Ben-Ze'ev A
Department of Molecular Genetics and Virology, Weizmann Institute of Science, Rehovot, Israel.
Electrophoresis. 1990 Mar;11(3):191-200. doi: 10.1002/elps.1150110302.
Two-dimensional gel electrophoresis was used to study the regulation of cytoskeletal protein synthesis during growth activation and development of the differentiated phenotype. We demonstrated a correlation between the state of organization and the expression of the respective cytoskeletal protein by showing that depolymerization of microtubules leads to a rapid decrease in new tubulin synthesis. We found that the synthesis of vimentin in both fibroblasts and epithelial cells correlates with extensive cell spreading on the substrate, while cytokeratin synthesis is maximal when cell to cell contacts are abundant. The analysis of cytoskeletal elements, involved directly in the formation of cell contacts, revealed that the level of vinculin synthesis is dependent on the extent of adherent type of cell contacts formed. Moreover, we found that the transient disappearance of vinculin from adhesion plaques of quiescent fibroblasts in response to serum factors was followed by an induction of vinculin mRNA and protein synthesis. The morphological changes associated with establishment of the differentiated phenotype were also found to include changes in the expression of the cytoskeletal-extracellular matrix complex. This was demonstrated in several differentiating systems: in 3T3 preadipocytes which change their shape from a fibroblastic to a spherical shape when stimulated to differentiate with adipogenic medium, we observed a decrease in mRNA levels and in the synthesis of fibronectin, beta-integrin, and the microfilament proteins, vinculin, alpha-actinin, tropomyosin and actin. The culturing of these cells on a certain extracellular matrix prevented the morphological changes occurring in the presence of adipogenic medium and blocked the shifts in cytoskeletal- and differentiation-related gene expression. Similar changes in the organization and expression of cytoskeletal proteins were identified during maturation of primary ovarian granulosa cell cultures, stimulated with gonadotropic hormones to form highly steroidogenic cells. The cell rounding and aggregation occurring during this process were associated with a decreased synthesis of vinculin, alpha-actinin, actin and the nonmuscle tropomyosins. The physiological relevance of these changes was suggested by the observation that the level of tropomyosin mRNA was lower in follicles of animals at late stages of granulosa cell maturation when compared to earlier stages. The expression of tissue-specific and cytoskeletal proteins was also determined in primary cultures of liver hepatocytes, maintained under conditions either favorable for growth or for expression of liver-specific functions. When DNA synthesis was elevated, cytoskeletal protein synthesis was high and that of liver-specific proteins was low.(ABSTRACT TRUNCATED AT 400 WORDS)
利用二维凝胶电泳研究分化表型生长激活和发育过程中细胞骨架蛋白合成的调控。通过证明微管解聚导致新微管蛋白合成迅速减少，我们展示了组织状态与各自细胞骨架蛋白表达之间的相关性。我们发现，成纤维细胞和上皮细胞中波形蛋白的合成与细胞在底物上的广泛铺展相关，而细胞角蛋白合成在细胞间接触丰富时达到最大值。对直接参与细胞接触形成的细胞骨架成分的分析表明，纽蛋白合成水平取决于形成的黏附型细胞接触的程度。此外，我们发现，静止成纤维细胞黏附斑中纽蛋白因血清因子而短暂消失后，会诱导纽蛋白mRNA和蛋白合成。与分化表型建立相关的形态学变化还包括细胞骨架-细胞外基质复合物表达的变化。这在几个分化系统中得到了证明：在3T3前脂肪细胞中，当用成脂培养基刺激其分化时，细胞形状从成纤维细胞样变为球形，我们观察到纤连蛋白、β-整合素以及微丝蛋白纽蛋白、α-辅肌动蛋白、原肌球蛋白和肌动蛋白的mRNA水平和合成减少。在特定细胞外基质上培养这些细胞可防止在成脂培养基存在下发生的形态学变化，并阻断细胞骨架和分化相关基因表达的改变。在用促性腺激素刺激原代卵巢颗粒细胞培养物成熟以形成高度类固醇生成细胞的过程中，也发现了细胞骨架蛋白组织和表达的类似变化。在此过程中发生 的细胞变圆和聚集与纽蛋白、α-辅肌动蛋白、肌动蛋白和非肌肉原肌球蛋白的合成减少有关。这些变化的生理相关性通过以下观察结果得到提示：与早期阶段相比，在颗粒细胞成熟后期动物卵泡中原肌球蛋白mRNA水平较低。还在原代培养的肝细胞中测定了组织特异性和细胞骨架蛋白的表达，这些肝细胞在有利于生长或有利于肝特异性功能表达的条件下维持。当DNA合成升高时，细胞骨架蛋白合成较高，而肝特异性蛋白合成较低。（摘要截选至400字）
