title: Over-expression of betaI tubulin in MDCK cells and incorporation of exogenous betaI tubulin into microtubules interferes with adhesion and spreading.
abstract: Little is known about the presence and distribution of tubulin isotypes in MDCK cells although essential epithelial functions in these monolayers are regulated by dynamic changes in the microtubule architecture. Using specific antibodies, we show here that the betaI, betaII, and betaIV isotypes are differentially distributed in the microtubules of these cells. Microtubules in subconfluent cells radiating from the perinuclear region contain betaI and betaII tubulins, while those extending to the cell edges are enriched in betaII. Confluent cells contain similar proportions of betaI and betaII along the entire microtubule length. betaIV is the less abundant isotype and shows a similar distribution to betaII. The effect of modifying tubulin isotype ratios in the microtubules that could affect their dynamics and function was analyzed by stably expressing in MDCK cells betaI tubulin from CHO cells. Three recombinant clones expressing different levels of the exogenous betaI tubulin were selected and subcloned. Clone 17-2 showed the highest expression of CHO beta1 tubulin. Total betaI tubulin levels (MDCK+CHO) in the clones were approximately 1.8 to 1.1-fold higher than in mock-transfected cells only expressing MDCK beta1 tubulin. In all the cells, betaII tubulin levels remained unchanged. The cells expressing CHO beta1 tubulin showed defective attachment, spreading, and delayed formation of adhesion sites at short times after plating, whereas mock-transfected cells attached and spread normally. Analysis of cytoskeletal fractions from clone 17-2 showed a MDCK betaI/CHO betaI ratio of 1.89 at 2 h that gradually decreased to 1.0 by 24 h. The ratio of the two isotypes in the soluble fraction remained unchanged, although with higher values than those found for the polymerized betaI tubulin. By 24 h, the transfected cells had regained normal spreading and formed a confluent monolayer. Our results show that excess levels of total betaI tubulin, resulting from the expression of the exogenous beta1 isotype, and incorporation of it into microtubules affect their stability and some cellular functions. As the levels return to normal, the cells recover their normal phenotype. Regulation of betaI tubulin levels implies the release of the MDCK betaI isotype from the microtubules into the soluble fraction where it would be degraded.
authors.affiliation: Department of Biología Celular, CINVESTAV del IPN, México D.F., México.
authors.forename: R
authors.initials: R
authors.lastname: Lezama
authors.forename: A
authors.initials: A
authors.lastname: Castillo
authors.forename: R F
authors.initials: RF
authors.lastname: Ludueña
authors.forename: I
authors.initials: I
authors.lastname: Meza
mesh_terms: D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion / Q000187:drug effects*; D006224:Cricetinae; D003595:Cytoplasmic Streaming / Q000187:drug effects*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008870:Microtubules / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014404:Tubulin / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D020033:Protein Isoforms; D011994:Recombinant Proteins; D014404:Tubulin