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

• 批准号: 8263959
• 负责人: Richard James McKenney
• 金额: $ 4.92万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/8263959
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Animals; Axonal Transport; Baculoviruses; Biochemical; Biological Assay; Caenorhabditis elegans; Carrier Proteins; Cells; Characteristics; Cilia; Complex; Defect; Dynein ATPase; Essential Genes; Family; Fellowship; Flagella; Genetic; Genetic Models; Goals; Health; Homeostasis; Human; In Vitro; Kinesin; Learning; Life; Measurement; Measures; Microtubules; Minus End of the Microtubule; Molecular; Molecular Motors; Monitor; Motion; Motor; Movement; Mutate; Mutation; Myosin ATPase; Nuclear; Organelles; Organism; Output; Polycystic Kidney Diseases; Production; Property; Protein Family; Proteins; Recombinants; Retinal Degeneration; Role; Structure; System; Time; Translating; Translational Research; Work; ciliopathy; cilium biogenesis; human disease; in vivo; infancy; insight; interest; member; public health relevance; single molecule

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. PUBLIC HEALTH RELEVANCE: Ciliopathies are a broad spectrum of human diseases caused by malfunctions of cell protrusions called cilia. Cilia are built and maintained by specialized transport proteins whose functions are incompletely understood. This project will investigate, in detail, one of those transport proteins to determine how it works as a molecular motor and how its motor functions are related to the normal functioning of cilia. )

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节动力蛋白是在活细胞内运输货物的大型且复杂的分子马达。动力蛋白主要有两类：特征较为明确的动力蛋白 1 家族和不太了解的动力蛋白 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 的作用仅限于在称为纤毛和鞭毛的特殊细胞延伸内运输。该提案的长期目标是在单分子水平上全面表征动力蛋白 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对于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 运动特性进行体外表征，以及在遗传模型秀丽隐杆线虫 (C. elegans) 中对这些测量特性进行体内操作。我们将从秀丽隐杆线虫中克隆出最小的动力蛋白 2 运动结构域，并使用杆状病毒系统表达该蛋白质。我们将结合生物化学和生物物理方法来表征动力蛋白 2 的酶促和运动特性。将在体外测量 ATP 酶、失速力、持续合成能力和速度等参数，并寻求突变来调节这些单分子特性。然后，我们将突变的动力蛋白 2 蛋白重新引入秀丽隐杆线虫的无效背景中。这将使我们能够确定不同的生物物理特性与动力蛋白 2 体内功能的关系。将首次在生命系统中分析单分子参数（例如力的产生和持续加工能力）的变化，以确定这些参数对正常运动功能的重要性。 公共卫生相关性：纤毛病是由称为纤毛的细胞突起功能障碍引起的一系列人类疾病。纤毛是由专门的转运蛋白构建和维持的，其功能尚不完全清楚。该项目将详细研究其中一种转运蛋白，以确定它如何作为分子马达发挥作用，以及其运动功能如何与纤毛的正常功能相关。 ）