RUI: Control Mechanisms for G1 Cyclin Subcellular Localization in Budding Yeast

在考虑调节基本细胞过程（如细胞分裂）的途径时，蛋白质进出细胞核的调节运动的不同机制是至关重要的。该项目的重点是鉴定和鉴定对蛋白质进出细胞核的调节运动重要的基因。这项工作将广泛影响我们对细胞核定位信号（NLS）和核输出信号（NES）依赖蛋白运动至关重要的细胞基因的理解，同时阐明出芽酵母G1型细胞周期蛋白的特殊情况下调节蛋白运动的机制。对NLS和NES活性重要的基因将使用基于绿色荧光蛋白（GFP）的报告器和自动化全基因组高通量显微镜进行鉴定。不同的NLS承载GFP报告基因的比较将被用来确定这些基因通常需要这个过程，以及那些基因专门为特定的NLS序列。这些基因与调节蛋白运动的生理相关性将在G1周期蛋白中得到解决。将进行定位、结合和遗传分析，以阐明细胞因子在G1周期蛋白Cln3的调节运动中的作用，并测量这种调节蛋白运动对细胞周期进程的影响。知识价值：这项工作将导致对出芽酵母G1周期蛋白调控定位机制的理解取得重大进展。这项研究还将确定信号序列依赖核细胞质运动的重要基因，这将对更广泛的科学界有价值。这项工作代表了PI重点的转变，并将以自动化高通量显微镜的形式引入研究实验室的新技术。为了促进这项研究，我们开展了合作，以获得罗德学院无法获得的专业知识和资源。更广泛的影响：这项工作将显著影响PI为本科研究人员提供重要和参与经验的能力。罗德学院和多伦多大学的合作，在洛克菲勒大学和弗吉尼亚大学的支持下，为不同背景的本科生研究人员提供了一个独特而宝贵的机会，不仅可以在使用先进技术的同时一起工作，而且可以对核-细胞质蛋白运输的基本生物学理解产生重大影响。来自美国科学界普遍代表性不足的群体的个人将被积极招募参与这项工作，特别是通过与田纳西州孟菲斯市的LeMoyne Owen学院（一所历史悠久的私立黑人小型文理学院）的安排。这项提议的工作特别适合本科研究人员。它利用清晰，直接和完善的方法来解决基本和必要的生物学问题，同时也向学生介绍功能基因组学领域的前沿技术。该项目将在学术和夏季资助本科生研究人员，增加罗德学院生物系的研究文化。学生和PI将通过在地区、国家和国际会议上的演讲，以及在备受尊敬的同行评审期刊上发表数据，向科学界广泛传播这项研究的结果。

Distinct mechanisms for regulated movement of proteins into and out of the nucleus are critical when considering pathways that regulate basic cellular processes, such as cell division. This project focuses on the identification and characterization of genes important for the regulated movement of proteins into and out of the nucleus. The work will broadly impact our understanding of the cellular genes critical for nuclear localization signal (NLS) and nuclear export signal (NES) dependent protein movement, while clarifying the mechanism of regulated protein movement in the specialized case of budding yeast G1 type cyclins. Genes important for NLS and NES activity will be identified using Green Fluorescent Protein (GFP) based reporters and automated genome wide high throughput microscopy. Comparisons of distinct NLS bearing GFP reporters will be used to identify those genes generally required for this process as well as those genes specialized for specific NLS sequences. The physiological relevance of these genes to regulated protein movement will be addressed for the G1 cyclins. Localization, binding, and genetic assays will be carried out to elucidate the role of cellular factors in regulated movement of the G1 cyclin Cln3 and to measure the impact that this regulatedprotein movement has on cell cycle progression. Intellectual Merit: This work will lead to significant progress in the understanding of the mechanisms of regulated localization of G1 cyclins in budding yeast. This research will also identify genes important for signal sequence dependent nucleo-cytoplasmic movement that will be of value to the broader scientific community. The work represents a shift in focus for the PI and will introduce new technologies in the form of automated high throughput microscopy into the research laboratory. To facilitate this research, collaborations have been developed to gain access to expertise and resources not available at Rhodes College.Broader Impacts: This work will significantly impact the ability of the PI to give significant and engaged experiences to undergraduate researchers. The collaboration of Rhodes College and the University of Toronto, with the support of The Rockefeller University and the University of Virginia, provides a unique and invaluable opportunity for undergraduate researchers of various backgrounds to not only work together while using advanced technologies, but to make a significant impact on the basic biological understanding of nucleo-cytoplasmic protein transport. Individuals from groups generally underrepresented in US science will be actively recruited for this work, specifically through arrangements with LeMoyne Owen College in Memphis, TN (a private historically black small liberal arts college). This proposed work is especially well suited for undergraduate researchers. It utilizes clear, straight forward and well established methods to address basic and essential biological questions while also introducing the students to cutting edge technology in the arena of functional genomics. This project will fund undergraduate researchers during academic and summer months, increasing the culture of research in the Department of Biology at Rhodes College. Students and the PI will disseminate results from this study broadly to the scientific community through presentations at regional, national, and international meetings, as well as publication of data in highly regarded peer-reviewed journals.