Gamete Membrane Adhesion and Fusion During Fertilization

 受精是一个古老的过程，其本质是两个单细胞之间的相互作用，最终产生一个细胞。一种性别的细胞--通常由鞭毛驱动- -接触另一种性别的细胞，一个或两个被激活，然后它们融合。我们仍然缺乏关于后生动物配子融合的分子和机制的基本信息，并且候选的脊椎动物配子融合蛋白仍然未被鉴定。单细胞双鞭毛衣原体是研究和理解受精进化发展的独特切入点。在衣原体中，高度运动的正配子和负配子之间的识别和粘附使它们聚集在一起，并触发两者直立细胞膜位点，专门用于融合，即交配结构。两种膜的附着和双层合并在遗传上是可区分的。融合前连接需要正接合结构上的FUS 1与负接合结构上的MAR 1相互作用。双分子层的合并依赖于负交配结构上广泛保守的HAP 2蛋白。两套蛋白质和HAP 2融合的这些要求已在原生生物和高等植物中得到证实，并涉及一些后生动物。疟原虫HAP 2已成为阻断传播的疟疾疫苗的靶点。对HAP 2功能结构域的新研究已经确定了胞质结构域中对靶向融合位点至关重要的基序和调节蛋白质融合功能的保守胞质基序。对衣原体转录组的一项新的全面分析已经确定了一组在配子中独特表达的基因，并揭示了一个以前未被认识的核膜蛋白家族，这些蛋白家族对合子原核融合至关重要。生物化学研究表明，MAR 1和HAP 2在负配子的交配结构膜的膜内相互作用，并且FUS 1、MAR 1和HAP 2在膜融合反应期间处于共同的复合物中。关于衣原体配子融合的细胞和分子基础的信息比其他任何生物都多。对于至少一种生物体，了解配子膜融合反应期间发生的分子事件将建立一个框架，用于解剖其他生物体中配子融合的基本原理，并将导致疫苗的开发，以阻止人类寄生虫病原体的受精，避孕药的开发，以及不孕症的治疗。本文提出的研究目的是测试HAP 2在膜融合反应期间作为融合蛋白发挥作用的模型。目的是确定融合所需的HAP 2结构域，并研究HAP 2-MAR 1相互作用是否对HAP 2的融合功能至关重要;研究有助于双层融合的专门FUS 1-MAR 1依赖性粘附连接的独特特性;并确定在异源系统中重建膜融合反应所需的最小蛋白质组。

 DESCRIPTION (provided by applicant): Fertilization is an ancient process whose essence is an interaction between two single cells that culminates in creation of one cell. A cell of one sex - typically driven by a flagellum - - contacts a cell of the opposite sex, one or both are activate, and then they fuse. We still lack fundamental information about the molecules and mechanisms that accomplish metazoan gamete fusion and a candidate vertebrate gamete fusion protein remains unidentified. The unicellular, biflagellated alga Chlamydomonas is a unique entry point into studying and understanding the evolutionary development of fertilization. In Chlamydomonas, recognition and adhesion between the highly motile plus and minus gametes bring them together and trigger both to erect cell membrane sites specialized for fusion, the mating structures. Attachment and bilayer merger of the two membranes are genetically distinguishable. Pre-fusion attachment requires interaction of FUS1 on the plus mating structure with MAR1 on the minus mating structure. Bilayer merger depends on the broadly conserved HAP2 protein on the minus mating structure. These requirements for two sets of proteins and for HAP2 in fusion have been confirmed in protists and higher plants and are implicated in some metazoans. Plasmodium HAP2 has become a target for a transmission-blocking malaria vaccine. New studies of functional domains of HAP2 have identified a motif in the cytoplasmic domain essential for targeting to the site of fusion and a conserved cytoplasmic motif that regulates the fusion function of the protein. A new comprehensive analysis of the Chlamydomonas transcriptome has identified a set of genes that are uniquely expressed in gametes and revealed a previously unrecognized family of nuclear envelope proteins essential for zygotic pronuclear fusion across domains of life. Biochemical studies show that MAR1 and HAP2 interact with each within the membrane of the mating structure membrane of minus gametes, and that FUS1, MAR1, and HAP2 are in a common complex during the membrane fusion reaction. More information is available about the cellular and molecular underpinning of gamete fusion in Chlamydomonas than in any other organism. Understanding, for at least one organism, the molecular events that occur during the gamete membrane fusion reaction will establish a framework for dissecting fundamental principles of gamete fusion in other organisms and will lead to development of vaccines to block fertilization of human parasitic pathogens, to development of contraceptives, and to treatments for infertility. The objective of the research proposed here is to test the model that HAP2 functions as a fusion protein during the membrane fusion reaction. The aims are to identify domains of HAP2 required for fusion and investigate whether the HAP2-MAR1 interaction is essential for the fusion function of HAP2; to examine the unique properties of the specialized FUS1-MAR1-dependent adhesion junction that contribute to bilayer fusion; and to identify the minimal set of proteins required to reconstitute the membrane fusion reaction in a heterologous system.