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共同的基本过程。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。我们建议继续分析接合的两个主要步骤--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、生育和疾病提供重要线索。在多细胞生物的发育过程中，细胞从致力于细胞分裂、缺乏特殊功能的增殖状态向细胞停止分裂、表达特殊功能的分化状态转变。增殖和分化是相互排斥的状态，有序的发展要求细胞在开启与分化相关的专门功能时关闭有丝分裂功能。事实上，癌症的一个特征是，当细胞重新获得不受限制的增殖能力时，它们往往会失去分化的功能。因此，有丝分裂和分化的协调至关重要。同样，当酵母细胞结合时，它们必须退出细胞周期，表达细胞和核融合所需的蛋白质。然而，由于基因表达在前一个细胞周期完成之前就开始了，而且接合所需的几种蛋白质具有其他有丝分裂功能，酵母细胞面临着必须防止接合过早激活的额外挑战。该项目的主要目标是确定过早激活交配功能的具体影响，并确定过早激活时有毒的基因/蛋白质。作为一个具体的例子，我们的目标是了解控制细胞融合的关键调节因子Fus2p的控制，它的定位受到细胞周期和接合的极其复杂的调节。因此，Fus2p的调控是有丝分裂和接合之间转换的中心范式。我们假设Fus2p定位的调节防止了对细胞周期完成的干扰，我们将通过识别Fus2p调节的下游途径来验证这一点。我们将研究在交配过程中增选的其他关键蛋白质的行为，以确定受调控的定位是否是防止细胞周期干扰的一般机制。在接合的顶峰，两个核膜融合形成一个单一的二倍体细胞核。由于核膜由两个膜组成，两个不同的融合事件以协调的方式发生，内膜的融合必须由未知的蛋白质催化。我们推测，Kar5p是一种新的结合诱导蛋白，在融合过程中将内外核膜偶联，促进内膜融合。核膜融合可能是内质网重塑的一个很好的范例，是一个关键的有丝分裂过程的例子，在接合过程中发挥不同的功能。与公共卫生相关：随着生物体的生长和发育，其细胞从增殖过渡到分化，当细胞分裂停止并获得专门功能时，细胞正在分裂，但缺乏专门功能。成功的发育需要细胞在尝试分裂时不开启专门化功能；癌症的一个标志是细胞在恢复不受限制的分裂能力时失去专门化功能。这个项目解决了模式生物面包师酵母的同样问题，它使用与人类细胞生物学、生育和疾病相关的类似人类基因，仔细地调节从细胞分裂到能够交配的转变。

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 a fundamental process common to all sexually reproducing organisms. Conjugation also has close parallels to cell fusion events during development. We propose to continue our analysis of genes required for two major steps in conjugation, cell fusion and nuclear envelope fusion. Many of the genes required for cell and nuclear fusion have homologs in all eukaryotic organisms and their study will provide important clues to human cell biology, fertility and disease. During development of multicellular organisms, cells transition from a proliferative state, devoted to cell division and lacking specialized functions, to a differentiated state, in which cell division ceases and specialized cell functions are expressed. Proliferation and differentiation are mutually exclusive states, and orderly development requires that cells shut down mitotic functions as they turn on the specialized functions related to differentiation. Indeed one hallmark of cancer is that cells tend to lose differentiated functions as they re- acquire the capacity for unrestrained proliferation. Thus the coordination of mitosis and differentiation is of vital importance. Similarly, when yeast cells conjugate, they must exit the cell cycle and express proteins required for cell and nuclear fusion. However, because gene expression begins before the completion of the previous cell cycle, and because several proteins required for conjugation have other mitotic functions, yeast cells face the additional challenge of having to prevent premature activation of conjugation. The major goal of this project is to identify the specific effects of premature activation of mating functions, and identify the genes/proteins that are toxic when prematurely activated. As a specific example, we aim to understand the controls governing a key regulator of cell fusion, Fus2p, whose localization is under extraordinarily complex regulation by both the cell cycle and conjugation. Fus2p's regulation therefore serves as a central paradigm for the transition between mitosis and conjugation. We hypothesize that the regulation of Fus2p localization prevents interference with cell-cycle completion, which we will test this by identifying the downstream pathways regulated by Fus2p. We will examine the behavior of other key proteins co-opted during mating to determine if regulated localization is a general mechanism to prevent cell-cycle interference,. At the culmination of conjugation, the two nuclear envelopes fuse to create a single diploid nucleus. Because the nuclear envelope is composed of two membranes, two distinct fusion events occur in a coordinated fashion, and fusion of the inner membranes must be catalyzed by as yet unknown proteins. We hypothesize that Kar5p, a novel conjugation-induced protein, couples the inner and outer nuclear envelopes during fusion and facilitates inner-membrane fusion. Nuclear envelope fusion may be excellent paradigm for ER remodeling, an example of a critical mitotic process co-opted to serve a different function during conjugation. Public Health Relevance: As organisms grow and develop their cells transition from proliferation, when they are dividing, but lack specialized functions, to differentiation, when cell division stops and they acquire specialized functions. Successful development requires that cells not turn on the specialized functions while they are trying divide; one hallmark of cancer is that cells lose their specialized functions as they regain the capacity for unrestrained division. This project addresses the same problem in a model organism, baker's yeast, which carefully regulates the transition from cell division to being able to mate, using genes similar to human genes with relevance to human cell biology, fertility and disease.