Tuning the Biophysical Properties of Dynein 2 for Intraflagellar Transport

描述(由申请人提供)：用于鞭毛内运输的细胞质动力蛋白2的生物物理参数的调整动力蛋白是在活细胞内运输货物的大型复杂分子马达。动力蛋白主要有两类，一类是特征较好的动力蛋白1家族，另一类是较少被理解的动力蛋白2家族。虽然动力蛋白1具有广泛的细胞内功能，但动力蛋白2‘S的作用仅限于在称为纤毛和鞭毛的特殊细胞延伸内运输。这项提议的长期目标是在单分子水平上充分描述动力蛋白2马达的特征，并提供一个遗传系统来操纵活体中的马达，以确定这些单分子特性的功能后果。由于纤毛与多囊肾病和视网膜变性等人类疾病有关，而动力蛋白2对这些细胞器的正常功能是必不可少的，因此这一提议直接关系到人类健康。该提案分为两个核心目标：在单分子水平上体外表征Dynein 2的运动特性，以及在遗传模型秀丽线虫(C.elegans)中对这些测量的特性进行体内操作。我们将从线虫中克隆出一个最小的dynein 2运动区，并利用杆状病毒系统表达该蛋白。我们将使用生物化学和生物物理方法相结合的方法来表征dynein 2的酶和运动特性。诸如ATPase、失速力、加工能力和速度等参数将在体外进行测量，并将寻求突变来调节这些单分子特性。然后，我们将把突变的dynein 2蛋白重新引入线虫的零背景中。这将使我们能够确定不同的生物物理性质如何与动力蛋白2‘S在体内发挥作用有关。单分子参数的变化，如作用力和加工性，将首次在生命系统中进行分析，以确定这些参数对正常运动功能的关键程度。

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.