Quantitative characterization of vacuole size in budding yeast

描述(申请人提供)：细胞器形态在功能上起着至关重要的作用，因此它受到细胞的严格调控。我建议使用萌芽酵母的液泡作为一个模型系统来研究细胞器大小的调节。液泡负责废物处理和储存细胞器，最近的研究表明，溶酶体(类似哺乳动物的细胞器)的功能与包括癌症和神经变性在内的许多疾病之间存在联系。对液泡的形态进行了定性描述，并发现了许多突变，这些突变极大地改变了液泡的大小。我将量化不同条件下酵母液泡的大小，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技术，使用外源或基因编码的蛋白质标记来可视化酵母空泡。酵母大小的测量将使用可用的软件和BASIC编程进行自动化。液泡大小控制的一种可能模式是，液泡膜的数量是细胞总脂质的固定比例。将比较野生型和蛋白质运输突变体的液泡表面积，这些突变体有望将脂类从液泡中转移出去，从而改变液泡与总细胞膜的比例。最后，将开发一种测定交配受体Ste2p内吞和降解速率的荧光分析方法，以探索这一过程与空泡大小的关系。 与公共健康相关：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(以及后来的人类细胞系)中的废物处理细胞器的研究将发现它们的形态与生长受体的降解之间的联系，生长受体与癌症有关。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的新诊断和治疗提供潜在的途径。

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.