Actin Assembly and Motility During Endocytosis in Yeast

描述（由申请人提供）：分支肌动蛋白丝的网络是高度动态的结构，并且假设它们的组装产生用于许多类型的细胞运动的力，包括细胞迁移、细胞内运输、内吞作用和细胞内病原体的运动。参与调节这些网络组装的蛋白质在整个真核生物中是保守的。使用酵母肌动蛋白补丁作为一个模型系统，这个建议的目的是测试预测的树突状成核模型的肌动蛋白网络组件，它描述了网络的分支肌动蛋白丝组装，并可以产生力，在体内。在这个模型中，一个中心角色是Arp 2/3复合体，它产生分支并为新细丝的形成提供种子。这些研究将侧重于了解Arp 2/3活性调节因子驱动酵母肌动蛋白补丁和内吞作用的组装和运动的分子机制。GFP标记的肌动蛋白补丁组件，在补丁进化的特定阶段进行可重复的运动，将进行研究。斑块的运动将在Arp 2/3调节基因携带突变的菌株中进行检查，并将使用高速显微镜结合计算机辅助颗粒跟踪和定量运动分析进行大量分析。此外，这些突变体中肌动蛋白网络的结构将通过电子显微镜进行分析。细胞迁移所需的力，移动内部的货物，从外部的物质和一些致病菌的运动是由肌动蛋白丝网络的聚合提供的。这些网络的组装的适当调节对于正常的细胞功能和多细胞生物体的正常发育是必不可少的，并且这些网络的失调通常伴随着疾病，如癌症。通过了解细胞中肌动蛋白网络的形成是如何调节的，我们将能够更好地理解细胞的正常功能以及它们的失调如何导致疾病。

DESCRIPTION (provided by applicant): Networks of branched actin filaments are highly dynamic structures and their assembly is hypothesized to generate the force for many types of cell motility including cell migration, intracellular transport, endocytosis and the movement of intracellular pathogens. The proteins involved in regulating the assembly of these networks have been conserved throughout eukaryotes. Using the yeast actin patch as a model system, this proposal aims to test predictions of the dendritic nucleation model of actin network assembly, which describes how networks of branched actin filaments are assembled and can generate force, in vivo. A central player in this model is the Arp2/3 complex, which generates branches and seeds the formation of new filaments. These studies will focus on understanding the molecular mechanisms by which regulators of Arp2/3 activity function to drive the assembly and movement of the yeast actin patch and endocytosis. The movement of GFP-labeled actin patch components, which undergo reproducible movements during specific phases of patch evolution, will be studied. The movement of patches will be examined in strains carrying mutations in Arp2/3 regulators and will be analyzed in large numbers using high-speed microscopy coupled with computer-aided particle tracking and quantitative motion analysis. In addition the architecture of the actin networks in these mutants will be analyzed by electron microscopy. The force required for cells to migrate, to move internal cargoes within, to inernalize materials from the outside and for the movement of some pathogenic bacteria is provided by the polymerization of networks of actin filaments. The proper regulation of the assembly of these networks is essential for normal cellular function and for normal development of mutlicellular organisms and the misregulation of these networks often accompanies diseases, like cancer. By understanding how the formation of actin networks is regulated in cells we will be able to better understand both the normal functions of cells and how their misregulation can lead to disease.