DeNofrio D, Hoock T C, Herman I M
Department of Anatomy and Cellular Biology, Tufts University Schools of Medicine, Boston, Massachusetts 02111.
J Cell Biol. 1989 Jul;109(1):191-202. doi: 10.1083/jcb.109.1.191.
We characterized the form and distribution of muscle and nonmuscle actin within retinal pericytes. Antibodies with demonstrable specificities for the actin isoforms were used in localization and immunoprecipitation experiments to identify those cellular domains that were enriched or deficient in one or several actin isoforms. Living pericyte behavior was monitored with phase-contract video microscopy before fixation to identify those cellular areas that might preferentially be stained with either of the fluorescent antiactins or phallotoxins. Antibody and phallotoxin staining of pericytes revealed that nonmuscle actin is present within membrane ruffles, pseudopods, and stress fibers. In contrast, muscle actin could be convincingly localized in stress fibers, but not within specific motile areas of pericyte cytoplasm. To confirm and quantitatively extend the results obtained by fluorescence microscopy, nonionic and ionic detergents were used to selectively extract the motile or immobilized (stress fiber-containing) regions of biosynthetically labeled pericyte cytoplasm. Immunoprecipitated actins that were present within these discrete cellular domains were subjected to isoelectric focusing in urea-polyacrylamide gels before fluorographic analysis. Scanning laser densitometry of the focused actins could not reveal any detectable alpha-actin within those beta- and gamma-actin-enriched motile regions extracted with nonionic detergents. Moreover, when pericyte stress fibers are completely dissolved by ionic detergent lysis, three actin isoforms can be quantified to be present in a ratio of 1:2.75:3 (alpha:beta:gamma). These biochemical findings on biosynthetically labeled and immunoprecipitated pericyte actins confirm the fluorescent localization studies. While the regulatory events governing this actin sorting are unknown, it seems possible that such events may play important roles in controlling cell shape, adhesion, or the promotion of localized cell spreading.
我们对视网膜周细胞内肌肉型和非肌肉型肌动蛋白的形态及分布进行了表征。利用对肌动蛋白亚型具有明确特异性的抗体进行定位和免疫沉淀实验，以确定那些富含或缺乏一种或几种肌动蛋白亚型的细胞区域。在固定之前，用相差视频显微镜监测活周细胞的行为，以确定那些可能优先被荧光抗肌动蛋白或鬼笔环肽染色的细胞区域。周细胞的抗体和鬼笔环肽染色显示，非肌肉型肌动蛋白存在于膜皱褶、伪足和应力纤维中。相比之下，肌肉型肌动蛋白可以令人信服地定位在应力纤维中，但不在周细胞细胞质的特定运动区域内。为了证实并定量扩展荧光显微镜获得的结果，使用非离子和离子去污剂选择性提取生物合成标记的周细胞细胞质的运动或固定（含应力纤维）区域。在进行荧光分析之前，对存在于这些离散细胞区域内的免疫沉淀肌动蛋白在尿素 - 聚丙烯酰胺凝胶中进行等电聚焦。聚焦肌动蛋白的扫描激光密度测定法未能在非离子去污剂提取的富含β - 和γ - 肌动蛋白的运动区域内检测到任何可检测到的α - 肌动蛋白。此外，当周细胞应力纤维通过离子去污剂裂解完全溶解时，可以定量三种肌动蛋白亚型，其比例为1:2.75:3（α:β:γ）。这些关于生物合成标记和免疫沉淀的周细胞肌动蛋白生化研究结果证实了荧光定位研究。虽然控制这种肌动蛋白分选的调节事件尚不清楚，但似乎这些事件可能在控制细胞形状、粘附或促进局部细胞铺展中起重要作用。
