STRUCTURAL ASSEMBLY IN MUSCLE AND NON-MUSCLE CELLS

我们研究的目的是为了了解心绞痛的发病机制。 非肌肉和发育期肌肉收缩结构的组装 细胞。现代技术的结合，包括显微注射， 荧光模拟细胞化学、数字图像处理和激光 光漂白将被用于直接从生活中获取信息 细胞。我们将首先微量注射荧光标记的肌动蛋白核化 并确定它们是否能够激活肌动蛋白 聚合反应。结果将表明，高水平的 活细胞中未聚合的肌动蛋白通过去激活肌动蛋白来维持 亚基，或通过封顶灯丝末端。接下来，我们将用 肌醇磷脂或蛋白激酶C激活剂，以确定其 可能在调节肌动蛋白聚合中的作用。我们还将确定 能够整合肌动蛋白亚基的内源位点，通过 显微注射荧光肌动蛋白亚基并提取细胞 短潜伏期。活动站点将通过比较来确定 注射肌动蛋白与全丝肌动蛋白的分布 使用比率成像技术。公司成立的能力和模式 形成主要的片状脂肪、收缩环和发育中的肌原纤维。 将会被分析。此外，合并到后部与 极化细胞的前部区域将被比较，以便检测 可能是含有肌动蛋白的皮质的流动或循环。最后，我们 将通过标记来研究应力纤维和肌原纤维的伸长 用荧光探针，并使用激光微束标记 靠近细长原纤维末端的参照点。运动的运动 相对于原纤维末端的斑点，将指示位置和 丝束的伸长是如何发生的。加在一起，这些 实验将提供有关这一机制的重要信息 肌动蛋白聚合和组装的调控 活细胞中含有肌动蛋白的结构。

The objective of our research is to understand the mechanism of the assembly of contractile structures in non-muscle and developing muscle cells. A combination of modern techniques, including microinjection, fluorescent analogue cytochemistry, digital image processing, and laser photobleaching will be used to obtain information directly from living cells. We will first microinject fluorescently labeled actin nucleation sites, and determine if they are capable of activating actin polymerization. The results will indicate whether the high level of unpolymerized actin in living cells is maintained by deactivating actin subunits, or by capping filament ends. We will next treat cells with phosphoinositides or protein kinase C activators, to determine their possible roles in regulating actin polymerization. We will also identify endogenous sites that are capable of incorporating actin subunits, by microinjecting fluorescent actin subunits and extracting cells after a short period of incubation. Active sites will be determined by comparing the distribution of injected actin with that of total filamentous actin using ratio imaging techniques. The ability and pattern of incorporation into leading lamellipodia, contractile rings, and developing myofibrils will be analyzed. Furthermore, the incorporation into posterior versus anterior regions of polarized cells will be compared, in order to detect a possible flux or cycling of the actin-containing cortex. Finally, we will study the elongation of stress fibers and myofibrils, by labeling them with fluorescent probes, and using laser microbeams to mark reference points near the ends of elongating fibrils. The movement of the spot, relative to the terminus of the fibril, will indicate where and how the elongation of filament bundles takes place. Together, these experiments will provide important information on the mechanisms of the regulation of actin polymerization and of the assembly of actin-containing structures in living cells.