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: 7911470
• 负责人: DEANNA S SMITH
• 金额: $ 1.72万
• 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 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7911470
• 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）：该项目的广泛目标是了解Lis 1蛋白的突变如何导致无脑畸形，一种儿童癫痫疾病。Lis 1对于发育中的大脑中的有丝分裂和迁移很重要。Lis 1与产生力的微管马达细胞质动力蛋白相互作用。虽然动力蛋白在有丝分裂和迁移细胞中是关键的，但它可能是最著名的轴突长距离逆行运输。轴突运输对于功能和存活都是至关重要的，并且是神经退行性疾病的促成因素。许多类型的癫痫对于靶向通道和受体的现有药物是难治的。Lis 1在成熟的神经系统中表达，Lis 1和动力蛋白之间的联系开辟了令人兴奋的可能性，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Lis 1在调节运输中的作用。拟议的实验将研究有助于这一功能的机制，部分指导下的新发现，Lis 1可以刺激动力蛋白在体外的酶活性。我们有以下具体目标： 目标1.识别和表征对动力蛋白刺激重要的Lis 1相互作用。鉴定被Lis 1截短突变体B破坏的相互作用。确定Lis 1结合蛋白是否与Lis 1协同刺激体外动力蛋白c.检查Lis 1错义突变对体外动力蛋白刺激的影响。 目标二。将影响动力蛋白刺激的因素与体内细胞器运输相关联。a.确定Cos-7细胞B中的Lis 1过表达表型是否需要动力蛋白。检查Ndel 1在Cos-7细胞中的溶酶体分布和运动中的作用。确定Lis 1错义突变体如何影响细胞器运输。测量成年Lis 1小鼠DRG神经元的逆行轴突运输 目标3。测量Lis 1对体外单个动力蛋白运动的影响。a.使用单马达珠运动性测定来确定Lis 1和Ndel 1是否影响动力蛋白运动性B。在测定中检测Ndel 1和Ndel 1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马达蛋白进行的。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马达，对神经元的健康和存活至关重要。Lis 1可以与动力蛋白结合，但其后果尚不清楚。Lis 1的突变会导致人类严重的大脑疾病。这项研究的目的是确定Lis 1突变是否会破坏基于动力蛋白的神经转运。