Basal body post-translational modifications promote resistance to ciliary force

基底体翻译后修饰促进对纤毛力的抵抗

• 批准号: 9760275
• 负责人: Anthony Junker
• 金额: $ 3.3万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 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 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9760275
• 关键词: Affect; Allergens; Asthma; Basal Cell; Binding; Binding Proteins; Body Regions; Cell physiology; Cells; Cellular Structures; Characteristics; Chronic Obstructive Airway Disease; Cilia; Cilium Microtubule; DNA Sequence Alteration; Disease; Enzymes; Genetic; Goals; Image Analysis; Impairment; Length; Measures; Mechanical Stress; Mediating; Microtubule Triplet; Microtubule-Associated Proteins; Microtubules; Modification; Molecular; Molecular Biology; Mucous body substance; Pathology; Positioning Attribute; Post-Translational Protein Processing; Primary Ciliary Dyskinesias; Property; Proteins; Radial; Regulation; Research Personnel; Research Project Grants; Resistance; Role; Rotation; Stress; Structure; Testing; Tetrahymena; Toxin; Translating; Tubulin; appendage; base; cell motility; cilium motility; experience; fascinate; fluid flow; hydrodynamic flow; imaging genetics; interdisciplinary approach; kinetosome; light microscopy; mechanical force; mucus clearance; pathogen; quantitative imaging; recruit; respiratory; respiratory airway clearance; response; skills; teacher

Project Summary/Abstract Motile cilia are microtubule based, cellular appendages that asymmetrically undulate to generate directed fluid flow. This fluid flow is vital for the conserved functions of cell motility and respiratory airway mucus clearance. Disruption of motile cilia contributes to pathologies such as chronic obstructive pulmonary disease (COPD), asthma and primary ciliary dyskinesia (PCD). Motile cilia are directly anchored to the cell by basal bodies (BB). BB are radially symmetric, cylinder shaped structures made up of nine-triplet microtubule blades. BBs must both resist and transmit mechanical forces from beating cilia to the cell for effective fluid flow. The Pearson lab identified proteins and the microtubule post-translational modification (PTM), glutamylation, to stabilize BBs against ciliary forces. While BBs are radially symmetric, the forces received by cilia are asymmetric. We find that BB PTM glutamylation localizes asymmetrically to BB regions predicted to experience the most mechanical force from cilia. Microtubule glycylation and glutamylation are competitive PTMs that modify the same residues of tubulin. PTMs are known to regulate microtubules by intrinsically controlling physical characteristic like bending or the binding of microtubule associated proteins. Moreover, tubulin glutamylation levels directly control protein activity. This gives rise to the fascinating possibility that BB stabilization is responsive to mechanical forces received from cilia. It is not known whether BB glycylation stabilizes BBs against ciliary forces. It is also unclear whether the competition between BB microtubule glutamylation and glycylation regulate BB stability. In Aim 1, I will use quantitative light microscopy to determine how PTM levels impact BB stability in response to ciliary stress. BB glutamylation asymmetrically localizes to BB domains that experience the greatest mechanical force from cilia. How this asymmetry is established and whether BB glutamylation and glycylation respond to changes in ciliary forces is unknown. In Aim 2, I will determine how BB PTMs asymmetrically localize and whether they respond to forces from ciliary beating. BB glutamylation stabilizes BBs against ciliary forces. Whether this stabilization is achieved through intrinsic BB microtubule regulation like bending or through BB stabilizing or destabilizing binding proteins is unknown. In Aim 3, I will determine whether microtubule PTMs affect BB bending and the localization of BB stabilizing or destabilizing proteins. My project will provide a mechanistic perspective on how cell structures interact with physical forces. I will measure how BBs are stabilized against forces from ciliary beating by employing quantitative imaging, genetic and molecular manipulation. By using multi-disciplinary approaches and quantitative analyses, I will hone skills that directly translate into my aspirations of being an independent investigator and teacher.

项目摘要/摘要 活动纤毛是以微管为基础的细胞附属物，不对称地波动以产生定向液体。 流。这种液体流动对于细胞运动和呼吸道粘液清除的保守功能是至关重要的。 运动纤毛的破坏会导致慢性阻塞性肺疾病(COPD)等病理变化， 哮喘和原发性睫状体运动障碍(PCD)。活动纤毛由基底体(BB)直接固定在细胞上。 BB是径向对称的圆柱形结构，由九个三重微管叶片组成。BBS必须 两者都能抵抗并将纤毛跳动产生的机械力传递给细胞，以实现有效的流体流动。 皮尔逊实验室发现了蛋白质和微管翻译后修饰(PTM)， 谷氨酰化，以稳定BBS对抗纤毛力。虽然BBS是径向对称的，但受到的力 纤毛是不对称的。我们发现，BB PTM谷氨酸化不对称地定位于BB区，预测 体验纤毛产生的最大机械力。微管甘氨酸化和谷氨酰化是竞争性的 修饰相同的微管蛋白残基的PTM。已知PTM通过内在地调节微管 控制物理特性，如弯曲或微管相关蛋白的结合。此外， 微管蛋白谷氨酰化水平直接控制蛋白质活性。这导致了一种迷人的可能性，即BB 稳定化是对纤毛产生的机械力的反应。 目前尚不清楚BB甘氨酸化是否能稳定BBS对抗睫状力。目前也不清楚是否 BB微管谷氨酸化和甘氨酸化之间的竞争调节BB的稳定性。在目标1中，我将使用 定量光学显微镜，以确定PTM水平如何影响BB在应对纤毛压力时的稳定性。BB 谷氨酰化不对称地定位于BB域，这些结构域经历了来自纤毛的最大机械力。 这种不对称性是如何建立的，以及BB谷氨酰化和甘氨酸化是否对纤毛的变化做出反应 力量是未知的。在目标2中，我将确定BB PTM如何不对称地局部化，以及它们是否响应 来自纤毛击打的力量。BB谷氨酸化稳定BBS对抗纤毛力。无论这种稳定是 通过内在的BB微管调节，如弯曲，或通过BB稳定或不稳定 结合蛋白是未知的。在目标3中，我将确定微管PTM是否影响BB弯曲和 BB稳定或不稳定蛋白的定位。 我的项目将提供一个关于细胞结构如何与物理力量相互作用的机械论观点。我 将通过使用定量成像来测量BB是如何稳定下来的，以抵御纤毛跳动的力量， 基因和分子操纵。通过使用多学科方法和量化分析，我将 磨练技能，这些技能直接转化为我想成为一名独立调查员和教师的愿望。