Actin Assembly and Motility During Endocytosis in Yeast

描述（由申请人提供）：分支肌动蛋白丝网络是高度动态的结构，它们的组装被假设为多种类型的细胞运动产生动力，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都是保守的。利用酵母肌动蛋白贴片作为模型系统，本研究旨在测试肌动蛋白网络组装的树突状成核模型的预测，该模型描述了分支肌动蛋白细丝的网络如何在体内组装并产生力。该模型的核心角色是Arp2/3复合体，它产生分支并为新细丝的形成埋下种子。这些研究将集中于了解Arp2/3活性调节因子驱动酵母肌动蛋白贴片的组装和运动以及内吞作用的分子机制。gfp标记的肌动蛋白贴片组分的运动，在贴片进化的特定阶段经历可重复的运动，将被研究。将在携带Arp2/3调节因子突变的菌株中检测斑块的运动，并将使用高速显微镜结合计算机辅助粒子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，将内部的货物移到内部，将外部的物质内化，以及一些致病菌的移动都是由肌动蛋白丝网络的聚合提供的。对这些网络组装的适当调节对于正常的细胞功能和多细胞生物的正常发育至关重要，而这些网络的错误调节通常伴随着癌症等疾病。通过了解细胞中肌动蛋白网络的形成是如何被调节的，我们将能够更好地理解细胞的正常功能以及它们的失调是如何导致疾病的。

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.