Quantitative characterization of vacuole size in budding yeast

描述（由申请人提供）：细胞器形态在功能中起着至关重要的作用，因此受到细胞的严格调节。我建议使用出芽酵母的液泡作为研究细胞器大小在细胞环境下调节的模型系统。液泡是负责废物处理和储存的细胞器，最近的研究表明，溶酶体（类似于哺乳动物的细胞器）的功能与许多疾病（包括癌症和神经变性）之间存在联系。液泡形态已定性描述和许多突变已被确定显著改变其大小。我将量化各种条件下酵母液泡大小，以确定细胞器在体内是如何调节的。我将使用三维荧光成像来可视化酵母液泡使用外源性或遗传编码的蛋白质标记。酵母大小的测量将自动使用可用的软件和基本编程。液泡大小控制的一个可能模型是液泡膜量是细胞总脂质的固定比例。将比较野生型和蛋白质转运突变体的液泡表面积，预计它们会将脂质从液泡中转移出去，从而改变液泡与总细胞膜之间的比例。最后，将开发一种荧光法检测内吞速率和交配受体Ste2p的降解速率，以探测这一过程与液泡大小之间的关系。

DESCRIPTION (provided by applicant): Organelle morphology plays a crucial role in function, and it is therefore tightly regulated by cells. I propose to use the vacuole of the budding yeast Saccharomyces cerevisiae as a model system for studying the regulation of organelle size in a cellular context. The vacuole is responsible for waste- processing and storage organelle, and recent research suggests a connection between the function of the lysosome (the analogous mammalian organelle) and many diseases including cancer and neurodegeneration. Vacuole morphology has been qualitatively described and many mutations have been identified which dramatically alter its size. I will quantify yeast vacuole size in various conditions to determine how the organelle is regulated in vivo. I will use three-dimensional fluorescence imaging to visualize yeast vacuoles using exogenous or genetically-encoded protein markers. Measurements of yeast size will be automated using available software and basic programming. A possible model for vacuole size control is that the vacuole membrane amount is a fixed proportion of the total cellular lipid. Vacuole surface area will be compared between wild-type and protein trafficking mutants, which are expected to divert lipid away from the vacuole and thus alter the ratio between vacuole and total cellular membrane. Lastly, a fluorescence assay for the rate of endocytosis and degradation of the mating receptor Ste2p will be developed to probe the relationship between this process and vacuole size. PUBLIC HEALTH RELEVANCE: The experiments I propose will increase our basic understanding of a fundamental problem in cell biology-How cells regulate the size of their organelles. My studies on the waste-processing organelles in yeast (and later in human cell lines) will find connections between their morphology and the degradation of growth receptors which is implicated in cancer. Such work will improve our basic understanding of the rise of disease and also provide potential avenues for new diagnostics and treatments based on organelle morphology.