Mechanisms of Nuclear and Cell Fusion in Yeast

酵母细胞核和细胞融合机制

• 批准号: 7163822
• 负责人: Mark David Rose
• 金额: $ 44.94万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-12-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7163822
• 关键词: Actins; Binding Sites; Biochemical; Biochemical Genetics; Biological; Cell Nucleus; Cell fusion; Cell physiology; Cell surface; Cells; Cellular biology; Chimeric Proteins; Complex; Consensus; Cues; Cytoskeleton; DNA Binding; Development; Diploid Cells; Disease; Dose; Ectopic Expression; Enzymes; Event; Exocytosis; Fertility; Fertilization; GTP-Binding Proteins; Gene Expression Regulation; Genes; Genetic; Genomics; Guanine Nucleotides; Haploidy; Histone Acetylation; Homologous Gene; Human; In Vitro; Kinesin; Light; Localized; Membrane; Membrane Fusion; Membrane Proteins; Methods; Microscopic; Microtubules; Mitogen-Activated Protein Kinases; Mitosis; Modeling; Motor; Movement; Mutation; Nature; Nuclear; Nuclear Envelope; Nuclear Fusion; Organelles; Organism; Partner in relationship; Pathway interactions; Phenotype; Pheromone; Phosphorylation; Phosphorylation Site; Play; Process; Proteins; Recruitment Activity; Role; Secretory Vesicles; Signaling Protein; Site; Testing; Time; Yeasts; amphiphysin; cell growth; in vitro Assay; in vivo; insight; intracellular protein transport; migration; mutant; novel; protein localization location; research study; response; rho; spindle pole body; transcription factor; zygote

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.

描述（由申请人提供）：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生物的基础和共同点。结合也与发育过程中的细胞融合事件密切相关。我们建议继续分析该途径中三个主要步骤所需的基因：细胞融合，核迁移和核膜融合。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;它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，分泌囊泡定位于细胞融合的初始位点，因为细胞彼此融合。我们推测，细胞融合englomerate后，本地化和调节胞吐的一个子集的分泌囊泡。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，Fus 3 p，保守的MAP激酶，和Fus 2 p，一个假定的Rho-GEF的作用。我们将测试的假设，Fus 3 p的磷酸化的保守的蛋白质，Bni 1 p，是至关重要的极化。我们将测试的假设，运输Fus 2 p和一个amphiphysin，Rvs 161 p，皮质，和随后的激活的Rho蛋白，是关键的调节事件的极化胞吐和细胞融合。 核迁移是受精和有丝分裂中依赖微管的基本过程。Kar 4p是一种转录因子，在交配过程中通过诱导Kar 3 p（一种驱动蛋白马达蛋白）的表达来促进核迁移。微阵列实验表明，Kar 4p在信息素反应中起着更广泛的作用，包括负调控有丝分裂表达的基因。了解Kar 4p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。Kar 4p调控基因的鉴定将鉴定新的候选细胞和核融合蛋白。 同型核膜融合是内质网重构和细胞器融合的范例。Kar 5 p和Prm 3 p是核融合所需的核膜蛋白，在交配过程中诱导，并定位于融合发生的纺锤体极体（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物。我们将使用多种显微镜方法来表征核膜融合的事件和拓扑结构。