Discovering the networks that control cell wall integrity, cell division, morphogenesis and adhesion in Candida albicans using high content microscopy assays.

高度动态的真菌细胞被膜在真菌的形态建成和毒力方面起着重要作用，是抵御化学物质攻击的第一道防线。微生物使用几种策略来维持细胞壁完整性（CWI），包括信号转导级联来监测细胞壁和应对环境挑战。启动CWI级联并延迟或抑制微生物细胞分裂的特定分子线索尚未得到充分研究。在过去的十年里，我的实验室在开发工具来探测真菌和细菌中的CWI策略方面取得了重大进展。我们将开发新的显微镜检测方法，以了解酵母糖基磷脂酰肌醇（GPI）锚定脂蛋白在响应精油成分和衍生物、激活CWI通路和调节形态发生和细胞分裂中的作用。从长远来看，我们将确定CWI途径、细胞分裂、形态学和毒力之间的相互关系，以响应众多抗真菌药物，为农业和医学上基于精油的抗真菌制剂的开发提供信息。我们最近开发的新型高含量检测方法使用相关原子力-定量成像-共聚焦激光扫描显微镜（AF-QI-CLSM）成像，以分别以纳米（nm）和皮牛顿（pN）分辨率同时监测活细胞包膜重塑、细胞壁弹性和表面粘附，同时真实的跟踪至少三个荧光蛋白（FP）标记的分子的组织和动力学。相关的数据提供了深入了解信号转导的细胞包膜和细胞中的亚致死水平的外源性物质的反应。缺乏关键CWI受体蛋白的白色念珠菌突变株将精确定位响应精油的途径。功能化的AFM针尖将探测细胞壁蛋白的定位、聚集和构象，而FP则跟踪参与应激反应和CWI信号传导的细胞内信号分子和细胞分裂蛋白。图像还将揭示形态学变化，如酵母-菌丝转变。同步辐射红外纳米光谱-原子力显微镜与高夫（UofM）将报告表面超微结构和细胞壁的生化组成与纳米精度。活细胞的相关AFM-QI-CLSM是一种全新的分析工具，广泛应用于细胞生物学，用于研究几乎任何细胞类型和压力。本研究将进一步加深我们对真菌CWI途径和细胞壁质量控制在分子水平上的理解。细胞壁是抗真菌剂的主要靶标，其适应能力允许真菌繁殖，这对农业和医学是一个重大损害。因此，了解精油对真菌CWI的影响将告知其作为环境安全的抗真菌替代品的使用。

The highly dynamic fungal cell envelope plays an important role in morphogenesis and virulence as the first line of defense for chemical challenge. Microbes use several strategies to maintain cell wall integrity (CWI), including signal-transduction cascades to monitor the cell wall and respond to environmental challenges. The specific molecular cues that initiate CWI cascades and delay or inhibit cell division in microbes are understudied. Over the past decade my lab has made significant strides in developing tools to probe CWI strategies in both fungi and bacteria.We will develop novel microscopy assays to understand the role of yeast glycosylphosphatidylinositol (GPI)-anchored lipoproteins in response to essential oil components and derivatives, activating CWI pathways and regulating morphogenesis and cell division. Over the long-term we will identify the interrelationship between CWI pathways, cell division, morphology and virulence in response to numerous antifungals to inform the development of essential oil-based antifungal formulations for agriculture and medicine.Our recently developed novel high-content assays use correlative atomic force-quantitative imaging-confocal laser scanning microscopy (AF-QI-CLSM) imaging to simultaneously monitor live cell envelope remodeling, cell wall elasticity and surface adhesion with nanometer (nm) and picoNewton (pN) resolution, respectively, while simultaneously tracking the organization and dynamics of at least three fluorescent protein (FP)-tagged molecules in real time. The correlative data provides insight into signal transduction across the cell envelope and into the cell in response to sub-lethal levels of xenobiotics. Mutant strains of Candida albicans deficient in key CWI receptor proteins will pinpoint the pathways that respond to essential oils. Functionalized AFM tips will probe the localization, clustering and conformation of cell wall proteins, while FPs track intracellular signaling molecules and cell division proteins involved in the stress response and CWI signaling. Images will also reveal morphological changes such as yeast-hyphal transitions. Synchrotron-infrared nano-spectroscopy-AFM with Gough (UofM) will report on surface ultrastructure and cell wall biochemical composition with nm precision. Correlative AFM-QI-CLSM of live cells is a completely novel analytical tool with broad application to cell biology for studying virtually any cell type and stress. This research will further our understanding of fungal CWI pathways and cell wall quality control at the molecular level. The cell wall is a primary target for antifungal agents, and its adaptive capabilities allow propagation of fungi that are a significant detriment to agriculture and medicine. Thus, understanding the impact of essential oils on fungal CWI will inform their use as environmentally safe antifungal alternatives.