Mechanisms of Nuclear and Cell Fusion in Yeast

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，交配是所有有性生殖生物共同的基本过程。在发育过程中，偶联也与细胞融合事件有密切的相似之处。我们建议继续我们的基因分析需要在两个主要步骤的结合，细胞融合和核膜融合。细胞和核融合所需的许多基因在所有真核生物中都有同源物，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，细胞从增殖状态（专注于细胞分裂，缺乏特化功能）过渡到分化状态（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状态，有序的发育要求细胞在开启与分化相关的特殊功能时关闭有丝分裂功能。事实上，癌症的一个特征是，当细胞重新获得无限制增殖的能力时，它们往往会失去分化的功能。因此有丝分裂和分化的协调是至关重要的。同样，当酵母细胞结合时，它们必须退出细胞周期并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，并且由于偶联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过早激活偶联的额外挑战。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，我们旨在了解控制细胞融合的关键调节因子Fus2p的控制，其定位受到细胞周期和结合的异常复杂的调节。因此，Fus2p的调控是有丝分裂和接合之间转变的中心范例。我们假设Fus2p定位的调节可以防止对细胞周期完成的干扰，我们将通过鉴定由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的行为，以确定调节定位是否是防止细胞周期干扰的一般机制。在结合的顶点，两个核包膜融合形成一个二倍体核。由于核膜由两层膜组成，两种不同的融合事件以协调的方式发生，而内膜的融合必须由未知的蛋白质催化。我们假设Kar5p是一种新的偶联诱导蛋白，在融合过程中偶联内外核膜，促进内膜融合。核膜融合可能是内质网重构的绝佳范例，这是一个关键的有丝分裂过程在接合过程中被选择服务于不同功能的例子。公共卫生相关性：随着生物体的生长和发育，它们的细胞从增殖（当它们分裂但缺乏特化功能时）过渡到分化（当细胞分裂停止并获得特化功能时）。成功的发育要求细胞在试图分裂时不开启特殊功能；癌症的一个特征是细胞失去了它们的特殊功能，因为它们重新获得了无限制分裂的能力。该项目在一种模式生物——面包酵母中解决了同样的问题，它使用与人类细胞生物学、生育和疾病相关的基因类似的人类基因，仔细调节从细胞分裂到能够交配的过渡。

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.