Examining the role of the Lissencephaly Protein, Lis1, in Dynein-Based 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，这是一种儿童癫痫症。Lis1对发育中的大脑有丝分裂和迁移很重要。Lis1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细胞和迁移细胞中起关键作用，但它最广为人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，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揭示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在调节运输中的作用。该实验将研究促进这一功能的机制，部分受Lis1在体外可以刺激动力蛋白酶活性的新发现的指导。我们的具体目标如下：

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.