The Biology of Motile Cilia

纤毛是基于微管的细胞延伸，在感知细胞外环境方面发挥关键作用， 处理发育信号并产生推进力和流体流动。它们也扮演着分泌者的角色 释放具有生物活性的囊泡外体的细胞器，参与细胞间的通讯和其他过程。 纤毛是古老而复杂的；在人类中，大约5%的基因与纤毛的形成/活动和缺陷有关。 导致复杂的症状或纤毛疾病。多年来，我的实验室一直对 活动纤毛的组装和功能，并在发现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-最近我们证明了纤毛是多肽能的来源 信号。在大多数研究中，我们使用双纤毛单细胞绿藻衣藻作为模型，因为 易于进行生化分析和大量的分子遗传学方法。在接下来的五年里 几年来，我们将继续关注两个广泛的领域，以解决我认为是纤毛生物学中的关键问题。 虽然表面上是不同的，但这两个领域是紧密相连的，我预计我们将能够 将它们结合在一起，产生对保守的、基于纤毛的基本途径的新见解。 1)纤毛运动：解剖产生纤毛跳动和动力的动力蛋白马达和控制系统 逆行鞭毛内转运(IFT)。我们计划重点解决三个主要问题。我们会解剖这个建筑群 轴丝和IFT动力蛋白在细胞质中合成和组装的途径 新设计的生化分级方法。建立纤毛是一个极其复杂的问题 大分子组装，我们将研究组装因子如何控制轴丝结合 位于双线微管上精确位置的动力蛋白外臂。我们还将研究轴丝动力蛋白马达。 A)确定对钙离子和氧化还原平衡变化的反应如何与曲率相结合 感知以产生运动性的综合变化，以及b)评估细胞如何感知睫状体搏动的强加变化 并通过增加动力蛋白调节器Lis1的睫状体内水平来做出反应。 2)纤毛形成和多肽能信号转导：多肽酰胺化酶(多肽甘氨酸- 酰胺化单加氧酶(PAM)及其酰胺化生物活性产物在纤毛组件和纤毛细胞中- 蜂窝通讯。我们最近证明，活性PAM存在于纤毛中，而PAM的丢失会导致 纤毛发生失败并干扰动力蛋白驱动的逆行IFT。此外，PAM产生的酰胺化 生物活性产物在纤毛衍生的囊泡外体中释放，其中一个起趋化调节剂的作用。 我们将在这些观察的基础上，确定与纤毛形成有关的新型酰胺化PAM产品。我们会 解剖导致睫状外体中调节酰胺化产物释放的途径，并确定 在何处/何时对前体进行处理。我们还将追查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不同调节。

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.