The Biology of Motile Cilia

运动纤毛的生物学

• 批准号: 10617255
• 负责人: Stephen M King
• 金额: $ 74.99万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10617255
• 关键词: Address; Area; Biochemical; Biology; Cell Communication; Cells; Chemotaxis; Chlamydomonas; Cilia; Cilium Microtubule; Complex; Cytoplasm; Defect; Development; Dynein ATPase; Environment; Enzymes; Failure; Fractionation; Genes; Green Algae; Human; Human Biology; Human Development; Laboratories; Location; Methods; Microtubules; Mixed Function Oxygenases; Modeling; Motor; Organelles; Oxidation-Reduction; Pathway interactions; Peptides; Play; Process; Regulation; Role; Signal Pathway; Signal Transduction; Source; Syndrome; System; amidation; arm; cell motility; ciliopathy; cilium biogenesis; cilium motility; extracellular; fluid flow; genetic approach; insight; interest; macromolecular assembly; molecular array; novel; receptor; response

Cilia are microtubule-based cellular extensions that play key roles in sensing the extracellular environment, processing developmental signals and generating propulsive force and fluid flow. They also act as secretory organelles releasing bioactive vesicular ectosomes involved in cell-cell communication and other processes. Cilia are ancient and complex; in humans, ~5% of all genes are involved in their formation/activity and defects result in complex syndromes or ciliopathies. For many years, my laboratory has been broadly interested in the assembly and function of motile cilia, and has a strong record of identifying new opportunities and pursuing them to reveal novel aspects of ciliary biology – most recently we demonstrated that cilia act as a source of peptidergic signals. For most studies, we utilize the biciliate unicellular green alga Chlamydomonas as a model due to the ease of biochemical analysis and large array of molecular genetic approaches available. Over the next five years, we will pursue two broad areas of focus to address what I consider key questions in ciliary biology. Although superficially distinct, these two areas are intimately connected, and I anticipate we will be able to integrate them to yield novel insights into conserved and essential cilia-based pathways. 1) Ciliary Motility: dissecting the dynein motors and control systems that generate ciliary beating and power retrograde intraflagellar transport (IFT). We plan to focus on three major issues. We will dissect the complex pathways by which axonemal and IFT dyneins are synthesized and assembled in cytoplasm employing our newly devised biochemical fractionation methods. Building a cilium is an immensely complex problem in macromolecular assembly and we will examine how assembly factors control the axonemal incorporation of outer dynein arms at precise locations on doublet microtubules. We will also study axonemal dynein motor regulation to a) determine how responses to alterations in Ca2+ and redox poise are combined with curvature sensing to yield integrated changes in motility, and b) assess how cells sense imposed changes in ciliary beating and respond by increasing intraciliary levels of the dynein regulator Lis1. 2) Cilia Formation and Peptidergic Signaling: studying the peptide amidating enzyme (peptidylglycine - amidating monooxygenase; PAM) and its amidated bioactive products in ciliary assembly and cilia-based cell- cell communication. We recently demonstrated that active PAM occurs in cilia and that PAM loss leads to the failure of ciliogenesis and disrupts dynein-driven retrograde IFT. Furthermore, PAM-generated amidated bioactive products are released in cilia-derived vesicular ectosomes and one acts as a chemotactic modulator. We will build on these observations to identify novel amidated PAM products involved in cilia formation. We will dissect the pathways leading to regulated amidated product release in ciliary ectosomes and determine where/when processing of the precursors occurs. We will also pursue the amidated product receptors and their downstream signaling pathways, which lead to differential regulation of the two motile cilia and chemotaxis.

纤毛是基于微管的细胞扩展，在感知细胞外环境中起关键作用， 处理信号并产生推进力和流体流动。他们也充当分泌物 细胞活性囊泡外神经体与细胞 - 细胞通信和其他过程有关的细胞器。 纤毛是古老而复杂的。在人类中，所有基因的形成/活性和缺陷都参与了所有基因 导致复杂的综合征或纤毛病。多年以来，我的实验室一直对 苏里亚妈妈的集会和功能，并有很大的记录来识别新机会并追求它们 为了揭示纤毛生物学的新方面 - 最近，我们证明了纤毛作为肽能的来源 信号。在大多数研究中，我们利用双细胞单细胞绿色藻类衣原体作为模型 可用的生化分析和大量分子遗传学方法的便利性。在接下来的五个 多年来，我们将追求两个广泛的重点领域，以解决我认为睫状生物学中的关键问题。 尽管超级截然不同，但这两个领域是密切联系的，我希望我们能够 将它们整合为将新颖的见解带入构成且基于纤毛的基于基础的途径。 1）睫状运动：剖析产生睫状动力和力量的动力蛋白电动机和控制系统 逆行内部运输（IFT）。我们计划专注于三个主要问题。我们将剖析综合体 轴突和IFT动力蛋白合成并在细胞质中组装的途径，并采用我们的途径 新设计的生化分级方法。建造纤毛是一个非常复杂的问题 大分子组件，我们将研究组装因子如何控制 在双线微管上的精确位置处的动力蛋白臂。我们还将研究轴突动力蛋白电机 调节a）确定如何将Ca2+和氧化还原毒药改变的反应与曲率结合 感知运动能力的综合变化，b）评估细胞如何施加睫状性跳动的变化 并通过增加动力蛋白调节剂LIS1的内部水平响应。 2）纤毛形成和肽吉尼信号传导：研究辣椒酶（肽基甘氨酸--- 纳入单加氧酶； PAM）及其在纤毛组装和基于纤毛的细胞中的生物活性产物 细胞通信。我们最近证明了活性PAM发生在纤毛中，PAM损失导致 纤毛发生的失败并破坏动力蛋白驱动的逆行IFT。此外，PAM生成的胺化 生物活性产物在纤毛衍生的囊泡生态体中释放，一种充当趋化调节剂。 我们将基于这些观察结果，以确定与纤毛形成有关的新型胺化PAM产品。我们将 剖析导致纤毛外生中受调节的胺化产物释放的途径，并确定 前体的处理时间/时发生。我们还将追求胺化的产品受体及其 下游信号通路，导致两位母纤毛和趋化性的差异调节。

项目成果

N-Terminal Processing and Modification of Ciliary Dyneins.

• DOI: 10.3390/cells12202492
• 发表时间: 2023-10-20
• 期刊: Cells
• 影响因子: 6

Inherently disordered regions of axonemal dynein assembly factors.

• DOI: 10.1002/cm.21789
• 发表时间: 2023-09
• 期刊: Cytoskeleton
• 影响因子: 2.9
• 作者: Stephen M King

Isolation of ciliary ectosomes and analysis of amidated peptide-mediated chemotaxis in Chlamydomonas.

衣藻中纤毛胞外体的分离和酰胺化肽介导的趋化性分析。

• DOI: 10.1016/bs.mcb.2022.09.009
• 发表时间: 2023
• 期刊: Methods in cell biology
• 作者: Luxmi,Raj;King,StephenM
• 通讯作者: King,StephenM

Methylation of ciliary dynein motors involves the essential cytosolic assembly factor DNAAF3/PF22.

纤毛动力蛋白马达的甲基化涉及必需的胞质组装因子 DNAAF3/PF22。

• DOI: 10.1073/pnas.2318522121
• 发表时间: 2024
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Sakato-Antoku,Miho;Patel-King,RamilaS;Balsbaugh,JeremyL;King,StephenM
• 通讯作者: King,StephenM