Characterization of Neurospora cell fusion events

脉孢菌细胞融合事件的表征

基本信息

批准号：
8000204
负责人：
Stephen J. FREE
金额：
$ 8.08万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2011-08-31
项目状态：
已结题

项目摘要

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 classicalgenetic mapping techniques. The affected genes will be identified using a PCRamplification and DNAsequencing 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. Keycell 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 andfor the differentiation of muscle, bone, and placenta. The proposed research will help us better understand cell fusion events.

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的吻合过程（营养菌丝细胞融合）粗糙脉孢菌。拟议研究的主要目标是识别和表征蛋白质介导细胞融合事件并确定它们如何发挥作用以促进细胞融合。将鉴定细胞融合所需的基因（由细胞融合缺陷突变体定义）并进行了表征。导致细胞融合缺陷表型的突变将被映射到通过经典遗传作图技术绘制脉孢菌遗传图谱的小区域。受影响的基因将使用 PCR 扩增和 DNA 测序策略进行鉴定。这种定位克隆的用途脉孢菌基因组 DNA 序列的发表使这种方法成为可能。这编码细胞融合蛋白的基因的同一性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。帮助我们确定哪些蛋白质在这些步骤中发挥作用，微分干涉对比显微镜和共焦显微镜延时视频记录系统将用于表征细胞融合突变体并阐明这些突变体中哪些细胞融合步骤被阻断。我们还将确定地点并跟踪细胞融合事件期间一些关键细胞融合蛋白的运动。的蜂窝位置细胞融合蛋白将在免疫定位实验中确定。关键细胞融合蛋白将是用绿色荧光蛋白标记以及这些标记蛋白在细胞融合过程中的运动事件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有关细胞融合事件如何策划的重要见解。公共卫生相关性：细胞融合事件对于受精过程和肌肉、骨骼和胎盘的分化。拟议的研究将帮助我们更好地了解细胞融合事件。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The ham-5, rcm-1 and rco-1 genes regulate hyphal fusion in Neurospora crassa.

DOI：
10.1099/mic.0.040147-0
发表时间：
2010-09
期刊：
Microbiology (Reading, England)
影响因子：
0
作者：
Aldabbous MS;Roca MG;Stout A;Huang IC;Read ND;Free SJ
通讯作者：
Free SJ

Trifluoromethanesulfonic acid-based proteomic analysis of cell wall and secreted proteins of the ascomycetous fungi Neurospora crassa and Candida albicans.

DOI：
10.1016/j.fgb.2009.06.005
发表时间：
2009-10
期刊：
FUNGAL GENETICS AND BIOLOGY
影响因子：
3
作者：
Maddi, Abhiram;Bowman, Shaun M.;Free, Stephen J.
通讯作者：
Free, Stephen J.

The Neurospora crassa dfg5 and dcw1 genes encode α-1,6-mannanases that function in the incorporation of glycoproteins into the cell wall.

DOI：
10.1371/journal.pone.0038872
发表时间：
2012
期刊：
PloS one
影响因子：
3.7
作者：
Maddi A;Fu C;Free SJ
通讯作者：
Free SJ

Characterization of GPIT-1 and GPIT-2, two auxiliary components of the Neurospora crassa GPI transamidase complex.

粗糙脉孢菌 GPI 转酰胺酶复合物的两个辅助成分 GPIT-1 和 GPIT-2 的表征。

DOI：
10.3852/09-022
发表时间：
2009
期刊：
Mycologia
影响因子：
2.8
作者：
Bowman,ShaunM;Piwowar,Amy;Arnone,EricD;Matsumoto,Reiko;Koudelka,GeraldB;Free,StephenJ
通讯作者：
Free,StephenJ

WSC-1 and HAM-7 are MAK-1 MAP kinase pathway sensors required for cell wall integrity and hyphal fusion in Neurospora crassa.