GAMETE MEMBRANE ADHESINO AND FUSION DURING FERTILIZATION

描述(申请人提供)：我的实验室的长期目标是了解受精过程中配子相互作用和细胞-细胞融合的细胞和分子机制。我们使用单细胞、双鞭毛藻衣藻进行研究，衣藻的受精高度服从细胞生物学、生化、分子生物学和遗传学方法。重要的是，在衣藻受精过程中，相互作用的雄配子和雌配子经历了几乎所有动物受精的细胞生物学事件。了解配子融合是未来人类生殖研究的基础。此外，配子融合对于寄生原虫引起的几种毁灭性人类疾病的昆虫传播至关重要，包括导致疟疾和昏睡病的有机体。了解寄生原生动物配子融合的分子机制将是努力减少它们造成的疾病影响的重要进展。不幸的是，尽管有许多不同的受精系统被研究，但我们对任何生物体中配子融合的分子理解仍然处于初级状态。本研究的目的是研究衣藻配子融合的细胞和分子机制，重点研究两种配子融合蛋白，一种是雌配子特异蛋白FUS1，另一种是我们在这一资助时期发现的一种新的雄配子特异蛋白质FusM。两者都定位于融合部位，是融合所必需的。与迄今为止在任何生物体中发现的所有其他配子融合蛋白不同，FusM是广泛保守的。虽然没有在双边动物中发现，但FusM家族成员存在于简单动物、高等植物和致病原虫中。在利用伯氏疟原虫(一种啮齿疟疾寄生虫)的靶向基因破坏的合作努力中，我们发现疟原虫FusM对于配子融合是必不可少的。因此，我们对衣藻的研究揭示了配子融合的一种保守机制。现在我们在每个衣藻配子上都有了对配子融合至关重要的蛋白质，我们处于一个独特的位置，可以对配子融合的基本细胞和分子机制有新的见解。我们建议鉴定FUS1和FusM的结合伙伴，确定这两个蛋白在配子膜黏附和融合中的分子功能，研究它们定位的分子机制，并表征它们在融合前、融合中和融合后的分子性质。

DESCRIPTION (provided by applicant): The long-term goals of my laboratory are to understand the cellular and molecular mechanisms that underlie gamete interactions and cell-cell fusion during fertilization. We use the unicellular, biflagellated alga Chlamydomonas for our studies, an organism whose fertilization is highly amenable to cell biological, biochemical, molecular biological and genetic approaches. Importantly, during Chlamydomonas fertilization, the interacting male and female gametes undergo the cell biological events that characterize fertilization in almost all animals. Understanding gamete fusion is needed to inform future studies on human reproduction. In addition, gamete fusion is essential for insect transmission of several devastating human diseases caused by parasitic protozoa, including the organisms that cause malaria and sleeping sickness. Knowing the molecular mechanisms of gamete fusion in parasitic protozoa would be an important advance in efforts to reduce the impact of the diseases they cause. Unfortunately, in spite of the many different fertilization systems being studied, our molecular understanding of gamete fusion in any organism remains rudimentary. The objectives of this proposal are to characterize the cellular and molecular mechanisms of gamete fusion in Chlamydomonas, focusing on two gamete fusion proteins, Fus1 a female gamete-specific protein, and a new male gamete-specific protein we discovered in this funding period, FusM. Both are localized at the sites of fusion and essential for fusion. Unlike all other gamete fusion proteins identified to date in any organism, FusM is widely conserved. Although not found in bilaterian animals, FusM family members are present in simple animals, higher plants, and pathogenic protozoa. In collaborative efforts using targeted gene disruption in Plasmodium berghei, a rodent malaria parasite, we discovered that the Plasmodium FusM is essential for gamete fusion. Thus, our studies on Chlamydomonas have revealed a conserved mechanism for gamete fusion. Now that we have a protein on each Chlamydomonas gamete that is essential for gamete fusion, we are in a unique position to make new insights into fundamental cellular and molecular mechanisms of gamete fusion. We propose to identify the binding partners of Fus1 and FusM, to determine the molecular functions of the proteins in gamete membrane adhesion and fusion, to investigate the molecular mechanisms of their localization, and to characterize their molecular properties before, during, and after fusion.