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了解。真菌是用于生产具有商业和医药价值的产品的微生物，而其他微生物则会引起疾病，并因作物破坏和储存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它们的使用的技术，并有助于制定战略，以控制它们在有害环境中的传播。真菌以两种不同的形式存在：球形的单细胞生物体和细长的多细胞生物体，它们被称为丝状真菌。这个项目的重点是丝状真菌的生长和细胞分裂。为了生长和定居它们的宿主，丝状真菌通过在生长的丝状物的顶端添加细胞材料来延伸到它们的环境中，它们通过沿着丝状物以均匀间隔构建被称为隔膜的交叉壁来分裂。该项目建立在该实验室以前NSF资助的研究的基础上，该研究确定了参与丝状真菌生长和细胞分裂的蛋白质，并开始确定哪些与生长相关的蛋白质形成与生长和分裂有关的物理复合体。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体，并将进一步定义这些复合体的功能。这项研究将由罗兹学院(孟菲斯，田纳西州)的两名资深科学家进行，他们与罗兹大学的本科生和孟菲斯地区历史上的黑人学院的学生合作。本科生将整体参与到这项工作中来，这一研究经验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。利用丝状真菌Nidulans作为模式生物，已经鉴定出与生长和细胞分裂有关的蛋白质，包括蛋白激酶C的同源物PKCA。该项目的主要研究人员之前的研究使用了包括荧光标记蛋白质的体内显微镜、免疫沉淀随后的质谱学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物理上相互作用的蛋白质，包括Forin SepA、三个Rho类型的GTP酶同源物、一个甲壳素合成酶同源物、一个葡聚糖合成酶和两个重要的支架蛋白--A.nidulans IQGAP Ortholog SepG和A.nidulans paxlin Ortholog PaxB--它们似乎在PKCA的分离位点定位中发挥了作用。该项目将确定这些蛋白质或其子集是否是PKCA底物，以及磷酸化事件是否在生长和细胞分裂中发挥重要作用。将通过确定PKCA的关键结构域以及促进它们之间蛋白质-蛋白质相互作用的复杂蛋白质来探索PKCA蛋白质复合体结合的机制。这项研究不仅对丝状真菌社区有价值，而且也将使更广泛的细胞生物学社区受益，因为它将阐明影响蛋白激酶C招募(和一般的蛋白质招募)到蛋白质复合体的因素以及蛋白质网络在招募后如何发挥作用。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.