Defining microtubule cytoskeleton regulatory pathways in development and disease

定义发育和疾病中的微管细胞骨架调控途径

• 批准号: 10466869
• 负责人: Kassandra Marie Ori-McKenney
• 金额: $ 36.71万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10466869
• 关键词: Affect; Architecture; Behavior; Binding; Binding Proteins; Biochemical; Biochemical Genetics; Biological Assay; Cell Culture Techniques; Cell physiology; Cells; Complement; Cytoskeletal Modeling; Cytoskeleton; Data; Development; Disease; Drosophila genus; Ensure; Exhibits; Goals; Human Pathology; Imaging Techniques; In Vitro; Individual; Intellectual functioning disability; Lead; Maintenance; Malignant Neoplasms; Microscopy; Microtubule-Associated Proteins; Microtubules; Molecular; Molecular Motors; Morphogenesis; Morphology; Motor; Neurodegenerative Disorders; Neurons; Output; Pathologic; Pathway interactions; Pattern; Peripheral Nervous System; Phosphorylation; Phosphotransferases; Play; Process; Proteins; Publishing; Regulation; Regulatory Pathway; Role; Signal Pathway; Structure; System; Total Internal Reflection Fluorescent; base; beta Tubulin; ex vivo imaging; experimental study; human disease; in vivo; insight; interdisciplinary approach; live cell imaging; reconstitution

Title: Defining microtubule cytoskeleton regulatory pathways in development and disease P.I. Kassandra Ori-McKenney Project Summary: Cellular architecture is governed by the organization of cytoskeletal networks and determines the functional output of a cell. It is therefore essential to understand the regulatory mechanisms of cytoskeleton organization as a cell develops, changes, or maintains its internal structure, because altering these processes can disrupt cell function and ultimately lead to pathological conditions. The DYRK1a kinase signaling pathway is implicated in intellectual disability disorders, neurodegenerative disease, and cancer. DYRK1a has important roles in a range of cellular processes; however, the downstream molecular mechanisms of this kinase are largely unknown. Our published and preliminary data have shown that DYRK1a regulates the microtubule cytoskeleton directly by phosphorylating β-tubulin, and indirectly through phosphorylation of multiple microtubule-associated proteins (MAPs). We have found that these MAPs exhibit diverse binding behaviors on the microtubule lattice and differentially affect microtubule motors, highlighting an essential role for MAPs in gating access to the lattice. The goal of this project is to dissect the multiple layers of regulation of microtubule- based processes by studying the biochemical and genetic relationships between three kinases, eight MAPs, and five motors both in vivo and in vitro. We aspire to construct a comprehensive network to elucidate the multiple ways in which disease-relevant kinases modulate the microtubule cytoskeleton during different cellular processes, from neuronal polarization to the establishment of specific dendritic morphologies to the maintenance of neuronal architecture. To accomplish these goals, we will use an interdisciplinary approach combining in vivo and ex vivo imaging techniques with in vitro biochemical assays. We will utilize the dendritic arborization neurons of the Drosophila peripheral nervous system to study neuronal morphogenesis, dendritic pruning, and polarized transport, combined with mammalian neuronal cell culture and expansion microscopy to analyze MAP localization patterns under various conditions and determine how MAPs differentially direct molecular motors to ensure proper targeting of cargoes to specific compartments. To complement these in vivo experiments, we plan to perform in vitro reconstitution experiments using TIRF microscopy with purified MAPs, microtubule motors, and kinases in order to elucidate their individual and collective effects on MAP binding, microtubule dynamics and microtubule-based transport. We endeavor to comprehensively dissect kinase-MAP networks at the protein level and at the cellular level in order to understand how these pathways contribute to a diversity of human pathologies.

标题：定义发育和疾病中的微管细胞骨架调控途径 P.I.卡桑德拉·奥里-麦肯尼 项目总结： 细胞结构由细胞骨架网络的组织所支配，并决定功能 单元格的输出。因此，了解细胞骨架组织的调节机制是至关重要的 随着细胞的发育、改变或维持其内部结构，因为改变这些过程可能会扰乱 细胞功能并最终导致病理状态。DYRK1a激酶信号通路被牵连 智力障碍、神经退行性疾病和癌症。DYRK1a在一个 一系列的细胞过程；然而，这种激酶的下游分子机制主要是 未知。我们已发表的和初步的数据表明，DYRK1a调节微管 细胞骨架直接通过磷酸化β-微管蛋白，间接通过磷酸化 微管相关蛋白(MAP)。我们发现这些映射在上表现出不同的绑定行为 微管晶格和微管马达的不同影响，突出了MAP在 对晶格的访问进行门禁。这个项目的目标是剖析微管的多层调控-- 通过研究三种酶之间的生化和遗传关系，八种图谱， 以及体内和体外的五个马达。我们希望建立一个全面的网络来阐明 疾病相关蛋白在不同细胞中对微管细胞骨架的多种调控方式 从神经元极化到特定树突形态的建立，再到 神经元结构的维护。为了实现这些目标，我们将使用跨学科的方法。 将体内和体外成像技术与体外生化检测相结合。我们将利用树枝状细胞 果蝇周围神经系统神经元树枝状突起的研究 修剪和极化运输，结合哺乳动物神经元细胞培养和扩增显微镜 分析各种情况下的地图本地化模式，确定地图如何进行差异化定向 分子马达，确保将货物正确定位到特定的车厢。在活体内补充这些 实验，我们计划使用提纯的MAP进行TIRF显微镜的体外重建实验， 微管马达和激酶为了阐明它们对MAP结合的单独和集体影响， 微管动力学和基于微管的转运。我们致力于全面剖析蛋白激酶图谱 蛋白质水平和细胞水平的网络，以了解这些途径如何对 人类病理的多样性。