Tuning the Biophysical Properties of Dynein 2 for Intraflagellar Transport

描述（由申请人提供）：鞭毛内运输的细胞质动力蛋白2的生物物理参数的调节动力蛋白是在活细胞内运输货物的大而复杂的分子马达。动力蛋白主要有两类，特征性较好的动力蛋白1家族和了解较少的动力蛋白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的作用仅限于在称为纤毛和鞭毛的专门细胞延伸内运输。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马达，并提供用于操纵活生物体中马达的遗传系统，以确定这些单分子性质的功能后果。由于纤毛与多囊性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至关重要，因此该提议与人类健康直接相关。该提案分为两个核心目标：在单分子水平上对动力蛋白2运动特性的体外表征和在遗传模型秀丽隐杆线虫（C.habditis elegans）中对这些测量特性的体内操纵。elegans）。我们将从C.并使用杆状病毒系统表达蛋白质。我们将结合生物化学和生物物理学的方法来表征动力蛋白2的酶和运动特性。将在体外测量诸如ATP酶、失速力、持续合成能力和速度等参数，并将寻求突变来调节这些单分子性质。然后，我们将突变的动力蛋白2蛋白重新引入到C中的空背景中。优雅这将使我们能够确定不同的生物物理特性如何与动力蛋白2的体内功能相关。单分子参数的变化，如力的产生和持续合成能力将首次在生命系统中进行测定，以确定这些参数对正常运动功能的重要性。

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.