Mechanisms of Nuclear and Cell Fusion in Yeast

酵母细胞核和细胞融合机制

• 批准号: 8685270
• 负责人: Mark David Rose
• 金额: $ 46.35万
• 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 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8685270
• 关键词: Actins; Animal Model; Behavior; Biochemical; Cell Cycle; Cell Nucleus; Cell Polarity; Cell division; Cell fusion; Cell membrane; Cell physiology; Cells; Cellular biology; Chimeric Proteins; Complex; Couples; Defect; Degradation Pathway; Development; Diploid Cells; Diploidy; Disease; Disease Progression; Effector Cell; Eukaryota; Event; Fertilization; Gene Expression; Genes; Genetic; Genomics; Haploid Cells; Hereditary Spastic Paraplegia; Homologous Gene; Human; Image; Malignant Neoplasms; Mediating; Meiosis; Membrane; Membrane Fusion; Membrane Protein Traffic; Membrane Proteins; Methods; Microscopy; Mitosis; Mitotic; Modeling; Molecular Chaperones; Molecular Genetics; Morphogenesis; Muscle Cells; Mutation; Myoblasts; Nuclear; Nuclear Envelope; Nuclear Fusion; Nuclear Outer Membrane; Organelles; Organism; Partner in relationship; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Play; Process; Property; Protein Kinase; Proteins; Recruitment Activity; Regulation; Reproduction; Resolution; Role; SNAP receptor; Saccharomyces cerevisiae; Shapes; Signal Transduction; Specificity; Structure; Suppressor-Effector T-Lymphocytes; Tissues; Transcription Coactivator; Ubiquitin; Undifferentiated; Vesicle; Yeasts; base; cancer cell; imaging modality; inorganic phosphate; multicatalytic endopeptidase complex; mutant; novel; precursor cell; premature; prevent; programs; protein complex; protein degradation; public health relevance; trait

DESCRIPTION (provided by applicant): Our long-term objectives are to define the pathway by which two cells fuse to become one. Fertilization is a fundamental process common to all sexually reproducing organisms, but cell fusion also occurs during development. We propose to continue analysis of genes required for two major steps in yeast mating, cell fusion and nuclear envelope fusion. Many genes required for cell and nuclear fusion have homologs in all eukaryotic organisms and their study will provide important clues to human cell biology and disease. During development, cells go from proliferation, devoted to cell division and lacking specialized functions, to differentiation, in which cell division ceases and specialized cell functions are expressed. Proliferation and differentiation are mutually exclusive; during development cells shut down mitosis as they turn on specialized cell functions. One hallmark of cancer cells is the loss of differentiated functions as cells re-acquire the capacity for unrestrained proliferation. Thus coordination of mitosis and differentiation is of vital importance Similarly, yeast cells exit the cell cycle and express specialized proteins as they differentiate ito mating cells. Gene expression begins before the completion of the previous cell cycle; cells must also prevent premature activation of mating. One way yeast cells block premature mating is by retaining a key activator of cell fusion, Fus2p, in the nucleus until mitosis is complete. We propose to determine how yeast cells couple nuclear retention to the cell cycle, focusing on a critical protein kinase, Cla4p, and a phosphatase, which together regulate Fus2p localization. We will next determine how cells reenter the mitotic pathway by targeting the degradation of the proteins induced during mating. We will also determine how a transcriptional activator of mating, Kar4p, is able to support a different regulatory program during entry into meiosis. Although numerous cell fusion events occur during development and disease, the mechanism of cell fusion is poorly understood and few cellular fusogens have been identified. We propose to use high-throughput imaging as part of a genomic screen to identify the cellular fusogen required for yeast mating. We will also use genetic and biochemical approaches to identify the downstream effectors of cell fusion, activated by Fus2p and Cdc42p, the highly conserved regulator of actin and cell polarity. We will use advanced imaging of membrane trafficking to identify the signaling mechanism(s) that regulate commitment and progression into cell fusion. At the culmination of mating, the nuclear envelopes fuse to form a single diploid nucleus. The nuclear envelope has two membranes, requiring two coordinated fusion events. Fusion of the outer membrane is related to ER fusion and mutations in this pathway have been identified in hereditary spastic paraplegia. How the inner membranes fuse is not known. We hypothesize that Kar5p, a conserved mating-induced protein, couples the inner and outer nuclear membranes during fusion and facilitates inner-membrane fusion. We will use advanced imaging and genetic methods to identify Kar5p's role(s) in nuclear membrane fusion.

描述（由申请人提供）：我们的长期目标是确定两个细胞融合成为一个细胞的途径。受精是所有有性生殖生物共同的基本过程，但细胞融合也发生在发育过程中。我们建议继续分析酵母交配，细胞融合和核膜融合的两个主要步骤所需的基因。许多细胞核融合所需的基因在所有真核生物中都有同源物，对它们的研究将为人类细胞生物学和疾病研究提供重要线索。 在发育过程中，细胞从增殖，致力于细胞分裂和缺乏专门的功能，分化，其中细胞分裂停止和表达专门的细胞功能。增殖和分化是相互排斥的;在发育过程中，细胞关闭有丝分裂，因为它们开启了专门的细胞功能。癌细胞的一个标志是随着细胞重新获得无限制增殖的能力而丧失分化功能。因此，有丝分裂和分化的协调是至关重要的。类似地，酵母细胞退出细胞周期，并在分化为交配细胞时表达专门的蛋白质。基因表达在前一个细胞周期完成之前就开始了;细胞还必须防止过早激活交配。酵母细胞阻止过早交配的一种方法是在细胞核中保留细胞融合的关键激活剂Fus 2 p，直到有丝分裂完成。我们 建议确定酵母细胞如何将核保留与细胞周期偶联，重点是一个关键的蛋白激酶，Cla 4p和磷酸酶，它们共同调节Fus 2 p定位。接下来，我们将通过靶向交配过程中诱导的蛋白质降解来确定细胞如何重新进入有丝分裂途径。我们还将确定如何转录激活交配，Kar 4p，是能够支持一个不同的调节程序进入减数分裂。 尽管在发育和疾病过程中发生了许多细胞融合事件，但对细胞融合的机制知之甚少，并且已经鉴定了很少的细胞融合剂。我们建议使用高通量成像作为基因组筛选的一部分，以确定酵母交配所需的细胞融合剂。我们还将使用遗传和生物化学方法来确定细胞融合的下游效应子，由Fus 2 p和Cdc 42 p激活，这是肌动蛋白和细胞极性的高度保守调节因子。我们将使用膜运输的先进成像来识别调节承诺和进展为细胞融合的信号传导机制。 在交配的高潮，核膜融合形成一个单一的二倍体核。核膜有两层膜，需要两个协调的融合事件。外膜的融合与ER融合有关，并且在遗传性痉挛性截瘫中已经确定了该途径的突变。内膜如何融合尚不清楚。我们假设Kar 5 p是一种保守的交配诱导蛋白，在融合过程中将内外核膜偶联并促进内膜融合。我们将使用先进的成像和遗传学方法来确定Kar 5 p在核膜融合中的作用。