McCarthy K M, Francis S A, McCormack J M, Lai J, Rogers R A, Skare I B, Lynch R D, Schneeberger E E
Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA.
J Cell Sci. 2000 Oct;113 Pt 19:3387-98. doi: 10.1242/jcs.113.19.3387.
Occludin and 18 distinct members of the claudin family are tetra-span transmembrane proteins that are localized in cell-specific tight junctions (TJs). A previous study showed that expression of chick occludin in Madin-Darby canine kidney (MDCK) cells raised transepithelial electrical resistance (TER) and, paradoxically, increased mannitol flux. In the present study, we employed epitope tagged canine occludin expression, under the control of the tetracycline repressible transactivator, to determine the extent to which the unexpected parallel increase in TER and mannitol flux was related to a structural mismatch between avian and canine occludins, which are only 50% identical. To determine whether the paradoxical changes in permeability was specific to occludin, we assessed the effect of over-expressing epitope tagged murine claudin-1. Our data revealed that over-expression of either of the epitope tagged mammalian tight junction proteins increased TER, mannitol and FITC-dextran flux. We observed a 2- and up to 5.6-fold over-expression of occludin-VSV-G and claudin-1-myc, respectively, with no change in ZO-1, endogenous occludin or claudin-1 expression. Confocal microscopy revealed that occludin-VSV-G, claudin-1-myc and ZO-1 co-localized at the TJ. In addition, claudin-1-myc formed aberrant strands along the lateral cell surface without an underlying ZO-1 scaffold. In fracture labeled replicas these strands consisted of claudin-1-myc with little accompanying occludin. These observations suggest that in epithelial cells claudin-1 can assemble into TJ strands without the participation of either ZO-1 or occludin. The proximity of the myc tag to the COOH-terminal YV sequence of claudin-1 appeared to interfere with its interaction with ZO-1, since over-expression of non-tagged claudin-1 increased TER but had a minimal effect on solute flux and no aberrant strands formed. From our data we conclude that differences in structure between avian and mammalian occludin do not account for the observed paradoxical increase in mannitol flux. Levels of ZO-1 remained unchanged despite substantial increases in induced TJ integral protein expression, suggesting that an imbalance between levels of ZO-1 and occludin or claudin-1 leads to altered regulation of pores through which non-charged solute flux occurs. We suggest that ion and solute flux are differentially regulated at the TJ.
闭合蛋白和紧密连接蛋白家族的18个不同成员是四跨膜蛋白，定位于细胞特异性紧密连接（TJ）中。先前的一项研究表明，在Madin-Darby犬肾（MDCK）细胞中表达鸡闭合蛋白会提高跨上皮电阻（TER），而矛盾的是，会增加甘露醇通量。在本研究中，我们利用在四环素可抑制反式激活因子控制下的表位标记犬闭合蛋白表达，来确定TER和甘露醇通量意外平行增加在多大程度上与禽类和犬类闭合蛋白之间的结构不匹配有关，它们的同源性仅为50%。为了确定通透性的矛盾变化是否是闭合蛋白特有的，我们评估了过表达表位标记的小鼠紧密连接蛋白-1的影响。我们的数据显示，过表达任何一种表位标记的哺乳动物紧密连接蛋白都会增加TER、甘露醇和异硫氰酸荧光素 - 葡聚糖通量。我们观察到闭合蛋白 - VSV - G和紧密连接蛋白 - 1 - myc分别过表达2倍和高达5.6倍，而ZO - 1、内源性闭合蛋白或紧密连接蛋白 - 1的表达没有变化。共聚焦显微镜显示，闭合蛋白 - VSV - G、紧密连接蛋白 - 1 - myc和ZO - 1在紧密连接处共定位。此外，紧密连接蛋白 - 1 - myc在细胞侧面形成异常的链，没有底层的ZO - 1支架。在断裂标记复制品中，这些链由紧密连接蛋白 - 1 - myc组成，几乎没有伴随的闭合蛋白。这些观察结果表明，在上皮细胞中，紧密连接蛋白 - 1可以在没有ZO - 1或闭合蛋白参与的情况下组装成紧密连接链。紧密连接蛋白 - 1的myc标签靠近COOH末端的YV序列似乎干扰了它与ZO - 1的相互作用，因为未标记的紧密连接蛋白 - 1过表达会增加TER，但对溶质通量影响最小，并且没有形成异常链。从我们的数据中我们得出结论，禽类和哺乳动物闭合蛋白之间的结构差异并不能解释观察到的甘露醇通量矛盾性增加。尽管诱导的紧密连接整合蛋白表达大幅增加，但ZO - 1的水平保持不变，这表明ZO - 1与闭合蛋白或紧密连接蛋白 - 1水平之间的不平衡导致了对不带电溶质通量通过的孔的调节改变。我们认为离子和溶质通量在紧密连接处受到不同的调节。
