The Biology of Motile Cilia

纤毛是基于微管的细胞延伸，在感知细胞外环境中发挥关键作用， 处理发育信号并产生推进力和流体流动。它们也可以分泌 释放生物活性囊泡外体的细胞器，参与细胞间通讯和其他过程。 纤毛是古老而复杂的;在人类中，约5%的基因参与其形成/活动和缺陷 导致复杂的综合征或纤毛病变。多年来，我的实验室一直对 活动纤毛的组装和功能，并在识别新机会和追求新机会方面有很好的记录 为了揭示纤毛生物学的新方面，最近我们证明纤毛作为肽能的来源， 信号.对于大多数研究，我们利用双纤毛单细胞绿色衣原体作为模型，由于 生物化学分析的简易性和大量可用的分子遗传学方法。在未来五 在接下来的几年里，我们将继续关注两个广泛的领域，以解决我认为纤毛生物学中的关键问题。 虽然表面上是不同的，这两个领域是密切相关的，我预计我们将能够 整合它们以产生对保守的和必需的基于纤毛的途径的新见解。 1)纤毛运动：解剖动力蛋白马达和产生纤毛跳动和动力的控制系统 鞭毛内逆行运输（IFT）。我们计划集中处理三个主要问题。我们会仔细分析 轴丝和IFT动力蛋白在细胞质中合成和组装的途径， 新设计的生化分离方法。建立纤毛是一个非常复杂的问题， 大分子组装，我们将研究如何组装因素控制轴丝纳入 动力蛋白的外臂位于双微管的精确位置。我们还将研究轴丝动力蛋白运动 调节以a）确定对Ca 2+和氧化还原平衡的改变的响应如何与曲率相结合 感测以产生运动性的综合变化，和B）评估细胞如何感测纤毛搏动中的强加变化 并通过增加动力蛋白调节剂Lis 1的睫状体内水平来响应。 2)纤毛的形成和肽能信号传导：肽酰胺化酶（肽基甘氨酸）的研究 酰胺化单加氧酶; PAM）及其酰胺化生物活性产物在纤毛组装和基于纤毛的细胞中的作用- 细胞通讯我们最近证明，活性PAM发生在纤毛中，PAM的损失导致纤毛脱落。 纤毛发生失败，并破坏动力蛋白驱动的逆行IFT。此外，PAM生成的酰胺化 生物活性产物在纤毛衍生的囊泡外体中释放，其中一种作为趋化调节剂。 我们将建立在这些观察，以确定新的酰胺化PAM产品参与纤毛形成。我们将 剖析导致纤毛外体中调节酰胺化产物释放的途径，并确定 在哪里/何时进行前体的加工。我们还将研究酰胺化产物受体及其 下游信号通路，导致两种运动纤毛和趋化性的差异调节。

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.