Characterization of constitutively active Arp2/3

我的目标是定量了解Arp2/3复合体的激活，Arp2/3复合体是一种在细胞运动中起作用的蛋白质组装工具。为了了解激活剂如何改变Arp2/3复合体的构象，以及这种构象变化如何在体内和体外影响肌动蛋白细丝的成核和分支，我提出了三条线的实验。 。利用半胱氨酸残基之间的二硫键交联形成裂解酵母Arp2/3复合体，以产生结构性活性和失活的Arp2/3复合体。我将比较天然Arp2/3复合体和这两种冷冻构象的光谱和单粒子荧光显微镜分析，以分析成核促进因子，如黄蜂和SCAR的VGA区，肌动蛋白单体，和预先存在的肌动蛋白细丝激活Arp2/3复合体的动力学和热力学。 。使用高分辨率3D荧光显微镜来表征一组突变对 Arp2/3复合体在裂解酵母中支持肌动蛋白细丝成核的能力。 使用模拟来研究参与核苷酸结合和激活的Arp2/3亚基机制。

My goal is a quantitative understanding of the activation of Arp2/3 complex, a protein assembly instrumental in cellular motility. I propose three lines of experiments to understand how activators change the conformation of Arp2/3 complex and how this conformational change influences actin filament nucleation and branching in vivo and in vitro. . Use disulfide crosslinking between cysteine residues engineered into fission yeast Arp2/3 complex to create constitutively active and inactive Arp2/3 complex. I will compare native Arp2/3 complex and these two frozen conformations in spectroscopic and single particle fluorescence microscopy assays to analyze the kinetics and thermodynamics of Arp2/3 complex activation by nucleation promoting factors such as the VGA regions of WASp and Scar, actin monomers, and pre-existing actin filaments. . Use high-resolution 3D fluorescence microscopy to characterize the effects of a panel of mutations on the ability of Arp2/3 complex to support actin filament nucleation in live fission yeast. Use simulations to study Arp2/3 subunit mechanisms involved in nucleotide binding and activation.