Conserved mechanisms of ciliary signaling and cell-cell fusion

我们建议用绿藻衣藻中的受精作为模式系统来研究 但对纤毛信号和配子膜的细胞和分子机制仍知之甚少 聚变反应。当相反交配型的衣藻配子混合在一起时，它们会附着在 通过纤毛上的互补黏附受体相互作用。黏附受体之间的相互作用 正配子上的SAG1和负配子上的SAD1启动了一条信号通路，该信号通路被传递到细胞 并触发细胞内cAMP的快速增加，从而激活配子进行细胞-细胞融合。规管 纤毛受体参与的纤毛环核苷酸在整个进化过程中几乎是保守的 脊椎动物中的所有纤毛信号事件，对纤毛受体参与的主要反应是 细胞器内cAMP的浓度。此外，在大多数脊椎动物的纤毛系统中， 纤毛cAMP的增加如何传导到细胞反应尚不清楚。我们发现一种蛋白质 衣藻中的GSPK在纤毛信号中的作用是一个以前未知的步骤。GSPK是 定位于细胞质，而不是纤毛，但GSPK的整个细胞补体在1 纤毛黏附受体接合后一分钟。此外，GSPK的磷酸化是在 CAMP的快速增加需要cAMP的增加和GSPK的作用。我们的发现为 研究纤毛受体参与与细胞和分子机制耦合的新策略 细胞质反应。我们将使用细胞生物学、生化和成像技术的组合来 研究GSPK功能的潜在机制。配子激活也激活顶端的勃起 来自每个配子质膜的局部突起，正负交配结构， 是受精过程中膜融合的部位..我们在理解 保守的配子膜融合反应的分子机制，我们现在对细胞- 衣藻的细胞融合比已知的任何其他系统都要多。1)我们证明了两者的双层合并 细胞是由真核生物第二类融合蛋白HAP2的三聚体形成驱动的，HAP2是一种古老的蛋白质，需要 在各个王国的有机体中受精。2)我们现在已经分离出细胞-细胞黏附蛋白S FUS1和MaR1位于每个细胞的融合部位。3)我们证明了黏附蛋白MaR1在负数 配子不仅与正配子上的FUS1结合，而且MaR1在生化和功能上也与 HAP2。4)我们发现FUS1-MaR1相互作用介导的膜黏附释放了被抑制的， HAP2的预融合形式，导致不可逆的结构重排进入融合驱动的HAP2三聚体。 5)在非洲的协作实验室研究和实地研究中，我们表明，以保守的融合为目标 疟原虫HAP2环抑制蚊子传播疟疾。我们的发现现在使一种 纤毛信号和配子融合的详细的、基于结构和功能的分子剖析。

We propose to use fertilization in the green alga Chlamydomonas as a model system to investigate conserved but still poorly understood cellular and molecular mechanisms of ciliary signaling and the gamete membrane fusion reaction. When Chlamydomonas gametes of opposite mating types are mixed together they adhere to each other by complementary adhesion receptors on their cilia. Interactions between the adhesion receptors SAG1 on plus gametes and SAD1 on minus gametes initiate a signaling pathway that is transmitted to the cell body and triggers a rapid increase in cellular cAMP that activates the gamete for cell-cell fusion. Regulation of ciiliary cyclic nucleotides by ciliary receptor engagement has been conserved throughout evolution and in almost all ciliary signaling events in vertebrates, the primary response to engagement of ciliary receptor is a change in the concentration of cAMP within the organelles. Moreover, in most vertebrate ciliary systems, the mechanisms that transmit the increase in ciliary cAMP to a cellular response are unclear. We have discovered that a protein kinase, GSPK, in Chlamydomonas functions at a previously unrecognized step in ciliary signaling. GSPK is localized in the cytoplasm, not the cilia, yet the entire cellular complement of GSPK is phosphorylated within 1 minute after engagement of the ciliary adhesion receptors. Furthermore, GSPK phosphorylation is upstream of the cAMP increase and GSPK function is required for the rapid increase in cAMP. Our findings set the stage for new strategies to investigate cellular and molecular mechanisms that couple ciliary receptor engagement to a cytoplasmic response. We will use a combination of cell biological, biochemical, and imaging strategies to investigate the mechanisms underlying GSPK function. Gamete activation also activates erection of apically localized protrusions, the plus and minus mating structures, from the plasma membrane of each gamete that are the sites of membrane fusion during fertilization..We have made exciting advances in understanding conserved molecular mechanisms of the gamete membrane fusion reaction, and we now know more about cell- cell fusion in Chlamydomonas than is known in any other system. 1) We showed that bilayer merger of the two cells is driven by trimer formation of eukaryotic class II fusion protein, HAP2, a ancient protein required for fertilization in organisms across kingdoms. 2) We have now identified the separate cell-cell adhesion proteins s FUS1 and MAR1 at the fusion site of each cell. 3) We demonstrated that the adhesion protein MAR1 on minus gametes not only binds to FUS1 on plus gametes, but MAR1 is also biochemically and functionally linked to HAP2. 4) We showed that membrane adhesion mediated by FUS1-MAR1 interactions releases the restrained, prefusion form of HAP2, leading to the irreversible structural rearrangements into the fusion-driving HAP2 trimer. 5) In collaborative laboratory studies and field studies in Africa, we showed that targeting the conserved fusion loop of Plasmodium HAP2 inhibits the mosquito transmission of malaria. Our discoveries now make possible a detailed, structure- and function-based molecular dissection of ciliary signaling and gamete fusion..