Cell Surface Recognition and Cell Interactions

描述(申请人提供)：我研究的长期目标是了解纤毛/鞭毛的细胞和分子特性，这是它们功能的基础。我的实验室使用双鞭毛绿藻衣藻作为模型系统来研究纤毛产生的信号和纤毛/鞭毛的缩短。在衣藻的交配反应中，负配子鞭毛上的黏附受体(凝集素)与正配子鞭毛上的同源凝集素结合，从而在两个细胞中激活纤毛产生的信号通路，激活配子以进行细胞-细胞融合形成受精卵。在合子形成后(以及在实验施加的压力期间)，衣藻细胞立即缩短并完全吸收它们的鞭毛。我们建议使用衣藻来剖析鞭毛内转运(IFT)机制的新功能和调节机制。由于几乎每个哺乳动物细胞都有一个初级纤毛，用于信号转导，也必须在细胞周期进入时被吸收，我们的研究将为人类发育和动态平衡所必需的基本细胞机制提供新的见解。在以前关于鞭毛黏附诱导信号的研究中，我们已经证明了一种调节蛋白，鞭毛蛋白酪氨酸激酶(PTK)，在该途径的早期被激活。此外，利用IFT中有条件缺陷的突变配子，我们提出了在完整的纤毛/鞭毛中需要IFT进行信号转导的证据。在目前的资助期间，我们发现该途径中的第二种调节蛋白，cGMP依赖的蛋白激酶，与鞭毛内的大组装有关，其形成需要IFT。令我们惊讶的是，新形成的大型组件中也含有IFT粒子。在这里，在我们对信号转导的研究中，我们提出了实验来测试这个模型，即除了在鞭毛组装和拆卸中所起的作用外，IFT机制还直接参与纤毛产生的信号转导，并将膜受体相互作用与配子激活联系起来。我们以前对鞭毛缩短的研究表明，极光样蛋白激酶(Calk)对于调节鞭毛缩短是必不可少的。在目前的资助期，我们有了一个令人惊讶的发现，鞭毛内的IFT运输和细胞体内IFT颗粒上的货物装载在缩短过程中受到调控。此外，我们还发现，细胞体中一种分解微管的蛋白质，一种解聚运动蛋白，被磷酸化并运输到鞭毛中，因为微管分解在缩短过程中被触发。我们的具体目标是剖析鞭毛内运输机制在鞭毛黏附诱导信号传递中的作用，研究将鞭毛黏附与配子激活相结合的分子，并研究鞭毛缩短的细胞和分子机制。 与公共卫生相关：初级纤毛在发育和动态平衡中发挥关键的信号作用，它们在细胞周期进入之前被吸收。然而，我们对纤毛产生信号或纤毛解体的细胞和分子机制知之甚少。研究衣藻中的鞭毛附着和鞭毛缩短将继续揭示这些显着细胞器的新的和基本的特性。

DESCRIPTION (provided by applicant): The long-term goals of my research are to understand the cellular and molecular properties of cilia/flagella that underlie their functions. My laboratory uses the biflagellated green alga Chlamydomonas reinhardtii as a model system to study cilium-generated signaling and ciliary/flagellar shortening. During the Chlamydomonas mating reaction, adhesion receptors (agglutinins) on the flagella of minus gametes bind to their cognate agglutinins on the flagella of plus gametes, thereby activating a cilium-generated signaling pathway in both cells that activates the gametes for cell-cell fusion to form a zygote. Immediately after zygote formation (and also during experimentally-imposed stress), Chlamydomonas cells shorten and completely resorb their flagella. We propose to use Chlamydomonas to dissect novel functions and mechanisms of regulation of the intraflagellar transport (IFT) machinery. Because almost every mammalian cell possesses a primary cilium that is used for signal transduction and also must be resorbed during cell cycle entry, our studies will provide novel insights into fundamental cellular mechanisms essential for human development and homeostasis. In previous studies of flagellar adhesion-induced signaling, we had shown that a regulatory protein, a flagellar protein tyrosine kinase (PTK), was activated early in the pathway. Moreover, using mutant gametes conditionally defective in IFT, we presented evidence that IFT is required for signal transduction in an intact cilium/flagellum. In the current funding period we discovered that a second regulatory protein in the pathway, a cGMP-dependent protein kinase, becomes associated with large assemblies within flagella whose formation requires IFT. To our surprise, the large, newly formed assemblies also contain IFT particles. Here, in our studies on signaling, we propose experiments to test the model that, in addition to its roles in flagellar assembly and disassembly, the IFT machinery participates directly in cilium-generated signaling and links membrane receptor interactions to gamete activation. Our previous studies on flagellar shortening showed that an aurora-like protein kinase (CALK) was essential for regulated shortening. In the current funding period, we made the surprising discovery that IFT trafficking within flagella and cargo loading onto IFT particles in the cell body are regulated during shortening. Moreover, we found that a protein that disassembles microtubules, a depolymerizing kinesin, in the cell body is phosphorylated and transported into flagella as microtubule disassembly is triggered during shortening. Our specific aims are to dissect the function of the intraflagellar transport machinery in flagellar adhesion-induced signaling, investigate the molecules that couple flagellar adhesion to gamete activation, and study the cellular and molecular mechanisms of flagellar shortening. PUBLIC HEALTH RELEVANCE: Primary cilia carry out key signaling roles in development and homeostasis and they are resorbed before cell cycle entry. Yet, we know little about the cellular and molecular mechanisms of cilium-generated signaling or ciliary disassembly. Studying flagellar adhesion and flagellar shortening in Chlamydomonas will continue to uncover novel and fundamental properties of these remarkable organelles.