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首页> 外文期刊>Cell motility and the cytoskeleton >Over-expression of betaI tubulin in MDCK cells and incorporation of exogenous betaI tubulin into microtubules interferes with adhesion and spreading.
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Over-expression of betaI tubulin in MDCK cells and incorporation of exogenous betaI tubulin into microtubules interferes with adhesion and spreading.

机译：MDCK细胞中betaI微管蛋白的过表达以及将外源betaI微管蛋白掺入微管会干扰粘附和扩散。

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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.
机译：关于微管蛋白同种型在MDCK细胞中的存在和分布知之甚少，尽管这些单层细胞的基本上皮功能是由微管结构的动态变化调节的。使用特异性抗体，我们在这里显示betaI，betaII和betaIV同种型在这些细胞的微管中差异分布。从核周区域辐射的亚汇合细胞中的微管含有betaI和betaII微管蛋白，而延伸到细胞边缘的微管则富含betaII。融合细胞沿整个微管长度包含相似比例的betaI和betaII。 betaIV是较不丰富的同种型，显示出与betaII类似的分布。通过在MDCK细胞中稳定表达CHO细胞中的betaI微管蛋白来分析微管中微管蛋白同种型比例改变的影响，从而影响其动力学和功能。选择表达不同水平的外源βI微管蛋白的三个重组克隆并亚克隆。克隆17-2显示了CHO beta1微管蛋白的最高表达。克隆中的总betaI微管蛋白水平（MDCK + CHO）比仅表达MDCK beta1微管蛋白的模拟转染细胞高约1.8至1.1倍。在所有细胞中，βII微管蛋白水平保持不变。表达CHO beta1微管蛋白的细胞在铺板后的短时间内显示出缺陷的附着，扩散和粘附位点的形成延迟，而模拟转染的细胞则附着并正常扩散。来自克隆17-2的细胞骨架部分的分析显示，在2小时时，MDCK betaI / CHO betaI比率为1.89，到24小时时逐渐下降至1.0。可溶性部分中两种同种型的比例保持不变，尽管其值高于聚合的βI微管蛋白的值。到24小时，转染的细胞恢复了正常铺展并形成了汇合的单层。我们的研究结果表明，由于外源性beta1同种型的表达而导致的总betaI微管蛋白含量过高，并将其掺入微管中会影响其稳定性和某些细胞功能。随着水平恢复正常，细胞恢复其正常表型。 betaI微管蛋白水平的调节意味着MDCK betaI同种型从微管释放到可溶级分中，在那里它将被降解。

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