Super-resolution optical microscopy studies of peroxisomal protein import in the yeast Saccharomyces cerevisiae

过氧化物酶体是一种单膜结合的细胞器，几乎存在于所有真核细胞中。过氧化物酶体的重要性在一组非常严重的代谢性疾病中得到强调，这些疾病是由过氧化物酶体生物发生因子的突变引起的。利用生物化学工具，在了解过氧化物酶体功能的基本原理方面取得了进展。然而，需要超分辨率光学STED（受激发射损耗）显微镜等技术来直接研究活细胞中的动态过程。由1905年DFG研究单位（过氧化物酶体转座子的结构和功能）发起，建立了波鸿鲁尔大学Erdmann教授和牛津大学Eggeling教授小组之间的合作，结合了两个小组在过氧化物酶体和显微镜方面的长期专门知识。在人类细胞中对过氧化物酶体进行了成功的STED显微镜研究。为了更好地了解单个过氧化物酶体蛋白的功能，我们需要精确地对它们进行基因修饰，以消除过氧化物酶体生物发生中的缺陷，正如Erdmann实验室在酵母菌株中实现的那样。目的是应用STED显微镜研究酵母中参与过氧化物酶体蛋白质输入的蛋白质的动态组织。首先建立染色和处理方案，优化数据采集和分析，为利用现有酵母菌株更好地了解过氧化物酶体蛋白输入和增殖的基本机制奠定基础。

Peroxisomes are single-membrane-bound organelles, which are ubiquitous in nearly all eukaryotic cells. The importance of peroxisomes is highlighted by a group of very severe metabolic diseases, which are caused by mutations in the peroxisomal biogenesis factors. Progress has been made in understanding the basic principles of peroxisomal functions using biochemical tools. However, techniques such as super-resolution optical STED (Stimulated Emission Depletion) microscopy are required to directly study dynamic processes in living cells. Initiated by the DFG Research Unit 1905 (Structure and function of the peroxisomal translocon), a collaboration between the groups of Prof. Erdmann at the Ruhr University of Bochum and Prof. Eggeling at the University of Oxford has been established, combining the long-standing expertise of both groups on peroxisomes and microscopy. Successful STED microscopy investigations on peroxisomes were performed in human cells. For a better understanding of the function of the individual peroxisomal proteins we need to precisely modify them genetically for defects in peroxisome biogenesis, as realized in yeast strains from the Erdmann lab. The aim is to apply STED microscopy to study the dynamic organization of proteins involved in peroxisomal protein import in yeast. First staining and handling protocols will be established as well as data acquisition and analysis will be optimized, which will lay the basis for using the available yeast strains to gain a better understanding of the basic mechanisms of peroxisomal protein import and proliferation.