Stevenson B R, Begg D A
Department of Anatomy and Cell Biology, University of Alberta, Edmonton, Canada.
J Cell Sci. 1994 Mar;107 ( Pt 3):367-75. doi: 10.1242/jcs.107.3.367.
The effects of different concentrations of the actin-disrupting drug cytochalasin D on tight junction permeability and distribution of actin filaments in MDCK epithelial cells were examined. Consistent with previous studies, 2 micrograms/ml cytochalasin D caused a significant decrease in transepithelial resistance, indicative of an increase in tight junction permeability. Surprisingly, increasing concentrations of cytochalasin D caused progressively smaller decreases in transepithelial resistance. The effects of cytochalasin D were reversible. Light microscopic analysis utilizing rhodamine-conjugated phalloidin demonstrated two distinct populations of actin filaments in MDCK cells: an apical peripheral ring of actin, presumably associated with the zonula adherens, and larger actin bundles more basally situated. When treated with 2 micrograms/ml cytochalasin D, both actin populations were severely disrupted and cells became flattened. Actin in the apical ring aggregated along cell boundaries, and these aggregates co-localized with similarly disrupted focal accumulations of the tight junction-associated protein ZO-1. The basal actin filament bundles also reorganized into focal aggregates. Increasing concentrations of cytochalasin D caused gradually less perturbation of the apical actin ring, consistent with the transepithelial resistance observations. However, the basal actin bundles were disrupted at all concentrations of cytochalasin D tested, demonstrating that the two actin populations are differentially sensitive to cytochalasin D and that apical actin filaments are more important in the regulation of tight junction permeability. Finally, treatment of cells with cytochalasin D inhibited the decrease in transepithelial resistance induced by the chelation of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)
研究了不同浓度的肌动蛋白破坏药物细胞松弛素D对MDCK上皮细胞紧密连接通透性和肌动蛋白丝分布的影响。与先前的研究一致，2微克/毫升的细胞松弛素D导致跨上皮电阻显著降低，表明紧密连接通透性增加。令人惊讶的是，细胞松弛素D浓度增加导致跨上皮电阻的降低逐渐变小。细胞松弛素D的作用是可逆的。利用罗丹明偶联鬼笔环肽进行的光学显微镜分析显示，MDCK细胞中有两种不同的肌动蛋白丝群体：一种是位于顶端周边的肌动蛋白环，可能与黏着小带有关，另一种是位于更基部的较大肌动蛋白束。用2微克/毫升细胞松弛素D处理时，两种肌动蛋白群体均受到严重破坏，细胞变扁平。顶端环中的肌动蛋白沿细胞边界聚集，这些聚集体与紧密连接相关蛋白ZO-1的类似破坏的局灶性聚集物共定位。基部肌动蛋白丝束也重新组织成局灶性聚集体。细胞松弛素D浓度增加导致顶端肌动蛋白环的扰动逐渐减少，这与跨上皮电阻的观察结果一致。然而，在所有测试的细胞松弛素D浓度下，基部肌动蛋白束均被破坏，表明这两种肌动蛋白群体对细胞松弛素D的敏感性不同，并且顶端肌动蛋白丝在紧密连接通透性调节中更重要。最后，用细胞松弛素D处理细胞可抑制细胞外Ca2+螯合诱导的跨上皮电阻降低。（摘要截短于250字）
