Characterization of Neurospora cell fusion events

脉孢菌细胞融合事件的表征

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/8000204
• 关键词: Adhesions; Affect; Anastomosis - action; Applications Grants; Cell Adhesion; Cell Wall; Cell fusion; Cell membrane; Cells; Chimeric Proteins; Chromosome Mapping; DNA Sequence; Elements; Enzymes; Eukaryotic Cell; Event; Fertilization; Genes; Genetic; Genomics; Goals; Green Fluorescent Proteins; Growth; Hyphae; Life Cycle Stages; Lipase; Lipids; Location; Maps; Mediating; Membrane Fusion; Microscope; Modeling; Molds; Movement; Muscle; Mutation; Neurospora; Neurospora crassa; Phase; Phenotype; Placenta; Process; Protein Secretion; Proteins; Publications; Research; Series; Signal Transduction; Signal Transduction Pathway; Techniques; Time; Video Recording; base; bone; insight; interest; mutant; positional cloning; pressure; protein function; public health relevance; research study

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根本有趣且在生物学上重要的过程。 我们对细胞融合的发生方式只有基本的理解。该赠款提案的重点是 表征丝状真菌中吻合的过程（营养菌丝细胞融合） Neurospora Crassa。拟议研究的主要目标是识别和表征蛋白质 功能以介导细胞融合事件并确定它们如何功能以促进细胞融合。 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 并描述了。负责细胞融合缺陷表型的突变将映射到 神经孢子遗传图的小区域通过经典化作用技术。受影响的基因 将使用Pcramplification和DNaseQuencing策略来识别。使用该位置克隆 神经孢子基因组DNA序列的出版使方法成为可能。这 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。 吻合术可以被描述为导致细胞融合事件的一系列步骤。帮助我们 确定哪些蛋白质功能是这些步骤中的每个步骤，差异干扰对比显微镜和 共聚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塞。我们还将确定位置 并遵循细胞融合事件中一些少数关键细胞融合蛋白的运动。细胞位置 细胞融合蛋白将在免疫定位实验中确定。 KeyCell Fusion蛋白将是 用绿色荧光蛋白标记和在细胞融合过程中这些标记蛋白的运动 事件将遵循共聚焦显微镜延时视频记录系统。我们预料到这一点 这些实验将为我们提供有关细胞融合事件如何精心策划的重要见解。 公共卫生相关性：细胞融合事件对于受精过程至关重要 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

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)
• 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

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

• DOI: 10.1371/journal.pone.0042374
• 发表时间: 2012
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Maddi A;Dettman A;Fu C;Seiler S;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