Hoock T C, Newcomb P M, Herman I M
Program in Cell, Molecular and Developmental Biology, Tufts University Health Science Schools, Boston, Massachusetts 02111.
J Cell Biol. 1991 Feb;112(4):653-64. doi: 10.1083/jcb.112.4.653.
Previous work in our laboratory has shown that microvascular pericytes sort muscle and nonmuscle actin isoforms into discrete cytoplasmic domains (Herman, I. M., and P. A. D'Amore. 1985. J. Cell Biol. 101:43-52; DeNofrio, D.T.C. Hoock, and I. M. Herman. J. Cell. Biol. 109:191-202). Specifically, muscle (alpha-smooth) actin is present on the stress fibers while nonmuscle actins (beta and gamma) are located on stress fibers and in regions of moving cytoplasm (e.g., ruffles, lamellae). To determine the form and function of beta actin in microvascular pericytes and endothelial cells recovering from injury, we prepared isoform-specific antibodies and cDNA probes for immunolocalization, Western and Northern blotting, as well as in situ hybridization. Anti-beta actin IgG was prepared by adsorption and release of beta actin-specific IgG from electrophoretically purified pericyte beta actin bound to nitrocellulose paper. Anti-beta actin IgGs prepared by this affinity selection procedure showed exclusive binding to beta actin present in crude cell lysates containing all three actin isoforms. For controls, we localized beta actin as a bright rim of staining beneath the erythrocyte plasma membrane. Anti-beta actin IgG, absorbed with beta actin bound to nitrocellulose, failed to stain erythrocytes. Simultaneous localization of beta actin with the entire F-actin pool was performed on microvascular pericytes or endothelial cells and 3T3 fibroblasts recovering from injury using anti-beta actin IgG in combination with fluorescent phalloidin. Results of these experiments revealed that pericyte beta actin is localized beneath the plasma membrane in association with filopods, pseudopods, and fan lamellae. Additionally, we observed bright focal fluorescence within fan lamellae and in association with the ends of stress fibers that are preferentially associated with the ventral plasmalemma. Whereas fluorescent phalloidin staining along the stress fibers is continuous, anti-beta actin IgG localization is discontinuous. When injured endothelial and 3T3 cells were stained through wound closure, we localized beta actin only in motile cytoplasm at the wound edge. Staining disappeared as cells became quiescent upon monolayer restoration. Appearance of beta actin at the wound edge correlated with a two- to threefold increase in steady-state levels of beta actin mRNA, which rose within 15-60 min after injury and returned to noninjury levels during monolayer restoration. In situ hybridization revealed that transcripts encoding beta actin were localized at the wound edge in association with the repositioned protein. Results of these experiments indicate that beta actin and its encoded mRNA are polarized at the membrane-cytoskeletal interface within regions of moving cytoplasm.
我们实验室之前的工作表明，微血管周细胞将肌肉型和非肌肉型肌动蛋白异构体分选到离散的细胞质区域（赫尔曼，I.M.，和P.A.德阿莫尔。1985年。《细胞生物学杂志》101:43 - 52；德诺弗里奥，D.T.、C.胡克，和I.M.赫尔曼。《细胞生物学杂志》109:191 - 202）。具体而言，肌肉型（α - 平滑肌）肌动蛋白存在于应力纤维上，而非肌肉型肌动蛋白（β和γ）则位于应力纤维以及细胞质移动的区域（如褶皱、片状伪足）。为了确定β - 肌动蛋白在微血管周细胞和从损伤中恢复的内皮细胞中的形式和功能，我们制备了异构体特异性抗体和cDNA探针，用于免疫定位、蛋白质免疫印迹和Northern印迹分析以及原位杂交。抗β - 肌动蛋白IgG是通过从结合在硝酸纤维素纸上的电泳纯化的周细胞β - 肌动蛋白中吸附和释放β - 肌动蛋白特异性IgG制备的。通过这种亲和选择程序制备的抗β - 肌动蛋白IgG显示出与含有所有三种肌动蛋白异构体的粗细胞裂解物中存在的β - 肌动蛋白特异性结合。作为对照，我们将β - 肌动蛋白定位为红细胞质膜下方明亮的染色边缘。用结合在硝酸纤维素上的β - 肌动蛋白吸收的抗β - 肌动蛋白IgG未能对红细胞进行染色。使用抗β - 肌动蛋白IgG与荧光鬼笔环肽结合，对从损伤中恢复的微血管周细胞、内皮细胞和3T3成纤维细胞进行β - 肌动蛋白与整个F - 肌动蛋白池同时定位。这些实验结果表明，周细胞β - 肌动蛋白定位于质膜下方，与丝状伪足、伪足和扇形片状伪足相关。此外，我们在扇形片状伪足内以及与优先与腹侧质膜相关的应力纤维末端观察到明亮的局灶性荧光。沿着应力纤维的荧光鬼笔环肽染色是连续的，而抗β - 肌动蛋白IgG的定位是不连续的。当通过伤口闭合对受伤的内皮细胞和3T3细胞进行染色时，我们仅在伤口边缘的运动细胞质中定位到β - 肌动蛋白。随着细胞在单层恢复后静止，染色消失。β - 肌动蛋白在伤口边缘的出现与β - 肌动蛋白mRNA稳态水平增加两到三倍相关，该水平在损伤后15 - 60分钟内升高，并在单层恢复期间恢复到未损伤水平。原位杂交显示，编码β - 肌动蛋白的转录本定位于伤口边缘，与重新定位的蛋白质相关。这些实验结果表明，β - 肌动蛋白及其编码的mRNA在移动细胞质区域内的膜 - 细胞骨架界面处极化。
