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。真菌是对工业、环境和农业都非常重要的微生物。其中许多被用来生产有商业价值的产品，而另一些则因其变质商品和感染作物而造成重大经济损失。了解它们的增长，推进涉及它们的使用的技术，并帮助制定战略，控制那些具有破坏性的东西的传播。真菌细胞的生长和发育主要集中在细胞壁，细胞壁是一种外层细胞结构，提供刚性和对潜在环境应激源的保护。如果没有健康的细胞壁，真菌的生长和定植宿主的能力就会受到严重影响。该项目建立在之前NSF资助的同一研究人员的研究基础上，该研究确定了参与真菌细胞壁合成和生长的候选蛋白质。除了发现更多与生长相关的蛋白质外，这项工作还将确定这些蛋白质中哪些在物理上相互作用，以及它们在生长过程中如何协同工作。这项研究将由两名资深科学家与罗兹学院(田纳西州孟菲斯)的本科生一起进行。本科生将完整地参与研究的各个方面，这将补充和扩展他们的课程，提供更完整的科学教育，并为他们的深造做好更好的准备。调查人员还将继续一个在之前的NSF奖项期间启动的项目，该项目为孟菲斯地区历史上曾就读的黑人学院的学生提供暑期研究职位。本项目研究了蛋白激酶C在丝状真菌生长中的作用，以及它与参与极化生长和发育的其他蛋白质的相互作用。这些研究人员和该领域的其他研究人员利用丝状真菌Nidulans作为模式生物，已经确定了定位于细胞壁合成位置的蛋白质，即生长细胞尖(菌丝尖端)和形成交叉壁(SEPTA)。其中一种蛋白质是PKCA(一种与蛋白激酶C同源的螺旋藻)，它是一种丝氨酸/苏氨酸激酶，与后生动物和酵母菌中描述的新的蛋白激酶C亚型最相似。虽然已经做了很多工作来描述参与生长的蛋白质，但这个项目将增加可以被认为是？PKCA模块？的内容。到更大的根结线虫生长网络复合体。使用荧光标记蛋白、免疫沉淀和酵母双杂交分析等技术，研究人员将揭示PKCA参与隔膜部位和菌丝尖端的蛋白质-蛋白质相互作用以及负责这些相互作用的PKCA基序。此外，研究人员还将利用与LC-MS/MS(LC-MS/MS)相结合的电子蛋白质组学工具，确定哪些复杂的蛋白质被PKCA磷酸化。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界也有价值，因为它将揭示影响蛋白质向复合体募集的因素以及这些蛋白质网络在募集后如何发挥作用。

This project seeks to increase our understanding of how fungi grow. Fungi are microorganisms of great importance to industry, the environment, and agriculture. Many are used to produce commercially valuable products, while others cause major financial losses due to their spoilage of goods and infecting crops. Understanding their growth advances technologies involving their use and aids in developing strategies to control spread of those that are damaging. Fungal cell growth and development are focused at the cell wall, which is an outer cellular structure that provides rigidity and protection from potential environmental stressors. Without a healthy cell wall, fungal growth and the ability to colonize its host are severely compromised. This project builds on previous NSF funded research by the same investigators, which identified candidate proteins involved in fungal cell wall synthesis and growth. In addition to discovering more growth-related proteins, this work will determine which of those proteins physically interact with one another and how they work together during growth. This research will be carried out by a pair of senior scientists working with undergraduate students at Rhodes College (Memphis, TN). Undergraduate students will be integrally involved in all aspects of the research, which will complement and extend their coursework providing a more complete scientific education and better preparing them for advanced studies. The investigators will also continue a program initiated during prior NSF awards, which provides summer research positions to students attending Historically Black Colleges in the Memphis region. This project investigates the function of protein kinase C in filamentous fungal growth and the interplay of its functions with those of other proteins involved in polarized growth and development. Using the filamentous fungus Aspergillus nidulans as a model organism, these researchers and others in the field have identified proteins that localize to sites of cell wall synthesis, namely growing cell apices (hyphal tips) and forming crosswalls (septa). One such protein is PkcA (an A. nidulans homolog of protein kinase C), which is a serine/threonine kinase most similar to the novel protein kinase C isoforms that have been well described in metazoans and yeasts. Although much has been done to describe proteins that participate in growth, this project will add what could be considered the ?PkcA module? to the larger A. nidulans growth network complex. Using techniques including fluorescence labeled proteins, immunoprecipitations, and yeast two-hybrid assays, the researchers will uncover the protein-protein interactions in which PkcA participates at septation sites and hyphal tips and the PkcA motifs responsible for these interactions. In addition, the researchers will determine which complexed proteins are phosphorylated by PkcA using in silico proteomics tools paired with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Not only will this research be of value to the filamentous fungi community, but also to the broader cell biology community, as it will shed light on factors that affect protein recruitment to complexes and how those protein networks function subsequent to recruitment.