DYNAMICS OF ACTIN STRUCTURES DURING CELL TRANSFORMATION

我们建议将联合收割机荧光类似物细胞化学光漂白， 利用图像处理技术研究肌动蛋白及其附件的行为 蛋白质在活细胞中的致癌转化过程中以及之后 导致含肌动蛋白的细胞分裂的各种处理， 结构. 肌动蛋白，α-辅肌动蛋白，黏着斑蛋白和原肌球蛋白将被 体外荧光标记并显微注射到活细胞中。 的 标记的分子将融入内源性结构， 来观察活细胞中特定分子的动态特性。 我们 将首先记录图像序列， 含肌动蛋白的结构。 蛋白质在各个部位的分布 各种蛋白质之间的结构和关系， 分析以确定中断机制。 类似意见 将在从治疗中恢复的细胞上进行， 转化，以确定结构组装的途径 以及参与启动过程的蛋白质。 接下来，我们将使用 激光微束在破坏过程中标记结构的各个部分 和重组。 漂白斑点的后续移动将产生 关于伸长或缩短的极性的信息，以及关于 可能发生拉伸或收缩。 此外该 将测量光漂白后荧光的恢复以确定 蛋白质在各种结构中的移动性和交换性。 最后，强烈的激光束将被用来切断应力纤维或破坏 粘附板，以研究粘附的可能要求 应力纤维的稳定性。 这些信息应该能帮助我们 了解含肌动蛋白的结构是如何组装的， 在各种条件下分解，以及转化如何与 也诱导含肌动蛋白结构变化的其他条件。

We propose to combine fluorescent analogue cytochemistry photobleaching, and image processing to study the behavior of actin and its accessory proteins in living cells during oncogenic transformation as well as after various treatments which result in the disruption of actin-containing structures. Actin, alpha-actinin, vinculin and tropomyosin will be fluorescently labeled in vitro and microinjected into living cells. The labeled molecules will incorporate into endogenous structures and allow us to see the dynamic properties of specific molecules in living cells. We will first record sequences of images during the disruption of actin-containing structures. The distribution of proteins in various parts of the structures and the relationship between various proteins will be analyzed to determine the mechanism of disruption. Similar observations will be performed on cells recovering from the treatments and from transformation, in order to determine the pathway of structural assembly and the proteins involved in he initiation process. Next, we will use laser microbeams to mark various parts of the structures during disruption and reassembly. The subsequent movement of the bleached spot will yield information on the polarity of elongation or shortening, and on the possible occurrence of stretching or contraction. In addition, the recovery of fluorescence after photobleaching will be measured to determine the mobility and exchangeability of proteins in various structures. Finally, intense laser beams will be used to sever stress fibers or disrupt adhesion plaques, in order to study the possible requirement of adhesion plaques for the stability of stress fibers. The information should help us to understand how actin-containing structures are assembled and disassembled under various conditions, and how transformation is related to other conditions which also induce changes in actin-containing structures.