Mechanisms of Nuclear and Cell Fusion in Yeast

描述(申请人提供)：我们的长期目标是确定两个单倍体酵母细胞融合成为一个二倍体细胞的途径。与受精有关，接合是所有有性生殖有机体的基本和共同之处。接合作用也与发育过程中的细胞融合事件有密切的相似之处。我们建议继续分析该途径中的三个主要步骤：细胞融合、核迁移和核膜融合所需的基因。细胞和核融合所需的许多基因在所有真核生物中都是保守的；他们的研究将为人类细胞生物学、生育和疾病提供重要线索。在酵母和高等生物中，当细胞彼此极化时，分泌小泡定位于细胞融合的初始位置。我们假设，细胞融合发生在局部和调控的分泌囊泡子集的胞吐之后。我们将使用遗传学、细胞生物学和生物化学的方法来阐明两个关键的细胞融合蛋白的作用，Fus3p是一种保守的MAP-Kinase，Fus2p是一种可能的Rho-Global。我们将检验这样的假设，即Fus3p对保守的福尔明Bni1p的磷酸化对极化至关重要。我们将检验这一假设，即Fus2p和两栖生物素Rvs161p的运输到皮质，以及随后的Rho蛋白的激活，是极化胞吐和细胞融合的关键调节事件。在受精和有丝分裂过程中，核迁移是一个基本的微管依赖过程。Kar4p是一种转录因子，通过诱导运动蛋白Kar3p的表达来促进交配过程中的核迁移。微阵列实验表明，Kar4p在信息素反应中发挥着更广泛的作用，包括负调控有丝分裂表达的基因。了解Kar4p的作用将有助于深入了解细胞如何修改细胞过程以响应发育线索。鉴定Kar4p调控基因将识别新的候选细胞和核融合蛋白。同型核膜融合是内质网重塑和细胞器融合的典范。Kar5p和Prm3p是核融合所必需的核膜蛋白，在交配过程中被诱导，并定位于发生融合的纺锤体极体(SPB)。我们建议研究它们在招募其他核融合蛋白到SPB中的作用，并表征膜融合所需的蛋白质复合体。我们将使用多种微观方法来表征核包膜聚变的事件和拓扑结构。

DESCRIPTION (provided by applicant): Our long-term objectives are to define the pathway by which two haploid yeast cells fuse to become one diploid cell. Related to fertilization, conjugation is fundamental and common to all sexually reproducing organisms. Conjugation also has close parallels to cell fusion events during development. We propose to continue the analysis of genes required for the three major steps in the pathway; cell fusion, nuclear migration and nuclear envelope fusion. Many of the genes required for cell and nuclear fusion are conserved in all eukaryotic organisms; their study will provide important clues to human cell biology, fertility and disease. In yeast and higher organisms, secretory vesicles localize to incipient sites of cell fusion, as ceils polarize toward each other. We hypothesize that cell fusion ensues after the localized and regulated exocytosis of a subset of secretory vesicles. We will use genetic, cell biological and biochemical methods to elucidate the roles of two key cell fusion proteins, Fus3p, a conserved MAP-kinase, and Fus2p, a putative Rho-GEF. We will test the hypothesis that Fus3p's phosphorylation of a conserved formin, Bni1p, is critical for polarization. We will test the hypothesis that transport of Fus2p and an amphiphysin, Rvs161p, to the cortex, and the subsequent activation of a Rho-protein, are key regulatory events for polarized exocytosis and cell fusion. Nuclear migration is a fundamental microtubule-dependent process in fertilization and mitosis. Kar4p is a transcription factor that promotes nuclear migration during mating by inducing the expression of Kar3p, a kinesin motor protein. Microarray experiments suggest that Kar4p plays a broader role in the pheromone response, including negatively regulating mitotically expressed genes. Understanding the role of Kar4p will provide insight into how cells modify cell processes in response to developmental cues. Identification of Kar4p regulated genes will identify new candidate cell and nuclear fusion proteins. Homotypic nuclear membrane fusion is a paradigm for ER remodeling and organelle fusion. Kar5p and Prm3p are nuclear envelope membrane proteins that are required for nuclear fusion, induced during mating, and localized to the spindle pole body (SPB), where fusion occurs. We propose to study their roles in recruiting other nuclear fusion proteins to the SPB and characterize the protein complex required for membrane fusion. We will use multiple microscopic methods to characterize the events and topology of nuclear envelope fusion.