Examining the role of the Lissencephaly Protein, Lis1, in Dynein-Based Transport

检查无脑畸形蛋白 Lis1 在基于动力蛋白的运输中的作用

• 批准号: 7544907
• 负责人: DEANNA S SMITH
• 金额: $ 24.63万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7544907
• 关键词: Adult; Affect; Axon; Axonal Transport; Binding; Binding Proteins; Biological Assay; Brain; Brain Diseases; Cells; Child; Childhood; Clinical; Cognitive; Data; Defect; Dendrites; Disease; Drug Delivery Systems; Dynein ATPase; Epilepsy; Genes; Goals; Health; Human; Immigration; In Vitro; Individual; Intervention; Intractable Epilepsy; LIS1 protein; Laboratories; Lead; Link; Lysosomes; Measures; Microtubules; Missense Mutation; Mitosis; Mitotic; Motor; Mus; Mutation; Nerve; Nervous system structure; Neurodegenerative Disorders; Neurons; Organelles; Phenotype; Proteins; Psychomotor Impairments; Research; Role; Seizures; Speed; Symptoms; Synapses; Testing; base; cell motility; experience; in vivo; lissencephaly; migration; mutant; neuronal cell body; novel; organelle movement; overexpression; protein expression; receptor; research study; retrograde transport; single molecule; trafficking

DESCRIPTION (provided by applicant): The broad goal of the project is to understand how mutations in the Lis1 protein cause lissencephaly, a childhood epilepsy disorder. Lis1 is important for mitosis and migration in the developing brain. Lis1 interacts with a force-generating microtubule motor, cytoplasmic dynein. Although dynein is critical in mitotic and migrating cells, it is perhaps best known for long distance retrograde trafficking in axons. Axon transport is critical for both function and survival and is a contributing factor in neurodegenerative diseases. Many types of epilepsy are intractable to available drugs that target channels and receptors. Lis1 is expressed in the mature nervous system, and the link between Lis1 and dynein opens up the exciting possibility that transport defects may contribute defective neurons and lead to seizures. Our preliminary data support a role for Lis1 in regulating transport. The proposed experiments will examine the mechanisms contributing to this function, guided in part by the novel finding that Lis1 can stimulate the enzymatic activity of dynein in vitro. We have the following specific aims: Aim 1. To identify and characterize Lis1 interactions important for dynein stimulation a. Identify interactions disrupted by Lis1 truncation mutants b. Determine if Lis1 binding proteins cooperate with Lis1 to stimulate dynein in vitro c. Examine the effect of Lis1 missense mutations on dynein stimulation in vitro. Aim 2. To correlate factors that impact dynein stimulation with organelle transport in vivo. a. Determine if dynein is required for the Lis1 overexpression phenotype in Cos-7 cells b. Examine a role for Ndel1 in lysosome distribution and motility in Cos-7 cells c. Determine how Lis1 missense mutants influence organelle transport d. Measure retrograde axon transport in DRG neurons from adult Lis1 mice Aim 3. To measure the impact of Lis1 on motility of individual dynein motors in vitro. a. Use single motor bead motility assays to determine if Lis1 and Ndel1 affect dynein motility b. Test Ndel1 and a phosphomutant for of Ndel1 in the assay. The movement of organelles and proteins within nerves is carried out by force-generating motor proteins. One of these, cytoplasmic dynein, is the principle motor involved in trafficking from synapses to cell bodies, and is crucial to the health and survival of neurons. Lis1 can bind to dynein, but the consequences of this are not clear. Mutations in Lis1 cause a severe brain disorder in humans. The goal of the proposed research is to determine if Lis1 mutations can disrupt dynein- based nerve transport.

描述（由申请人提供）：该项目的总体目标是了解 Lis1 蛋白的突变如何导致无脑畸形（一种儿童癫痫病）。 Lis1 对于大脑发育中的有丝分裂和迁移非常重要。 Lis1 与产生力的微管马达、细胞质动力蛋白相互作用。尽管动力蛋白在有丝分裂和迁移细胞中至关重要，但它最出名的可能是轴突的长距离逆行运输。轴突运输对于功能和生存都至关重要，并且是神经退行性疾病的一个促成因素。许多类型的癫痫对于针对通道和受体的现有药物来说是难以治疗的。 Lis1 在成熟的神经系统中表达，Lis1 和动力蛋白之间的联系开启了一个令人兴奋的可能性，即运输缺陷可能导致有缺陷的神经元并导致癫痫发作。我们的初步数据支持 Lis1 在调节运输方面的作用。拟议的实验将研究促成此功能的机制，部分是受到 Lis1 可以在体外刺激动力蛋白酶活性的新发现的指导。我们有以下具体目标： 目标 1. 识别和表征对动力蛋白刺激很重要的 Lis1 相互作用鉴定被 Lis1 截短突变体破坏的相互作用 b．确定 Lis1 结合蛋白是否与 Lis1 配合在体外刺激动力蛋白 c．检查 Lis1 错义突变对体外动力蛋白刺激的影响。 目标 2. 将影响动力蛋白刺激的因素与体内细胞器运输相关联。一个。确定 Cos-7 细胞中 Lis1 过表达表型是否需要动力蛋白 b．检查 Ndel1 在 Cos-7 细胞溶酶体分布和运动中的作用 c．确定 Lis1 错义突变体如何影响细胞器运输 d．测量成年 Lis1 小鼠 DRG 神经元的逆行轴突运输 目标 3. 测量 Lis1 对体外单个动力蛋白马达运动的影响。一个。使用单运动珠运动测定来确定 Lis1 和 Ndel1 是否影响动力蛋白运动 b．在测定中测试 Ndel1 和 Ndel1 的磷酸突变体。 神经内细胞器和蛋白质的运动是由产生力的运动蛋白进行的。其中之一，细胞质动力蛋白，是参与从突触到细胞体运输的主要运动，对于神经元的健康和生存至关重要。 Lis1 可以与动力蛋白结合，但其后果尚不清楚。 Lis1 突变会导致人类严重的脑部疾病。拟议研究的目标是确定 Lis1 突变是否会破坏基于动力蛋白的神经运输。