Basal body post-translational modifications promote resistance to ciliary force

项目总结/摘要 运动纤毛是基于微管的细胞附属物，其不对称地起伏以产生定向流体 流这种流体流动对于细胞运动和呼吸道粘液清除的保守功能至关重要。 运动纤毛的破坏有助于诸如慢性阻塞性肺病（COPD）， 哮喘和原发性纤毛运动障碍（PCD）。活动纤毛通过基体（BB）直接锚定到细胞。 BB是由九个三联体微管叶片组成的径向对称的圆柱形结构。BB必须 两者都抵抗并将来自跳动纤毛的机械力传递到细胞以实现有效的流体流动。 皮尔逊实验室鉴定了蛋白质和微管翻译后修饰（PTM）， 谷氨酰化，以稳定BB抵抗纤毛力。虽然BB是径向对称的，但 纤毛不对称。我们发现BB PTM谷氨酰化不对称地定位于预测为 感受到来自纤毛的最大机械力。微管的甘氨酰化和谷氨酰化是竞争性的 修饰微管蛋白相同残基的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，委员会认为， 微管蛋白谷氨酰化水平直接控制蛋白质活性。这就产生了一种有趣的可能性， 稳定性响应于从纤毛接收的机械力。 BB糖基化是否能稳定BB抵抗纤毛力尚不清楚。目前还不清楚是否 BB微管谷氨酰化和甘氨酰化之间的竞争调节BB稳定性。在目标1中，我将使用 定量光学显微术以确定PTM水平如何影响响应于纤毛应激的BB稳定性。BB 谷氨酰化不对称地定位于经历来自纤毛的最大机械力的BB结构域。 这种不对称性是如何建立的，以及BB谷氨酰化和甘氨酰化是否对纤毛的变化有反应。 力量未知。在目标2中，我将确定BB PTM如何不对称定位以及它们是否响应 纤毛跳动的力量BB谷氨酰化稳定BB抵抗纤毛力。无论这种稳定是 通过内在的BB微管调节（如弯曲）或通过BB稳定或不稳定来实现 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， BB稳定或去稳定蛋白的定位。 我的项目将提供一个关于细胞结构如何与物理力相互作用的机械观点。我 将通过采用定量成像来测量BB如何稳定对抗来自睫状体跳动的力， 基因和分子操控通过运用多学科方法和定量分析， 磨练技能，直接转化为我成为一名独立调查员和教师的愿望。

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.