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: 8013510
• 负责人: DEANNA S SMITH
• 金额: $ 24.14万
• 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 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8013510
• 关键词: Adult; Affect; Axon; Binding; Binding Proteins; Biological Assay; Brain; Brain Diseases; Cells; Childhood; Data; Defect; Disease; Drug Delivery Systems; Dynein ATPase; Epilepsy; Goals; Health; Human; Immigration; In Vitro; Individual; Intractable Epilepsy; LIS1 protein; Lead; Link; Lysosomes; Measures; Microtubules; Missense Mutation; Mitosis; Mitotic; Motor; Mus; Mutation; Nerve; Nervous system structure; Neurodegenerative Disorders; Neurons; Organelles; Phenotype; Proteins; Research; Role; Seizures; Synapses; Testing; base; cell motility; in vivo; lissencephaly; mutant; neglect; neuronal cell body; novel; organelle movement; overexpression; receptor; research study; 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）：该项目的广泛目标是了解LIS1蛋白中的突变如何引起lissencephaly，这是一种儿童癫痫病。 LIS1对于发育中的大脑中的有丝分裂和迁移很重要。 Lis1与生成力的微管运动，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，但它可能以轴突中的长距离逆行运输而闻名。轴突转运对于功能和生存都是至关重要的，并且是神经退行性疾病的促成因素。许多类型的癫痫症与针对靶向通道和受体的可用药物很棘手。 Lis1在成熟的神经系统中表达，Lis1和Dynein之间的联系打开了令人兴奋的可能性，即传输缺陷可能导致神经元有缺陷并导致癫痫发作。我们的初步数据支持LIS1在调节运输中的作用。提出的实验将检查对此功能的机制，部分原因是Lis1可以刺激体外动力蛋白的酶活性。我们有以下具体目标： 目的1。确定和表征对动力蛋白刺激重要的LIS1相互作用。确定lis1截断突变体破坏的相互作用b。确定LIS1结合蛋白是否与LIS1合作以在体外刺激动力蛋白。检查LIS1错义突变对体外动力蛋白刺激的影响。 目的2。将影响动力蛋白刺激的因素与体内的细胞器转运相关联。一个。确定cos-7细胞中LIS1过表达表型是否需要动力蛋白。检查NDEL1在COS-7细胞中溶酶体分布和运动性中的作用c。确定LIS1错义突变体如何影响细胞器转运d。测量成年Lis1小鼠DRG神经元中的逆行轴突转运 目的3。测量Lis1对单个动力蛋白电动机在体外运动的影响。一个。使用单运动珠运动测定法确定Lis1和Ndel1是否影响Dynein运动b。在测定中测试NDEL1和NDEL1的磷化剂。 细胞器和蛋白质在神经内的运动是通过产生力的运动蛋白来进行的。其中之一是细胞质动力蛋白，是从突触到细胞体进行运输的主要动力，对神经元的健康和存活至关重要。 LIS1可以与动力蛋白结合，但是尚不清楚的后果。 LIS1突变会导致人类严重的脑部疾病。拟议的研究的目的是确定LIS1突变是否会破坏基于动力蛋白的神经传输。