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(由申请者提供)：该项目的主要目标是了解Lis1蛋白的突变如何导致儿童癫痫障碍--无脑畸形。在发育中的大脑中，Lis1对有丝分裂和迁移很重要。Lis1与一种产生力量的微管马达--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，但它最为人所知的可能是轴突的长距离逆行运输。轴突运输对功能和生存都至关重要，也是神经退行性疾病的一个促成因素。许多类型的癫痫对靶向通道和受体的现有药物是难以治愈的。Lis1在成熟的神经系统中表达，而Lis1和dynein之间的联系开启了一种令人兴奋的可能性，即运输缺陷可能导致神经元缺陷并导致癫痫发作。我们的初步数据支持Lis1在调节运输方面的作用。拟议中的实验将检验有助于这一功能的机制，部分是由新的发现指导的，即LiS1可以在体外刺激动力蛋白的酶活性。我们有以下具体目标： 目的1.鉴定和表征在动力蛋白刺激中起重要作用的L is1相互作用a.鉴定被L is1截断突变体破坏的相互作用b.确定L is1结合蛋白是否与L is1在体外协同刺激动力蛋白c.检测L is1错义突变对动力蛋白刺激的影响。 目的2.探讨影响动力蛋白刺激的因素与体内细胞器转运的关系。A.确定在Cos-7细胞中是否需要dynein来表达Lis1的表型b.研究Ndel1在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小鼠背根神经节神经元的逆行轴突运输 目的3.体外检测Lis1对单个动力蛋白运动的影响。B.检测Ndel1和Ndel1的磷酸化突变体。神经内细胞器和蛋白质的运动是由产生力量的马达蛋白质完成的。其中，细胞质动力蛋白是参与突触到细胞体运输的主要马达，对神经元的健康和生存至关重要。Lis1可以与dynein结合，但这一点的后果尚不清楚。Lis1基因的突变会导致人类严重的大脑疾病。这项拟议的研究的目标是确定Lis1突变是否会扰乱基于动力蛋白的神经运输。