Amato P A, Taylor D L
J Cell Biol. 1986 Mar;102(3):1074-84. doi: 10.1083/jcb.102.3.1074.
The mechanism of actin incorporation into and association with stress fibers of 3T3 and WI38 fibroblasts was examined by fluorescent analog cytochemistry, fluorescence recovery after photobleaching (FRAP), image analysis, and immunoelectron microscopy. Microinjected, fluorescein-labeled actin (AF-actin) became associated with stress fibers as early as 5 min post-injection. There was no detectable cellular polarity in the association of AF-actin with pre-existing stress fibers relative to perinuclear or peripheral regions. The rate of incorporation was quantified by image analysis of images generated with a two-dimensional photon counting microchannel plate camera. After equilibration of up to 2 h post-injection, FRAP demonstrated that actin subunits exchanged rapidly between filaments in stress fibers and the surrounding cytoplasm. When co-injected with rhodamine-labeled bovine serum albumin as a control, only actin was detected in the phase-dense stress fibers. The control protein was excluded from fibers and any linear fluorescence of the control was demonstrated as a pathlength artifact. The incorporation of AF-actin into stress fibers was studied by immunoelectron microscopy using anti-fluorescein as the primary antibody and goat anti-rabbit IgG coupled to peroxidase as the secondary antibody. At 5 min post-injection, reaction product was localized periodically in some fibers with a periodicity of approximately 0.75 microns. In large diameter fibers at 5 min post-injection, the analog was seen first on the surface of fibers, with individual filaments resolvable within the core. In the same cell, thinner diameter fibers were labeled uniformly throughout the diameter. By 20 min post-injection, most fibers were uniformly labeled. We conclude that the rate of actin subunit exchange in vivo is extremely rapid with molecular incorporation into actin filaments of stress fibers occurring as early as a few minutes post-injection. Exchange appears to first occur in filaments along the surface of stress fibers and then into more central regions in a periodic manner. We suggest that the periodic localization of actin at very early time points is due to a local microheterogeneity in which microdomains of fast vs. slower incorporation result from the periodic localization of actin-binding protein, such as alpha-actinin, along the length of the fiber.
通过荧光类似物细胞化学、光漂白后荧光恢复（FRAP）、图像分析和免疫电子显微镜检查了肌动蛋白掺入3T3和WI38成纤维细胞的应力纤维并与之结合的机制。显微注射的荧光素标记肌动蛋白（AF-肌动蛋白）在注射后5分钟就开始与应力纤维结合。相对于核周或周边区域，AF-肌动蛋白与预先存在的应力纤维的结合中没有可检测到的细胞极性。通过使用二维光子计数微通道板相机生成的图像进行图像分析来量化掺入率。注射后长达2小时的平衡后，FRAP表明肌动蛋白亚基在应力纤维中的细丝与周围细胞质之间快速交换。当与罗丹明标记的牛血清白蛋白作为对照共同注射时，在相致密的应力纤维中仅检测到肌动蛋白。对照蛋白被排除在纤维之外，对照的任何线性荧光都被证明是光程伪像。使用抗荧光素作为一抗和与过氧化物酶偶联的山羊抗兔IgG作为二抗，通过免疫电子显微镜研究了AF-肌动蛋白掺入应力纤维的情况。注射后5分钟，反应产物周期性地定位在一些纤维中，周期约为0.75微米。在注射后5分钟的大直径纤维中，类似物首先出现在纤维表面，核心内可分辨出单个细丝。在同一细胞中，较细直径的纤维在整个直径上均匀标记。注射后20分钟，大多数纤维被均匀标记。我们得出结论，体内肌动蛋白亚基交换的速率极快，分子在注射后几分钟就开始掺入应力纤维的肌动蛋白细丝中。交换似乎首先发生在应力纤维表面的细丝中，然后以周期性方式进入更中心的区域。我们认为，肌动蛋白在非常早期时间点的周期性定位是由于局部微异质性，其中快速与较慢掺入的微区是由肌动蛋白结合蛋白（如α-辅肌动蛋白）沿纤维长度的周期性定位导致的。
