Meza I, Sabanero M, Stefani E, Cereijido M
J Cell Biochem. 1982;18(4):407-21. doi: 10.1002/jcb.1982.240180403.
In MDCK cell monolayers the opening and resealing of occluding junctions can be induced by removal and restoration of calcium to the external medium. The overall changes in permeability of the occluding junctions in the monolayer can be monitored by the drop and recovery of the total transepithelial electrical resistance. We have investigated the effects of cytochalasin B (CB) on this process. When CB is added to sealed monolayers there is a gradual drop in the electrical resistance across the monolayer. This drop is accompanied by a slow disorganization of the microfilament pattern of these cells, including a disturbance of a ring of cortical microfilaments that is normally associated with the junctions. Cells in open monolayers treated with CB will not reseal and have an altered filament distribution. These cells do not have a continuous cortical ring. We have used a voltage scanning technique that uses a microelectrode to measure the resistance at selected points along the junction which surrounds a single cell. In untreated, closed monolayers, the junction is heterogeneous with alternating points of high and low conductance. In closed monolayers treated with CB, although there are low conductance points, we have observed an increased frequency of high conductance points that correlates with the change in the overall conductance. The frequency of high conductance points along the junction and the overall conductance both increase with time of exposure to CB. In an effort to understand the molecular basis for the permeability changes induced by EGTA and CB, we have looked for differences in the protein components of the cell membranes of open, closed, and CB-treated MDCK monolayers. This was done by radioiodinating the surface membrane proteins under control and experimental conditions that bring about permeability changes. No significant differences in the labeled protein patterns were found under these conditions. These results suggest that the permeability changes involve only a structural rearrangement of membrane components. In additions we have observed that about 36% of the surface label remains bound to the insoluble cytoskeletons obtained from cells in control and experimental conditions that alter the permeability of the tight junctions. The iodinated proteins attached to the CS include polypeptides with Mr of greater than or equal to 120K daltons as well as peptides with Mr = 56K, 50K, 36K, and 18K daltons.
在MDCK细胞单层中，通过去除和恢复细胞外培养基中的钙，可以诱导封闭连接的打开和重新封闭。单层中封闭连接通透性的总体变化可以通过跨上皮总电阻的下降和恢复来监测。我们研究了细胞松弛素B（CB）对这一过程的影响。当将CB添加到密封的单层中时，单层上的电阻会逐渐下降。这种下降伴随着这些细胞微丝模式的缓慢紊乱，包括通常与连接相关的皮质微丝环的紊乱。用CB处理的开放单层中的细胞不会重新封闭，并且丝状分布会发生改变。这些细胞没有连续的皮质环。我们使用了一种电压扫描技术，该技术使用微电极测量围绕单个细胞的连接沿线选定位置的电阻。在未处理的封闭单层中，连接是异质的，具有高电导和低电导交替的点。在用CB处理的封闭单层中，虽然存在低电导点，但我们观察到高电导点的频率增加，这与总体电导的变化相关。沿着连接的高电导点的频率和总体电导都随着暴露于CB的时间而增加。为了理解EGTA和CB诱导的通透性变化的分子基础，我们寻找了开放、封闭和CB处理的MDCK单层细胞膜蛋白质成分的差异。这是通过在导致通透性变化的对照和实验条件下对表面膜蛋白进行放射性碘化来完成的。在这些条件下，未发现标记蛋白模式有显著差异。这些结果表明，通透性变化仅涉及膜成分的结构重排。此外，我们观察到，在改变紧密连接通透性的对照和实验条件下，约36%的表面标记物仍与从细胞中获得的不溶性细胞骨架结合。附着在CS上的碘化蛋白质包括分子量大于或等于120千道尔顿的多肽以及分子量分别为56K、50K、36K和18K道尔顿的肽。
