Over-expression of βI tubulin in MDCK cells and incorporation of exogenous βI tubulin into microtubules interferes with adhesion and spreading

R. Lezama, A. Castillo, R. F. Ludueña, I. Meza

Research output: Contribution to journalArticle

6 Citations (Scopus)

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 βI, βII, and βIV isotypes are differentially distributed in the microtubules of these cells. Microtubules in subconfluent cells radiating from the perinuclear region contain βI and βII tubulins, while those extending to the cell edges are enriched in βII. Confluent cells contain similar proportions of βI and βII along the entire microtubule length. βIV is the less abundant isotype and shows a similar distribution to βII. 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 βI tubulin from CHO cells. Three recombinant clones expressing different levels of the exogenous βI tubulin were selected and subcloned. Clone 17-2 showed the highest expression of CHO β1 tubulin. Total βI tubulin levels (MDCK+CHO) in the clones were approximately 1.8 to 1.1-fold higher than in mock-transfected cells only expressing MDCK β1 tubulin. In all the cells, βII tubulin levels remained unchanged. The cells expressing CHO β1 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 βI/CHO βI 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 βI tubulin. By 24 h, the transfected cells had regained normal spreading and formed a confluent monolayer. Our results show that excess levels of total βI tubulin, resulting from the expression of the exogenous β1 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 βI tubulin levels implies the release of the MDCK βI isotype from the microtubules into the soluble fraction where it would be degraded.

Original languageEnglish (US)
Pages (from-to)147-160
Number of pages14
JournalCell Motility and the Cytoskeleton
Volume50
Issue number3
DOIs
StatePublished - 2001

Fingerprint

Madin Darby Canine Kidney Cells
Tubulin
Microtubules
Clone Cells
CHO Cells

Keywords

  • Epithelial cell functions
  • MDCK cells
  • Tubulin isotypes ratio

ASJC Scopus subject areas

  • Cell Biology

Cite this

Over-expression of βI tubulin in MDCK cells and incorporation of exogenous βI tubulin into microtubules interferes with adhesion and spreading. / Lezama, R.; Castillo, A.; Ludueña, R. F.; Meza, I.

In: Cell Motility and the Cytoskeleton, Vol. 50, No. 3, 2001, p. 147-160.

Research output: Contribution to journalArticle

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AU - Ludueña, R. F.

AU - Meza, I.

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AB - 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 βI, βII, and βIV isotypes are differentially distributed in the microtubules of these cells. Microtubules in subconfluent cells radiating from the perinuclear region contain βI and βII tubulins, while those extending to the cell edges are enriched in βII. Confluent cells contain similar proportions of βI and βII along the entire microtubule length. βIV is the less abundant isotype and shows a similar distribution to βII. 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 βI tubulin from CHO cells. Three recombinant clones expressing different levels of the exogenous βI tubulin were selected and subcloned. Clone 17-2 showed the highest expression of CHO β1 tubulin. Total βI tubulin levels (MDCK+CHO) in the clones were approximately 1.8 to 1.1-fold higher than in mock-transfected cells only expressing MDCK β1 tubulin. In all the cells, βII tubulin levels remained unchanged. The cells expressing CHO β1 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 βI/CHO βI 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 βI tubulin. By 24 h, the transfected cells had regained normal spreading and formed a confluent monolayer. Our results show that excess levels of total βI tubulin, resulting from the expression of the exogenous β1 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 βI tubulin levels implies the release of the MDCK βI isotype from the microtubules into the soluble fraction where it would be degraded.

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