Peterson M D, Bement W M, Mooseker M S
Department Cell Biology, Yale University, New Haven, CT 06511-8112.
J Cell Sci. 1993 Jun;105 ( Pt 2):461-72. doi: 10.1242/jcs.105.2.461.
In the companion paper (M. D. Peterson and M. S. Mooseker (1993). J. Cell Sci. 105, 445-460) we describe a method for modeling brush border assembly in the Caco-2BBe clones. In this study we have examined the molecular changes accompanying cell contact-induced brush border assembly. A subset of brush border proteins was tracked throughout brush border assembly by immunoblotting and by immunofluorescent localization using laser scanning confocal microscopy. Actin, fodrin, villin and presumptive unconventional myosin immunogens were distributed at the periphery of depolarized cells. All proteins partitioned primarily with the membrane fraction upon differential sedimentation of depolarized cell lysates; the fractionation patterns were comparable to those of confluent cells. After a monolayer had formed, each protein showed a redistribution to the apical domain in a discrete sequence. Actin and villin began to shift apically at 2 d, while fodrin and the unconventional myosin immunogens did not redistribute until 3 d. Enterocyte-like localization was observed by 5 d for all proteins. Sucrase-isomaltase was not reliably detectable until 9 d by immunofluorescence, after brush border assembly was complete. Quantitative immunoblot analysis of total cell extracts demonstrated an average 10-fold increase in villin levels, while fodrin levels appeared to remain unchanged. Three putative unconventional myosin immunogens of 140 kDa, 130 kDa, and 110 kDa have been detected previously in the C2BBe cells with a head-specific monoclonal antibody to avian brush border myosin I (M. D. Peterson and M. S. Mooseker (1992) J. Cell Sci. 102, 581-600). Each of these immunogens displayed distinct expression patterns during brush border assembly. The 140 kDa species decreased by half, while the 130 kDa immunogen(s) did not change in any consistent fashion. The 110 kDa protein, presumed to be human brush border myosin I, rose on average 8-fold. A ribonuclease protection assay was also performed using a probe for human brush border myosin I. Equal amounts of total RNA from depolarized and confluent cells were assayed; the level of protected product was approximately 9-fold greater in the confluent cells. The expression patterns of the brush border proteins, coupled with the correlation to the ultrastructural features during brush border assembly in C2BBe cells, show that differentiation of the C2BBe cells closely resembles the changes that occur during human fetal intestinal differentiation.
在配套论文中（M.D. 彼得森和M.S. 穆斯克（1993年）。《细胞科学杂志》105卷，445 - 460页），我们描述了一种在Caco - 2BBe克隆中模拟刷状缘组装的方法。在本研究中，我们研究了伴随细胞接触诱导的刷状缘组装的分子变化。通过免疫印迹以及使用激光扫描共聚焦显微镜进行免疫荧光定位，在整个刷状缘组装过程中追踪了一部分刷状缘蛋白。肌动蛋白、血影蛋白、绒毛蛋白和推定的非常规肌球蛋白免疫原分布在去极化细胞的周边。在对去极化细胞裂解物进行差速沉降时，所有蛋白质主要与膜部分一起分离；分级分离模式与汇合细胞的模式相当。单层形成后，每种蛋白质都以离散的顺序重新分布到顶端区域。肌动蛋白和绒毛蛋白在第2天开始向顶端移动，而血影蛋白和非常规肌球蛋白免疫原直到第3天才重新分布。到第5天时，所有蛋白质都观察到类似肠上皮细胞的定位。在刷状缘组装完成后，直到第9天通过免疫荧光才能可靠地检测到蔗糖酶 - 异麦芽糖酶。对总细胞提取物进行的定量免疫印迹分析表明，绒毛蛋白水平平均增加了10倍，而血影蛋白水平似乎保持不变。先前在C2BBe细胞中用针对禽刷状缘肌球蛋白I的头部特异性单克隆抗体检测到了三种推定的分子量为140 kDa、130 kDa和110 kDa的非常规肌球蛋白免疫原（M.D. 彼得森和M.S. 穆斯克（1992年）。《细胞科学杂志》102卷，581 - 600页）。在刷状缘组装过程中，这些免疫原中的每一种都表现出不同的表达模式。140 kDa的种类减少了一半，而130 kDa的免疫原没有以任何一致的方式变化。推定为人刷状缘肌球蛋白I的110 kDa蛋白质平均增加了8倍。还使用针对人刷状缘肌球蛋白I的探针进行了核糖核酸酶保护试验。对去极化细胞和汇合细胞的等量总RNA进行了检测；在汇合细胞中受保护产物的水平大约高9倍。刷状缘蛋白的表达模式，以及与C2BBe细胞刷状缘组装过程中超微结构特征的相关性，表明C2BBe细胞的分化与人类胎儿肠道分化过程中发生的变化非常相似。
