POWRE: Characterization of the Yeast Cell Wall Protein FLO11

酵母细胞壁还没有被很好地理解，但它是一个越来越重要的研究课题。 真菌是植物和动物（包括人类）的重要病原体，真菌细胞壁是真菌疾病治疗的潜在靶点。此外，了解酵母细胞壁的结构和生物发生对于了解酵母发育过程，如交配，细胞分裂，入侵和假菌丝形成至关重要。 Dranginis博士工作的长期目标是在分子水平上了解酵母的发育。最近，她发现了一个受交配型基因座（MAT）调控的基因，该基因编码一种细胞壁蛋白Flo 11 p。Flo 11 p引起称为絮凝的细胞粘附，并且是酵母中入侵和丝状体形成所必需的。FLO 11受MAP激酶信号转导通路调节，该通路调节信息素应答和入侵/入侵应答。转录因子Ste 12 p和Tec 1 p是该途径的下游效应子，用于入侵反应。 值得注意的是，FLO 11是唯一一个被Ste 12 p和Tec 1 p激活的基因，它是入侵所必需的。FLO 11是S.啤酒。FLO 11是入侵和假菌丝形成的第一个特异性细胞表面要求，因此是一种重要的细胞壁蛋白。这个POWRE奖是为了支持研究休假，以促进Dranginis博士和亨特学院的Peter Lipke博士之间的合作。 Lipke博士是真菌细胞壁领域的著名专家。 这次合作将为Dranginis博士提供一个机会，扩大她对Flo 11 p的研究，同时掌握一个对她来说是新的领域，即，除了酵母细胞壁外，还可以使用亨特学院丰富的设施，特别是带有共聚焦显微镜的生物成像设施。 这些设施是Dranginis博士的家乡圣约翰大学所没有的。提出了一个合作项目，具体目标如下：1。构建改良的绿色荧光蛋白（GFP）-FLO 11基因融合体，应用共聚焦显微镜研究Flo 11 p在酵母中的亚细胞定位.利用共聚焦显微镜观察含有GFP-FLO 11融合基因的单倍体细胞在营养生长、交配、侵入生长和假菌丝生长过程中的变化。为了研究Flo 11 p在交配细胞和受精卵以及随后分裂的二倍体细胞中的分布，将含有融合蛋白的单倍体细胞与未标记的细胞交配。随着新形成的二倍体开始分裂，将跟踪标记的分布，提供关于细胞壁中蛋白质的流动性和交配后细胞壁的重排的信息，以及关于二倍体细胞壁中单倍体特异性蛋白质的周转的问题。为了研究Flo 11 p在单倍体细胞侵袭性生长期间的定位，将细胞生长在盖玻片上并通过共聚焦显微镜检查。Flo 11 p是入侵所必需的，其亚细胞分布将有助于阐明入侵的机制.利用激光共聚焦显微镜观察GFP-FLO 11融合基因在二倍体细胞中的营养生长、假菌丝生长和孢子形成过程。初步认为Flo 11的功能是引起假菌丝链中细胞的粘附。Flo 11 p集中在假菌丝中的母芽连接处的假设将通过检查在盖玻片上生长为假菌丝的含有GFP-FLO 11融合体的二倍体细胞来检验。为了研究在孢子形成过程中FLO 11表达的调节，将含有GFP-FLO 11融合体的二倍体细胞培养在孢子形成培养基中并间隔检查。

The yeast cell wall is not well understood, but is an increasingly important subject of research. Fungi include important pathogens of plants and animals, including man. Fungal cell walls represent good potential targets for therapeutic intervention in fungal diseases. In addition, understanding the structure and biogenesis of the yeast cell wall is crucial for understanding the yeast developmental processes such as mating, cell division, invasion, and pseudohyphae formation. The long term goal of Dr. Dranginis' work is to understand development in yeast at a molecular level. Recently, she discovered a gene that is regulated by the mating-type locus (MAT) and which encodes a cell wall protein, Flo11p. Flo11p causes cell adhesion of the type known as flocculation, and is critically required for invasion and filament formation in yeast. FLO11 is regulated by a MAP kinase signal transduction pathway which regulates both pheromone response and the invasion/filamentation response. The transcription factors Ste12p and Tec1p are downstream effectors of this pathway for the invasion response. Remarkably, FLO11 is the only gene whose activation by Ste12p and Tec1p is required for invasion. FLO11 is the first (and so far, only) downstream effector gene of the filamentation response pathway in S. cerevisiae. FLO11 is the first specific cell surface requirement for invasion and the formation of pseudohyphae, and is therefore an important cell wall protein. This POWRE award is to support a research leave to facilitate a collaboration between Dr. Dranginis and Dr. Peter Lipke of Hunter College. Dr. Lipke is a renowned expert in the area of the fungal cell wall. This collaboration will provide an opportunity for Dr. Dranginis to expand her studies of Flo11p while developing mastery of an area that is new to her, i.e., the yeast cell wall, as well as access to the rich facilities of Hunter College, particularly the BioImaging Facility with its confocal microscope. These are facilities that are not available at Dr. Dranginis' home institution, St. John's University. A collaborative project is proposed with the following specific aims: 1. To construct improved Green Fluorescent Protein (GFP)-FLO11 gene fusions suitable for usewith confocal microscopy to study the subcellular localization of Flo11p in yeast.2. To use confocal microscopy to study haploid cells containing the GFP-FLO11 fusions duringvegetative growth, mating, and invasive growth and pseudohyphal growth. In order to study thedistribution of Flo11p in mating cells and in the zygote and the subsequently dividing diploidcell, haploid cells containing the fusion proteins will be mated with unlabelled cells. The distribution of the label will be followed as the newly formed diploid begins to divide, providing information about the mobility of proteins in the cell wall and the rearrangement of the cell wall after mating, as well as to the question of turnover of haploid-specific proteins in the diploid cell wall. To study the localization of Flo11p during invasive growth of haploid cells, cells will be grown on cover slips and examined by confocal microscopy. The subcellular distribution of Flo11p, which is absolutely required for invasion, will help illuminate the mechanism of invasion.3. To use confocal microscopy to study diploid cells containing the GFP-FLO11 fusions duringvegetative growth, pseudohyphal growth and sporulation. It is proposed that Flo11 functions tocause adhesion of the cells in the pseudohyphal chains. The hypothesis that Flo11p isconcentrated at the mother-bud junction in pseudohyphal filaments will be tested by examiningdiploid cells containing the GFP-FLO11 fusions growing as pseudohyphae on coverslips. Tostudy modulation of FLO11 expression during sporulation, diploid cells containing the GFP-FLO11fusions will be cultured in sporulation media and examined at intervals.