CAREER: Structural Studies of a Highly Conserved Dynein Light Chain and its Role in Dynein Assembly and Cargo Recruitment

细胞质动力蛋白是一种运动蛋白复合物，在有丝分裂过程中的染色体分离、囊泡货物的运动、胞内细胞器的定位以及卵母细胞发育过程中细胞极性的建立等许多过程中都是必不可少的。本项目主要研究果蝇动力蛋白轻链LC8在动力蛋白组装和货物识别中的作用。LC8是高度保守的，在果蝇和人类之间有94%的序列同源性。动力蛋白中间链IC74介导重链和轻链之间的相互作用，LC8和IC74的组装可能是形成功能复合物的关键步骤。为了阐明LC8/IC74识别的基础，配合物的结构将用核磁共振光谱确定。燕子蛋白Swa是一种RNA结合蛋白，在发育中的卵母细胞中建立细胞极性至关重要。Swa与LC8的相互作用已在果蝇中得到证实。这种相互作用中结合位点的鉴定将通过有限的蛋白质水解和交联研究来确定。更广泛的影响：LC8的结构研究及其与IC74和Swa的相互作用将对理解动力蛋白马达复合物的组装及其对货物的附着有重要贡献。这项工作将需要许多本科生的实质性贡献。该项目还将涉及到结构生物学和生物物理学中当前主题的课程开发，这些主题已成为许多生物学研究的核心。

Cytoplasmic dynein is a motor protein complex that is essential in a number of processes including chromosome segregation during mitosis, movement of vesicular cargo, positioning of intracellular organelles, and the establishment of cell polarity in oocyte development. This project focuses on characterizing the role of dynein light chain LC8 from Drosophila in dynein assembly and cargo recognition. LC8 is highly conserved, with 94% sequence identity between Drosophila and human. The dynein intermediate chain IC74 mediates the interaction between the heavy and light chains, and the assembly of LC8 and IC74 is likely to be a key step in formation of a functional complex. To elucidate the basis for LC8/IC74 recognition, the structure of the complex will be determined using NMR spectroscopy. The swallow protein, Swa, an example of cargo, is an RNA binding protein, which is essential in establishing cell polarity in developing oocytes. The interaction of Swa with LC8 has been demonstrated in Drosophila. Identification of binding sites in this interaction will be determined by limited proteolysis and cross-linking studies.Broader Impact: Structural studies of LC8 and its interactions with IC74 and Swa will be a significant contribution to understanding both the assembly of the dynein motor complex and its attachment to cargo. This work will entail substantive contributions from a number of undergraduates. The project will also involve development of a class on the current topics in structural biology and biophysics, which have become central to much of biological research.