal intercellular junction biogenesis if intracellular stores are perturbed by TG. The results indicate the presence of calcium-sensitive intracellular mechanisms involved in the sorting and cytoskeletal stabilization of both tight junction and desmosomes and suggest a role for calcium-dependent signaling pathways at an early (possibly common) step in polarized epithelial biogenesis.

Highlights

  • The ability of epithelial tissues such as the kidney and intestine to carry out vectorial transport of solutes and water is critically dependent on the maintenance of a permeability barrier to passive diffusion and upon the polarized distribution of

  • On the basis of our previous finding that, during the MDCK cell calcium switch, impressive global and local increases in intracellular Ca2ϩ occur during junction formation (Nigam et al, 1992), we have hypothesized that intracellular calcium is involved in signaling pathways leading to intercellular junction biogenesis

  • We had previously observed that buffering these changes in intracellular calcium with dimethyl BAPTA-AM disrupted several measures of tight junction assembly including the development of transepithelial electrical resistance (TER) and the sorting of the tight junction protein ZO-1 (Stuart et al, 1994)

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Summary

EXPERIMENTAL PROCEDURES

Materials—Culture media was from Life Technologies, Inc. (Grand Island, NY). Plasticware was from Falcon (Lincoln Park, IL) except that Transwells were from Costar (Cambridge, MA). The cells were solubilized in 300 ␮l of modified RIPA buffer designed to preserve protein-protein interactions (150 mM NaCl, 10 mM PIPES, pH 7.5, 1% Triton X-100, 0.5% sodium deoxycholate, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM iodoacetamide, and 20 ␮g/ml each of leupeptin, antipain, and pepstatin-A). With this buffer, most if not all, currently known proteins of the tight junction complex, can be coimmunoprecipitated with anti-ZO-1 antibodies (Stuart and Nigam, 1995). Filters were probed with anti-ZO-1 hybridoma supernatant and anti-desmoplakin antibody

RESULTS
Dependence of Epithelial Biogenesis on Calcium Stores
DISCUSSION
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