QUANTIFYING CELLULAR COMPLEX COMPOSITION IN VIVO

体内细胞复合物成分的量化

• 批准号: 8463051
• 负责人: MICHAEL L NONET
• 金额: $ 18.34万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8463051
• 关键词: Apoptosis; Architecture; Autophagocytosis; Behavior; Binding; Biological; Biological Assay; Biological Models; Biological Process; Brain; Caenorhabditis elegans; Calcium Channel; Calibration; Cell Culture Techniques; Cell Nucleus; Cell division; Cell physiology; Cells; Cellular Structures; Centrosome; Chimeric Proteins; Chromosomes; Cilia; Clathrin; Complex; Data; Development; Diffuse; Disease; Eukaryota; Eukaryotic Cell; Exocytosis; Fluorescence; Fluorescence Microscopy; Functional Imaging; Gene Expression; Genes; Genetic; Genetic Models; Genome; Genomics; Grant; Human; Individual; Kinetochores; Label; Lamins; Light; Location; Malignant Neoplasms; Measurement; Methodology; Methods; Modeling; Molecular; Molecular Analysis; Molecular Genetics; Mus; Nematoda; Nerve; Neurons; Nobel Prize; Noise; Nuclear; Nuclear Envelope; Nuclear Pore; Organism; Play; Positioning Attribute; Process; Proteins; Proteomics; RNA Interference; Relative (related person); Role; Saccharomyces cerevisiae; Signal Transduction; Site; Source; Stroke; Structure; Subcellular structure; Synapses; System; Techniques; Technology; Technology Transfer; Transgenes; Transgenic Animals; Transgenic Organisms; Vesicle; Western Blotting; Yeasts; Zebrafish; base; cell motility; cell type; cholinergic synapse; coated pit; cytomatrix; human disease; in vivo; insight; molecular assembly/self assembly; mutant; neurotransmitter release; polarized cell; presynaptic; promoter; receptor; research study; response; stoichiometry; therapy design; therapy development; tool

DESCRIPTION (provided by applicant): Modern molecular biological approaches have provided enormous insight into a vast array of cell biological mechanisms from polarized cell division to neuronal cell migration, autophagy, neuronal exocytosis and countless others. While many molecular players participating in these processes have been elucidated defining the more mechanistic and structural aspects of these processes has been more difficult. Importantly, these mechanistic insights provide the cellular basis for our understanding of disease and serve as the primary guides for developing therapy. One systemic problem in developing mechanistic insight has been the difficulty in obtaining quantitative data about the organization and stoichiometry of components in many cellular complexes. For example, over a dozen proteins have been documented to localize to the cytomatrix of synaptic active zones, a structural presynaptic feature essential for vesicular neurotransmitter release site from neurons in the brain. Furthermore, proteomic analysis of synaptic active zones suggests that the number of distinct proteins associated with presynaptic release sites is greater than one hundred. Defining the structure and organization of such complex subcellular assemblies is a daunting challenge, but it is essential if we wish to understand the basic cellular process of neurotransmitter release in the brain at a mechanistic level. Here we propose to develop methodology for assessing quantitatively the composition of various components of subcellular assemblies in vivo. Multiple methods for performing such quantitative measurements have previously been described, but none are easy to implement for the analysis of structures in complex multicellular organisms. Herein we propose to create in vivo internal calibration standards that can be used in combination with fluorescent protein fusions to quantify levels of specific proteins present at specific sites in vivo. Specifically, we propose to use GFP-LacI bound to LacO sites integrated in known copy number to specific sites on chromosomes as a calibration curve to quantify the numbers of molecules found in subcellular assemblies in vivo. We propose to develop the system in yeast, transfer the technology to C. elegans, and then apply the technology to characterize the relative and absolute stoichiometry of a half dozen proteins found at presynaptic active zones. The system we propose to develop should be applicable to any molecular genetic model system that can be manipulated using transgenic techniques including mouse and zebrafish, as well as human cell culture.

描述（由申请人提供）：现代分子生物学方法已经为从极化细胞分裂到神经元细胞迁移、自噬、神经元胞吐和无数其他细胞生物学机制的大量细胞生物学机制提供了巨大的洞察力。虽然参与这些过程的许多分子参与者已经阐明，但定义这些过程的更多机制和结构方面更加困难。重要的是，这些机制的见解为我们理解疾病提供了细胞基础，并作为开发治疗的主要指南。发展机械论的一个系统性问题是，在许多细胞复合体中，很难获得有关组分的组织和化学计量的定量数据。例如，已经记录了十几种蛋白质定位于突触活性区的细胞基质，这是脑中神经元的囊泡神经递质释放位点所必需的突触前结构特征。此外，突触活性区的蛋白质组学分析表明，与突触前释放位点相关的不同蛋白质的数量大于100。定义如此复杂的亚细胞组装体的结构和组织是一项艰巨的挑战，但如果我们希望在机械水平上了解大脑中神经递质释放的基本细胞过程，这是必不可少的。在这里，我们建议开发的方法，用于定量评估体内亚细胞组件的各种成分的组合物。先前已经描述了用于执行这种定量测量的多种方法，但是没有一种方法容易实现用于分析复杂多细胞生物体中的结构。在此，我们提出创建体内内部校准标准品，其可以与荧光蛋白融合物组合使用，以量化存在于体内特定位点的特定蛋白质的水平。具体而言，我们建议使用GFP-LacI绑定到LacO网站集成在已知的拷贝数的染色体上的特定网站作为校准曲线，以量化在体内亚细胞组件中发现的分子的数量。我们建议在酵母中开发该系统，将该技术转移到C。elegans，然后应用该技术来表征在突触前活动区发现的六种蛋白质的相对和绝对化学计量。我们建议开发的系统应该适用于任何分子遗传模型系统，可以使用转基因技术，包括小鼠和斑马鱼，以及人类细胞培养。