Mechanisms of Nuclear and Cell Fusion in Yeast

描述（由申请人提供）：我们的长期目标是定义两个细胞融合成一个细胞的途径。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的两个主要步骤，细胞融合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有同源基因，它们的研究将为人类细胞生物学和疾病提供重要线索。在发育过程中，细胞从增殖到分化，细胞分裂，缺乏特化功能，细胞分裂停止，细胞特化功能表达。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，因为它们开启了特殊的细胞功能。癌细胞的一个特征是随着细胞重新获得无限制增殖的能力而失去分化功能。因此，有丝分裂和分化的协调是至关重要的。类似地，酵母细胞在分化成交配细胞的过程中，也会退出细胞周期并表达专门的蛋白质。基因表达在前一个细胞周期结束之前就开始了；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因子Fus2p，直到有丝分裂完成。我们

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.