Tuning the Biophysical Properties of Dynein 2 for Intraflagellar Transport

调节动力蛋白 2 的生物物理特性以实现鞭毛内运输

基本信息

批准号：
8413037
负责人：
Richard James McKenney
金额：
$ 5.22万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-02-01 至 2014-01-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Tuning of the Biophysical Parameters of Cytoplasmic Dynein 2 for Intraflagellar Transport Dyneins are large and complex molecular motors that transport cargo inside of living cells. There are two main classes of dyneins, the more well characterized dynein 1 family and the less understood dynein 2 family. While dynein 1 has a broad range of intracellular functions, dynein 2's role is restricted to transport within specialized cell extensions called cilia and flagella. The long-term objective of this proposal is to fully characterize the dynein 2 motor at the single molecule level and provide a genetic system for manipulating the motor in living organisms in order to determine the functional consequences of these single molecule properties. Because cilia are implicated in human diseases such as polycystic kidney disease and retinal degeneration, and dynein 2 is essential for normal functioning of these organelles, this proposal is directly relevant to human health. The proposal is divided into two core aims: the in vitro characterization of the dynein 2 motor properties at the single molecule level and the in vivo manipulation of these measured properties in the genetic model Caenorhabditis elegans (C. elegans). We will clone out a minimal dynein 2 motor domain from C. elegans and express the protein using the baculovirus system. We will characterize the enzymatic and motile properties of dynein 2 using a combination of biochemical and biophysical approaches. Parameters such as ATPase, stall force, processivity and velocity will be measured in vitro and mutations will be sought to modulate these single molecule properties. We will then reintroduce the mutated dynein 2 protein into a null background in C. elegans. This will allow us to determine how distinct biophysical properties relate to dynein 2's functions in vivo. Changes in single molecule parameters such as force production and processivity will be assayed for the first time in a living system to determine how critical those parameters are for normal motor function.

描述（由申请人提供）：用于氟叶氏菌输运动力蛋白的细胞质动力蛋白2的生物物理参数调整是大而复杂的分子电动机，可将货物运输到活细胞内。有两种主要类动力素类别，一个更富有特征的动力蛋白1家族和知识较少的Dynein 2家族。虽然Dynein 1具有广泛的细胞内功能，但Dynein 2的作用仅限于在称为Cilia和Flagella的专用细胞扩展中运输。该提案的长期目标是在单分子水平上充分表征动力蛋白2电动机，并提供一种遗传系统来操纵活生物体中的运动，以确定这些单个分子特性的功能后果。因为纤毛与多囊性肾脏疾病和视网膜变性有关的人类疾病与人类疾病有关，而动力蛋白2对于这些细胞器的正常功能至关重要，因此该建议与人类健康直接相关。该提案分为两个核心目的：单分子水平上动力蛋白2运动性能的体外表征，以及在遗传模型Caenorhabditis elegans（C. elegrans）中对这些测得的特性的体内操纵。我们将从秀丽隐杆线虫中取出一个最小的动力蛋白2运动结构域，并使用杆状病毒系统表达蛋白质。我们将使用生物化学和生物物理方法的组合来表征动力蛋白2的酶促性和运动性能。将在体外测量ATPase，摊位力，加工性和速度等参数，并寻求突变以调节这些单分子特性。然后，我们将将突变的动力蛋白2蛋白重新引入秀丽隐杆线虫中的无效背景。这将使我们能够确定不同的生物物理特性与体内的动力蛋白2功能如何相关。单分子参数（例如力产生和加工性）的变化将在生活系统中首次分析，以确定这些参数对于正常运动功能的关键程度。