RUI: Protein-Protein Interactions of Protein Kinase C During Polarized Growth in Filamentous Fungi

这个项目旨在增加我们对真菌如何生长的理解。真菌是一种微生物，用于生产具有商业价值和药用价值的产品，而其他真菌则引起疾病，并由于作物受损和储存的食品变质而造成重大经济损失。了解它们的增长有助于推进涉及使用它们的技术，并有助于制定控制它们在有害环境中传播的战略。真菌以两种不同的形式存在；球形的单细胞生物和细长的多细胞生物，它们被称为丝状真菌。本项目主要研究丝状真菌的生长和细胞分裂。丝状真菌为了在宿主体内生长和定植，它们通过在生长的丝状末端添加细胞物质来延伸到宿主的环境中，它们通过沿着丝状末端以均匀间隔的间隔构建被称为隔的横壁来分裂。该项目建立在之前由NSF资助的实验室研究的基础上，该实验室确定了丝状真菌生长和细胞分裂中涉及的蛋白质，并开始确定哪些与生长相关的蛋白质形成了参与生长和分裂的物理复合物。正在进行的工作将通过识别参与其中的新蛋白质来扩大生长和细胞分裂蛋白质复合物，并将进一步确定这些复合物的功能。这项研究将由罗德学院（田纳西州孟菲斯市）的两名资深科学家与在罗德学院就读的本科生和在孟菲斯地区就读历史悠久的黑人学院的学生一起进行。本科生将全面参与工作，这一研究经历将加强他们的科学教育。本项目研究蛋白激酶C在丝状真菌生长和细胞分裂（分离）中的作用。以丝状真菌中性曲霉（Aspergillus nidulans）为模式生物，鉴定了与生长和细胞分裂有关的蛋白，包括中性曲霉蛋白激酶C的同源物PkcA。该项目的主要研究人员先前的研究使用了包括荧光标记蛋白质的体内显微镜，免疫沉淀，质谱和蛋白质组学分析以及酵母双杂交分析等技术来鉴定几种与PkcA物理相互作用的蛋白质，包括formin SepA，三个rho型GTPase同源物，一个几丁质合成酶同源物，一个葡聚糖合成酶。以及两个重要的支架蛋白——A. nidulans IQGAP同源蛋白SepG和A. nidulans paxillin同源蛋白PaxB——它们似乎在PkcA定位到分裂位点中起作用。该项目将确定这些蛋白或一个亚群是否是PkcA底物，以及磷酸化事件是否在生长和细胞分裂中起重要作用。通过鉴定PkcA的关键结构域和促进它们之间蛋白-蛋白相互作用的复杂蛋白，将探索PkcA蛋白复合物结合的机制。这项研究不仅对丝状真菌群落有价值，而且对更广泛的细胞生物学群落也有好处，因为它将揭示影响蛋白激酶C向蛋白质复合体募集（以及一般的蛋白质募集）的因素，以及蛋白质网络在募集后如何发挥作用。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project seeks to increase our understanding of how fungi grow. Fungi are microorganisms which are used to produce commercially and medicinally valuable products, while others cause disease and result in major financial losses due to crop damage and spoilage of stored foods. Understanding their growth helps to advance technologies involving their use and aids in developing strategies to control their spread in harmful contexts. Fungi exist in two different forms; spherical, single-celled organisms and elongated, multicellular organisms, which are called filamentous fungi. This project focuses on growth and cell division in filamentous fungi. In order to grow and colonize their hosts, filamentous fungi extend into their environments by adding cellular materials at the tips of growing filaments, and they divide by constructing cross-walls, called septa, at evenly spaced intervals along the filaments. This project builds on previous NSF funded research in this laboratory which identified proteins involved in filamentous fungal growth and cell division, and began to determine which growth-related proteins form physical complexes that are involved in growth and division. Ongoing work will expand growth and cell division protein complexes by identifying new proteins which are involved, and it will further define how these complexes function. This research will be carried out by two senior scientists at Rhodes College (Memphis, TN) working with undergraduate students enrolled at Rhodes and students attending Historically Black Colleges in the Memphis region. Undergraduate students will be integrally involved in the work, and this research experience will strengthen their scientific education. This project investigates the function of protein kinase C in filamentous fungal growth and cell division (septation). Using the filamentous fungus Aspergillus nidulans as a model organism proteins involved in growth and cell division, including the A. nidulans homolog of protein kinase C, PkcA, have been identified. Previous research by the primary investigators of this project used techniques including in vivo microscopy of fluorescence labeled proteins, immunoprecipitations followed by mass spectrometry and proteomics analyses, and yeast two-hybrid assays to identify several proteins that physically interact with PkcA including the formin SepA, three Rho-type GTPase orthologs, a chitin synthase ortholog, a glucan synthase, and two important scaffold proteins - A. nidulans IQGAP ortholog SepG and an A. nidulans paxillin ortholog PaxB - which appear to have roles in PkcA’s localization to septation sites. This project will determine if these proteins or a subset are PkcA substrates, and if phosphorylation events play important roles in growth and cell division. The mechanisms by which PkcA protein complexes coalesce will be explored by identifying key domains of PkcA and complexed proteins that facilitate protein-protein interactions among them. Not only will this research be of value to the filamentous fungi community, but it will also benefit the broader cell biology community, as it will shed light on the factors that affect protein kinase C recruitment (and protein recruitment in general) to protein complexes and how protein networks function subsequent to recruitment.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.