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细胞融合成为一个细胞的途径。受精是所有有性生殖生物体共同的基本过程，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，致力于细胞分裂，缺乏特殊功能，到分化，细胞分裂停止，特殊细胞功能表现出来。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。癌细胞的一个特点是，随着细胞重新获得不受限制的增殖能力，分化功能的丧失。因此，有丝分裂和分化的协调至关重要。同样，酵母细胞退出细胞周期，在分化为交配细胞时表达特定的蛋白质。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地激活交配。酵母细胞阻止过早交配的一种方法是将细胞融合的关键激活物Fus2p保留在细胞核中，直到有丝分裂完成。我们 建议确定酵母细胞如何将核滞留与细胞周期相结合，重点关注关键的蛋白激酶Cla4p和磷酸酶，它们共同调节Fus2p的定位。接下来，我们将以交配过程中诱导的蛋白质降解为目标，确定细胞如何重新进入有丝分裂途径。我们还将确定交配的转录激活因子Kar4p在进入减数分裂过程中如何支持不同的调控程序。虽然在发育和疾病过程中会发生大量的细胞融合事件，但对细胞融合的机制了解很少，也很少鉴定细胞融合原。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FusoGen。我们还将使用遗传和生化方法来确定细胞融合的下游效应器，由Fus2p和CDC42p激活，CDC42p是肌动蛋白和细胞极性的高度保守的调节因子。我们将使用先进的膜转运成像来确定调节细胞融合的承诺和进展的信号机制(S)。在交配达到顶峰时，核膜融合形成单一的二倍体细胞核。核膜有两层膜，需要两次协调的融合事件。外膜融合与内质网融合有关，在遗传性痉挛截瘫中发现了这一途径的突变。内膜如何融合尚不清楚。我们推测，Kar5p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，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Kar5p(S)在核膜融合中的作用。

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.