Characterization of Neurospora cell fusion events

脉孢菌细胞融合事件的表征

• 批准号: 7131222
• 负责人: Stephen J. FREE
• 金额: $ 19.16万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-25 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7131222
• 关键词: Neurospora; cell fusion; confocal scanning microscopy; fungal genetics; fungal proteins; gene deletion mutation; genetic mapping; green fluorescent proteins; membrane fusion; positional cloning; protein localization; protein structure function; secretory protein; video microscopy

DESCRIPTION (provided by applicant): Fusion between two eukaryotic cells is a fundamentally interesting and biologically important process. We have only a rudimentary understanding of how cell fusion occurs. This grant proposal focuses on characterizing the process of anastomosis (vegetative hyphal cell fusion) in the filamentous fungus Neurospora crassa. The major goals of the proposed research are to identify and characterize proteins that function to mediate the cell fusion event and to determine how they function to facilitate cell fusion. The genes that are required for cell fusion, as defined by cell fusion-defective mutants, will be identified and characterized. The mutations responsible for the cell fusion-defective phenotype will be mapped to small regions of the Neurospora genetic map by classical genetic mapping techniques. The affected genes will be identified using a PCR amplification and DNA sequencing strategy. The use of this positional cloning approach has been made possible by the publication of the Neurospora genomic DNA sequence. The identity of the genes encoding cell fusion proteins will be verified by gene disruption experiments. Anastomosis can be characterized as a series of steps leading to a cell fusion event. To help us determine which proteins function is each of these steps, differential interference contrast microscope and confocal microscope time-lapse video recording systems will be used to characterize the cell fusion mutants and elucidate which cell fusion steps are blocked within these mutants. We will also determine the location and follow the movement of a few key cell fusion proteins during cell fusion events. The cellular location of cell fusion proteins will be determined in immunolocalization experiments. Key cell fusion proteins will be tagged with the green fluorescent protein and the movement of these tagged proteins during cell fusion events will be followed with the confocal microscope time-lapse video recording system. We anticipate that these experiments will provide us with important insights into how cell fusion events are orchestrated. Public Health Relevance: Cell fusion events are critical to the process of fertilization and for the differentiation of muscle, bone, and placenta. The proposed research will help us better understand cell fusion events.

描述（由申请人提供）：两个真核细胞之间的融合是一个非常有趣且具有生物学意义的过程。我们对细胞融合如何发生只有初步的了解。该拨款提案的重点是描述丝状真菌粗糙脉孢菌的吻合（营养菌丝细胞融合）过程。拟议研究的主要目标是识别和表征介导细胞融合事件的蛋白质，并确定它们如何发挥促进细胞融合的作用。细胞融合所需的基因（由细胞融合缺陷突变体定义）将被鉴定和表征。导致细胞融合缺陷表型的突变将通过经典的遗传作图技术被映射到脉孢菌遗传图谱的小区域。将使用 PCR 扩增和 DNA 测序策略来识别受影响的基因。脉孢菌基因组 DNA 序列的公布使得这种定位克隆方法的使用成为可能。编码细胞融合蛋白的基因的身份将通过基因破坏实验来验证。吻合可被表征为导致细胞融合事件的一系列步骤。为了帮助我们确定哪些蛋白质在这些步骤中发挥作用，将使用微分干涉显微镜和共焦显微镜延时视频记录系统来表征细胞融合突变体，并阐明哪些细胞融合步骤在这些突变体中被阻断。我们还将确定细胞融合事件期间一些关键细胞融合蛋白的位置并跟踪其运动。细胞融合蛋白的细胞位置将在免疫定位实验中确定。关键的细胞融合蛋白将用绿色荧光蛋白进行标记，并且这些标记蛋白在细胞融合事件期间的运动将通过共焦显微镜延时视频记录系统进行跟踪。我们预计这些实验将为我们提供关于如何精心安排细胞融合事件的重要见解。公共健康相关性：细胞融合事件对于受精过程以及肌肉、骨骼和胎盘的分化至关重要。拟议的研究将帮助我们更好地了解细胞融合事件。