Fath K R, Obenauf S D, Burgess D R
Department of Cell Biology and Anatomy, University of Miami School of Medicine, Florida 33101.
Development. 1990 Jun;109(2):449-59. doi: 10.1242/dev.109.2.449.
We have explored the development of the brush border in adult chicken enterocytes by analyzing the cytoskeletal protein and mRNA levels as enterocytes arise from crypt stem cells and differentiate as they move toward the villus. At the base of the crypt, a small population of cells contain a rudimentary terminal web and a few short microvilli with long rootlets. These microvilli appear to arise from bundles of actin filaments which nucleate on the plasma membrane. The microvilli apparently elongate via the addition of membrane supplied by vesicles that fuse with the microvillus and extend the membrane around the actin core. Actin, villin, myosin, tropomyosin and spectrin, but not myosin I (previously called 110 kD; see Mooseker and Coleman, J. Cell Biol. 108, 2395-2400, 1989) are already concentrated in the luminal cytoplasm of crypt cells, as seen by immunofluorescence. Using quantitative densitometry of cDNA-hybridized RNA blots from cells isolated from crypts, villus middle (mid), or villus tip (tip), we found a 2- to 3-fold increase in villin, calmodulin and tropomyosin steady-state mRNA levels; an increase parallel to morphological brush border development. Actin, spectrin and myosin mRNA levels did not change significantly. ELISA of total crypt, mid and tip cell lysates show that there are no significant changes in actin, myosin, spectrin, tropomyosin, myosin I, villin or alpha-actinin protein levels as the brush border develops. The G-/F-actin ratio also did not change with brush border assembly. We conclude that, although the brush border is not fully assembled in immature enterocytes, the major cytoskeletal proteins are present in their full concentration and already localized within the apical cytoplasm. Therefore brush border formation may involve reorganization of a pool of existing cytoskeletal proteins mediated by the expression or regulation of an unidentified key protein(s).
我们通过分析细胞骨架蛋白和mRNA水平，探讨了成年鸡肠上皮细胞中刷状缘的发育过程，因为肠上皮细胞由隐窝干细胞产生，并在向绒毛移动的过程中分化。在隐窝底部，一小部分细胞含有原始的终末网和一些带有长根丝的短微绒毛。这些微绒毛似乎起源于在质膜上成核的肌动蛋白丝束。微绒毛显然是通过与微绒毛融合并围绕肌动蛋白核心延伸膜的囊泡提供的膜的添加而延长的。通过免疫荧光观察，肌动蛋白、绒毛蛋白、肌球蛋白、原肌球蛋白和血影蛋白，但不包括肌球蛋白I（以前称为110 kD；见Mooseker和Coleman，《细胞生物学杂志》108，2395 - 2400，1989）已经集中在隐窝细胞的腔质中。使用对从隐窝、绒毛中部（mid）或绒毛顶端（tip）分离的细胞的cDNA杂交RNA印迹进行定量光密度分析，我们发现绒毛蛋白、钙调蛋白和原肌球蛋白的稳态mRNA水平增加了2至3倍；这种增加与形态学上刷状缘的发育平行。肌动蛋白、血影蛋白和肌球蛋白的mRNA水平没有显著变化。对隐窝、中部和顶端细胞总裂解物的ELISA显示，随着刷状缘的发育，肌动蛋白、肌球蛋白、血影蛋白、原肌球蛋白、肌球蛋白I、绒毛蛋白或α - 辅肌动蛋白的蛋白质水平没有显著变化。G - /F - 肌动蛋白比率也没有随着刷状缘的组装而改变。我们得出结论，虽然刷状缘在未成熟的肠上皮细胞中没有完全组装，但主要的细胞骨架蛋白已以其全部浓度存在，并且已经定位在顶端细胞质内。因此，刷状缘的形成可能涉及由一种未确定的关键蛋白的表达或调节介导的现有细胞骨架蛋白池的重组。
