Florian P, Schöneberg T, Schulzke J D, Fromm M, Gitter A H
Institut für Klinische Physiologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, 12200 Berlin, Germany.
J Physiol. 2002 Dec 1;545(2):485-99. doi: 10.1113/jphysiol.2002.031161.
Restitution of single-cell defects, a frequent event in epithelia with high turnover, is poorly understood. Morphological and functional changes were recorded, using intravital time-lapse video microscopy, confocal fluorescence microscopy, and conductance scanning techniques. After artificial single-cell loss from an HT-29/B6 colonic cell monolayer, the basal ends of adjacent cells extended. Concurrently, the local conductive leak associated with the defect sealed with an exponential time course (from 0.48 +/- 0.05 microS 2 min post lesion to 0.17 +/- 0.02 microS 8 min post lesion, n = 17). Between 3 and 10 min post lesion, a band of actin arose around the gap, which colocalized with a ring of ZO-1 and occludin. Hence, tight junction proteins bound to the actin band facing the gap, and competent tight junctions assembled in the adjoining cell membranes. Closure and sealing were inhibited when actin polymerization was blocked by cytochalasin D, delayed following decrease of myosin-ATPase activity by butanedione monoxime, and blocked after myosin light chain kinase inhibition by ML-7. The Rho-associated protein kinase inhibitor Y-27632 did not affect restitution. After loosening of intercellular contacts in low Ca(2+) Ringer solution, the time course of restitution was not significantly altered. Albeit epithelial conductivity was 12-fold higher in low Ca(2+) Ringer solution than in controls, under both conditions the repaired epithelium assumed the same conductivity as distant intact epithelium. In conclusion, epithelial restitution of single-cell defects comprises rapid closure by an actinomyosin 'purse-string' mechanism and simultaneous formation of a functional barrier from tight junction proteins also associated with the purse string.
单细胞缺陷修复是上皮组织中常见的现象，上皮组织更新频繁，但人们对此了解甚少。我们使用活体延时视频显微镜、共聚焦荧光显微镜和电导扫描技术记录了形态和功能的变化。在HT - 29/B6结肠细胞单层中人为造成单细胞缺失后，相邻细胞的基底端会延伸。同时，与缺陷相关的局部传导性泄漏以指数时间进程封闭（损伤后2分钟时为0.48±0.05微西门子，损伤后8分钟时为0.17±0.02微西门子，n = 17）。损伤后3至10分钟，间隙周围出现一条肌动蛋白带，其与ZO - 1和闭合蛋白环共定位。因此，紧密连接蛋白与面向间隙的肌动蛋白带结合，在相邻细胞膜中组装成有功能的紧密连接。当细胞松弛素D阻断肌动蛋白聚合时，封闭和密封受到抑制；丁二酮肟降低肌球蛋白 - ATP酶活性后，修复延迟；ML - 7抑制肌球蛋白轻链激酶后，修复被阻断。Rho相关蛋白激酶抑制剂Y - 27632不影响修复。在低钙林格溶液中细胞间连接松弛后，修复的时间进程没有明显改变。尽管在低钙林格溶液中上皮导电性比对照组高12倍，但在两种条件下，修复后的上皮都与远处完整上皮具有相同的导电性。总之，单细胞缺陷上皮修复包括通过肌动球蛋白“束带”机制快速封闭，以及同时由与束带相关的紧密连接蛋白形成功能屏障。
