Yap A S, Stevenson B R, Abel K C, Cragoe E J, Manley S W
Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Brisbane, Australia.
Exp Cell Res. 1995 Jun;218(2):540-50. doi: 10.1006/excr.1995.1189.
Vectorial transport in the thyroid epithelium requires an efficient barrier against passive paracellular flux, a role which is principally performed by the tight junction (zonula occludens). There is increasing evidence that tight junction integrity is determined by integral and peripheral membrane proteins which interact with the cell cytoskeleton. Although the contribution of the actin cytoskeleton to tight junction physiology has been intensively studied, less is known about possible interactions with microtubules. In the present study we used electrophysiological and immunohistochemical approaches to investigate the contribution of microtubules to the paracellular barrier in cultured thyroid cell monolayers which displayed a high transepithelial electrical resistance (6000-9000 ohm.cm2). Colchicine (1 microM) caused a progressive fall in electrical resistance to < 10% of baseline after 6 h and depolarization of the transepithelial electrical potential difference consistent with a significant increase in paracellular permeability. The effect of colchicine on TER was not affected by agents which inhibit the major apical conductances of thyroid cells but was reversed upon removal of the drug. Immunofluorescent staining for tubulin combined with confocal laser scanning microscopy demonstrated that thyroid cells possessed a dense microtubule network extending throughout the cytoplasm which was destroyed by colchicine. Colchicine also produced changes in the localization of the tight junction-associated protein, ZO-1: its normally continuous junctional distribution was disrupted by striking discontinuities and the appearance of many fine strands which extended into the cytoplasm. A similar disruption in E-cadherin staining was also observed, but colchicine did not affect the distribution of vinculin associated with adherens junctions nor the integrity of the perijunctional actin ring. We conclude that microtubules are necessary for the functional and structural integrity of tight junctions in this electrically tight, transporting epithelium.
甲状腺上皮细胞中的矢量运输需要一个有效的屏障来阻止被动的细胞旁通量，这一作用主要由紧密连接（闭锁小带）来执行。越来越多的证据表明，紧密连接的完整性由与细胞细胞骨架相互作用的整合膜蛋白和外周膜蛋白决定。尽管肌动蛋白细胞骨架对紧密连接生理学的贡献已得到深入研究，但关于其与微管可能的相互作用却知之甚少。在本研究中，我们使用电生理学和免疫组织化学方法来研究微管对培养的甲状腺细胞单层中细胞旁屏障的贡献，这些细胞单层表现出高跨上皮电阻（6000 - 9000欧姆·平方厘米）。秋水仙碱（1微摩尔）导致电阻在6小时后逐渐下降至基线的<10%，并且跨上皮电位差去极化，这与细胞旁通透性的显著增加一致。秋水仙碱对跨上皮电阻的影响不受抑制甲状腺细胞主要顶端电导的药物的影响，但在去除药物后可逆转。用微管蛋白进行免疫荧光染色并结合共聚焦激光扫描显微镜显示，甲状腺细胞拥有一个密集的微管网络，延伸至整个细胞质，而秋水仙碱可破坏该网络。秋水仙碱还使紧密连接相关蛋白ZO - 1的定位发生变化：其正常连续的连接分布被明显的间断以及延伸至细胞质的许多细链的出现所破坏。在E - 钙黏蛋白染色中也观察到类似的破坏，但秋水仙碱不影响与黏附连接相关的纽蛋白的分布，也不影响连接周围肌动蛋白环的完整性。我们得出结论，在这种电紧密的、具有运输功能的上皮细胞中，微管对于紧密连接的功能和结构完整性是必需的。
