Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane glycoprotein that forms Cl- channels. Previous work has shown that when some CF-associated mutants of CFTR are expressed in heterologous cells, their glycosylation is incomplete. That observation led to the hypothesis that such mutants are not delivered to the plasma membrane where they can mediate Cl- transport. Testing this hypothesis requires localization of CFTR in nonrecombinant cells and a specific determination of whether CFTR is in the apical membrane of normal and CF epithelia. To test the hypothesis, we used primary cultures of airway epithelia grown on permeable supports because they polarize and express the CF defect in apical Cl- permeability. Moreover, their dysfunction contributes to disease. We developed a semiquantitative assay, using nonpermeabilized epithelia, an antibody directed against an extracellular epitope of CFTR, and large (1 microns) fluorescent beads which bound to secondary antibodies. We observed specific binding to airway epithelia from non-CF subjects, indicating that CFTR is located in the apical membrane. In contrast, there was no specific binding to the apical membrane of CF airway epithelia. These data were supported by qualitative studies using confocal microscopy: the most prominent immunostaining was in the apical region of non-CF cells and in cytoplasmic regions of CF cells. The results indicate that CFTR is either missing from the apical membrane of these CF cells or it is present at a much reduced level. The data support the proposed defective delivery of some CF-associated mutants to the plasma membrane and explain the lack of apical Cl- permeability in most CF airway epithelia.

The phenotypic effects of selectively decreasing the levels of S100 beta in cultured glial cells were analyzed. Two separate antisense approaches were utilized for inhibition of S100 beta production: analysis of clonal isolates of rat C6 glioma cells containing an S100 beta antisense gene under the control of a dexamethasone-inducible promoter, and analysis of C6 cells treated with S100 beta antisense oligodeoxynucleotides. Both antisense methods resulted in a decrease in S100 beta levels in the cell, as measured by RIA. The inhibition of S100 beta production correlated with three alterations in cellular phenotype: (a) a flattened cell morphology; (b) a more organized microfilament network; and (c) a decrease in cell growth rate. The studies describe here provide direct evidence for an involvement of S100 beta in glial cell structure and function, and suggest potential in vivo roles for S100 beta in regulation of glial cell morphology, cytoskeletal organization, and cell proliferation.

To investigate the function of calmodulin (CaM) in the mitotic apparatus, the effect of microinjected CaM and chemically modified CaMs on nocodazole-induced depolymerization of spindle microtubules was examined. When metaphase PtK1 cells were microinjected with CaM or a CaM-TRITC conjugate, kinetochore microtubules (kMTs) were protected from the effect of nocodazole. The ability of microinjected CaM to subsequently protect kMTs from the depolymerizing effect of nocodazole was dose dependent, and was effective for approximately 45 min, with protection decreasing if nocodazole treatment was delayed for more than 60 min after injection of CaM. The CaM-TRITC conjugate, similar to native CaM, displayed the ability to activate bovine brain CaM-dependent adenylate cyclase in a Ca++-dependent manner and showed a Ca++-dependent mobility shift when subjected to PAGE. A heat-altered CaM-TRITC conjugate also protected kMTs from the effect of nocodazole. However, this modified CaM was not able to activate adenylate cyclase nor did it display a Ca++-dependent mobility shift when electrophoresed. In a permeabilized cell model system, both CaM analogs were observed to bind to the spindle in a Ca++-independent manner. In contrast, a performic acid-oxidized CaM did not have a protective effect on spindle structure when microinjected into metaphase cells before nocodazole treatment. The oxidized CaM did not activate adenylate cyclase and did not exhibit Ca++-dependent mobility on polyacrylamide gels. These results are interpreted as supporting the hypothesis that CaM binds to the mitotic spindle in a Ca++-independent manner and that CaM may serve in the spindle, at least in part, to stabilize kMTs.

Indirect immunofluorescence was used to determine the distribution of calmodulin in the mitotic apparatus of rat kangaroo PtK2 and Chinese hamster ovary (CHO) cells. The distribution of calmodulin in PtK2 cells was compared to the distribution of tubulin, also as revealed by indirect immunofluorescence. During mitosis, calmodulin was found to be a dynamic component of the mitotic apparatus. Calmodulin first appeared in association with the forming mitotic apparatus during midprophase. In metaphase and anaphase, calmodulin was found between the spindle poles and the chromosomes. While tubulin was found in the interzonal region throughout anaphase, calmodulin appeared in the interzone region only at late anaphase. The interzonal calmodulin of late anaphase condensed during telophase into two small regions, one on each side of the midbody. Calmodulin was not detected in the cleavage furrow. In view of the differences in the localization of calmodulin, tubulin, and actin in the mitotic apparatus, experiments were designed to determine the effects of various antimitotic drugs on calmodulin localization. Cytochalasin B, an inhibitor of actin microfilaments, had no apparent effect on calmodulin or tubulin localization in the mitotic apparatus of CHO cells. Microtubule inhibitors, such as colcemid and N2O, altered the appearance of tubulin- and calmodulin-specific fluorescence in mitotic CHO cells. Cold temperature (0 degrees C) altered tubulin-specific fluorescence of metaphase PtK2 cells but did not alter calmodulin-specific fluorescence. From these studies, it is concluded that calmodulin is more closely associated with the kinetichore-to-pole microtubules than other components of the mitotic apparatus.