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在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，这种生物的受精高度适应细胞生物学、生物化学、分子生物学和遗传学方法。重要的是，在衣藻受精过程中，相互作用的雄性和雌性配子经历了几乎所有动物受精特征的细胞生物学事件。了解配子融合对未来人类生殖的研究至关重要。此外，配子融合对于由寄生原生动物（包括引起疟疾和昏睡病的生物体）引起的几种破坏性人类疾病的昆虫传播至关重要。了解寄生原生动物配子融合的分子机制将是减少它们引起的疾病影响的重要进展。不幸的是，尽管研究了许多不同的受精系统，我们对任何生物配子融合的分子理解仍然是初级的。本论文的目的是表征衣藻配子融合的细胞和分子机制，重点研究了两种配子融合蛋白，Fus1是一种雌性配子特异性蛋白，FusM是我们在资助期间发现的一种新的雄性配子特异性蛋白。两者都位于核聚变的部位，对核聚变至关重要。与迄今为止在任何生物体中鉴定的所有其他配子融合蛋白不同，FusM广泛保守。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，高等植物和致病性原生动物中。在对伯氏疟原虫（一种啮齿动物疟疾寄生虫）进行靶向基因破坏的合作研究中，我们发现FusM疟原虫对配子融合至关重要。因此，我们对衣藻的研究揭示了配子融合的保守机制。现在我们在每个衣藻配子上都有一种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认识。我们拟鉴定Fus1和FusM的结合伙伴，确定其在配子膜粘附和融合中的分子功能，探讨其定位的分子机制，并对其融合前、融合中和融合后的分子特性进行表征。

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.