Apical recruitment of CFTR in T-84 cells is dependent on cAMP and microtubules but not Ca2+ or microfilaments.
Previous studies from this laboratory have demonstrated that chloride transport induced by forskolin, but not ionomycin, in T84 cells is highly dependent on an intact microtubular network. Using an antibody raised against a region of the R domain of CFTR, we now show by indirect immunofluorescence that forskolin causes relocation of CFTR to the apical domain of T84 cells. T84 cells grown on transparent filters were incubated with agonists and/or cytoskeletal inhibitors prior to fixation, permeabilization, and staining with the antibody. A 30 second stimulation with forskolin (10 microM) caused a twofold increase in relative fluorescence intensity at the apical surface. In contrast, a 30 second exposure to ionomycin (2 microM), had no effect on the distribution of CFTR-related fluorescence. Incubation of the cells with nocodazole (33 microM), a microtubule disrupting agent, prevented the forskolin-induced rise in CFTR fluorescence at the apical surface. However, incubation of the cells with cytochalasin D, an actin inhibitor, was without effect on forskolin-related re-distribution of CFTR-associated fluorescence. In double label experiments using antibodies against both beta-tubulin and actin, CFTR-related fluorescence was found to co-localize with the microtubule network, but not with actin filaments. These observations are consistent with the microtubule-dependent acute recruitment of CFTR to the apical plasma membrane of T84 cells in response to elevations in intracellular cAMP.
Tousson
A
A
Department of Cell Biology, University of Alabama at Birmingham 35294, USA.
Fuller
C M
CM
Benos
D J
DJ
D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000037:antagonists & inhibitors / Q000378:metabolism; D000911:Antibodies, Monoclonal; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000276:immunology / Q000378:metabolism*; D015638:Cytochalasin D / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D014404:Tubulin / Q000378:metabolism
D000199:Actins; D000911:Antibodies, Monoclonal; C505032:CFTR protein, human; D007476:Ionophores; D014404:Tubulin; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D015638:Cytochalasin D; D015759:Ionomycin; D000242:Cyclic AMP; D015739:Nocodazole; D002118:Calcium