Quantitative characterization of vacuole size in budding yeast

芽殖酵母液泡大小的定量表征

• 批准号: 8152156
• 负责人: Yee-Hung Mark Chan
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8152156
• 关键词: Area; Automation; Biological Assay; Biological Models; Cell Size; Cells; Cellular Membrane; Cellular biology; Computer software; Data; Development; Diagnostic; Disease; Endocytosis; Equilibrium; Fluorescence; Fluorescence Microscopy; Foundations; Goals; Growth; Homeostasis; Human Cell Line; Image; Image Analysis; Individual; Label; Life; Lipids; Lysosomes; Malignant Neoplasms; Mammalian Cell; Measurement; Measures; Membrane; Methods; Microscopy; Modeling; Morphology; Mutation; Nerve Degeneration; Nutrient; Observer Variation; Organelles; Partner in relationship; Play; Process; Proteins; Receptor Down-Regulation; Regulation; Research; Role; Saccharomyces cerevisiae; Saccharomycetales; Shapes; Staining method; Stains; Surface; Testing; Vacuole; Vesicle; Work; Yeasts; base; cell growth; fluorescence imaging; improved; in vivo; mutant; novel diagnostics; programs; protein transport; public health relevance; receptor; receptor downregulation; research study; tool; trafficking; tumorigenesis; wasting

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.

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控。我建议使用的芽殖酵母酿酒酵母的液泡作为模型系统，研究在细胞内的细胞器大小的调节。液泡负责废物处理和储存细胞器，最近的研究表明溶酶体（类似哺乳动物细胞器）的功能与许多疾病（包括癌症和神经变性）之间存在联系。已经定性描述了微球形态，并且已经鉴定了许多突变，这些突变显著改变了微球的大小。我将在各种条件下定量酵母液泡的大小，以确定如何在体内调节细胞器。我将使用三维荧光成像可视化酵母空泡使用外源或遗传编码的蛋白质标记。酵母大小的测量将使用可用的软件和基本编程自动化。液泡大小控制的一个可能模型是液泡膜量占细胞总脂质的固定比例。将在野生型和蛋白质运输突变体之间比较空泡表面积，预期其将脂质从空泡转移，从而改变空泡和总细胞膜之间的比率。最后，将开发用于交配受体Ste 2 p的内吞和降解速率的荧光测定，以探测该过程与液泡大小之间的关系。 公共卫生相关性：我提出的实验将增加我们对细胞生物学中一个基本问题的基本理解-细胞如何调节其细胞器的大小。我对酵母中废物处理细胞器的研究（以及后来在人类细胞系中的研究）将发现它们的形态与生长受体的降解之间的联系，而生长受体的降解与癌症有关。这些工作将提高我们对疾病发生的基本认识，并为基于细胞器形态学的新诊断和治疗提供潜在途径。