Symons M H, Mitchison T J
Department of Pharmacology, University of California, San Francisco 94143.
J Cell Biol. 1991 Aug;114(3):503-13. doi: 10.1083/jcb.114.3.503.
We have investigated the spatial control of actin polymerization in fibroblasts using rhodamine-labeled muscle actin in; (a) microinjection experiments to follow actin dynamics in intact cells, and (b) incubation with permeabilized cells to study incorporation sites. Rhodamine-actin was microinjected into NIH-3T3 cells which were then fixed and stained with fluorescein-phalloidin to visualize total actin filaments. The incorporation of newly polymerized actin was assayed using rhodamine/fluorescein ratio-imaging. The results indicated initial incorporation of the injected actin near the tip and subsequent transport towards the base of lamellipodia at rates greater than 4.5 microns/min. Furthermore, both fluorescein- and rhodamine-intensity profiles across lamellipodia revealed a decreasing density of actin filaments from tip to base. From this observation and the presence of centripetal flux of polymerized actin we infer that the actin cytoskeleton partially disassembles before it reaches the base of the lamellipodium. In permeabilized cells we found that, in agreement with the injection studies, rhodamine-actin incorporated predominantly in a narrow strip of less than 1-microns wide, located at the tip of lamellipodia. The critical concentration for the rhodamine-actin incorporation (0.15 microM) and its inhibition by CapZ, a barbed-end capping protein, indicated that the nucleation sites for actin polymerization most likely consist of free barbed ends of actin filaments. Because any potential monomer-sequestering system is bypassed by addition of exogenous rhodamine-actin to the permeabilized cells, these observations indicate that the localization of actin incorporation in intact cells is determined, at least in part, by the presence of specific elongation and/or nucleation sites at the tips of lamellipodia and not solely by localized desequestration of subunits. We propose that the availability of the incorporation sites at the tips of lamellipodia is because of capping activities which preferentially inhibit barbed-end incorporation elsewhere in the cell, but leave barbed ends at the tips of lamellipodia free to add subunits.
我们利用罗丹明标记的肌肉肌动蛋白，通过以下方式研究了成纤维细胞中肌动蛋白聚合的空间控制：(a) 进行显微注射实验以追踪完整细胞中的肌动蛋白动态，以及 (b) 与通透细胞孵育以研究掺入位点。将罗丹明 - 肌动蛋白显微注射到NIH - 3T3细胞中，然后固定并用荧光素 - 鬼笔环肽染色以可视化总肌动蛋白丝。使用罗丹明/荧光素比率成像测定新聚合肌动蛋白的掺入情况。结果表明，注射的肌动蛋白最初在片足尖端附近掺入，随后以大于4.5微米/分钟的速率向片足基部运输。此外，横跨片足的荧光素和罗丹明强度分布均显示，从尖端到基部肌动蛋白丝的密度逐渐降低。基于这一观察结果以及聚合肌动蛋白向心通量的存在，我们推断肌动蛋白细胞骨架在到达片足基部之前会部分解体。在通透细胞中，我们发现，与注射研究结果一致，罗丹明 - 肌动蛋白主要掺入位于片足尖端小于1微米宽的窄带区域。罗丹明 - 肌动蛋白掺入的临界浓度（0.15微摩尔）及其被一种带刺末端封端蛋白CapZ抑制的现象表明，肌动蛋白聚合的成核位点很可能由肌动蛋白丝的游离带刺末端组成。由于向通透细胞中添加外源罗丹明 - 肌动蛋白绕过了任何潜在的单体隔离系统，这些观察结果表明，完整细胞中肌动蛋白掺入的定位至少部分是由片足尖端特定的延伸和/或成核位点的存在决定的，而不仅仅是由亚基的局部解隔离决定的。我们提出，片足尖端掺入位点的可用性是由于封端活性，这种活性优先抑制细胞其他部位的带刺末端掺入，但使片足尖端的带刺末端能够自由添加亚基。
