QUANTIFYING CELLULAR COMPLEX COMPOSITION IN VIVO

体内细胞复合物成分的量化

基本信息

批准号：
8397006
负责人：
MICHAEL L NONET
金额：
$ 22.8万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-06-01 至 2014-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8397006
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

项目摘要

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. PUBLIC HEALTH RELEVANCE: This grant proposes to develop methodology and tools for a model system that will enable better quantification of components of subcellular assemblies in vivo. Model systems are used in the study of basic cellular processes that underlie human disease. Understanding of cellular mechanisms is a fundamental requirement for designing therapy. The contributions of this model system to understanding disease include the discoveries of several biological processes each of which was recently recognized by a Nobel prize. The first discovery is that of programmed cell death which plays critical roles in cellular responses to stroke. The second is the discovery of the process of RNA interference that is widely recognized as very promising methodology to treat human diseases such as cancer that result from the mis-expression of genes.

描述（由申请人提供）：现代分子生物学方法为从极化细胞分裂到神经元细胞迁移，自噬，神经元外胞菌病和其他无数的细胞生物学机制提供了巨大的洞察力。尽管许多参与这些过程的分子参与者已经阐明了定义这些过程的机械和结构方面的更加困难。重要的是，这些机械见解为我们理解疾病的理解提供了细胞基础，并作为发展治疗的主要指南。在开发机理洞察力时，一个系统性问题是难以获得许多细胞复合物中有关组件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，以定位于突触活动区的细胞膜，这是大脑神经元的囊泡神经递质释放位点必不可少的结构性突触前特征。此外，突触活动区域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数量大于一百。定义这种复杂的亚细胞组件的结构和组织是一个艰巨的挑战，但是如果我们希望了解机械水平上神经递质释放的基本细胞过程，这一点至关重要。在这里，我们建议开发用于定量评估体内亚细胞组件各个组成部分的方法。先前已经描述了进行此类定量测量的多种方法，但是没有一个容易实施复杂多细胞生物中结构的分析。本文中，我们建议创建体内内部校准标准标准，这些标准可以与荧光蛋白融合结合使用，以量化体内特定位点存在的特定蛋白质水平。具体而言，我们建议将与已知拷贝数集成到染色体上特定位点的LACO位点结合的GFP-LACI作为校准曲线，以量化体内亚细胞组件中发现的分子数量。我们建议在酵母中开发系统，将技术转移到秀丽隐杆线虫，然后应用该技术来表征在突触前活性区域发现的六个蛋白质的相对和绝对化学测定法。我们建议开发的系统应适用于可以使用包括小鼠和斑马鱼以及人类细胞培养的转基因技术来操纵的任何分子遗传模型系统。公共卫生相关性：该赠款提议为模型系统开发方法和工具，以便更好地量化体内亚细胞组件的组件。模型系统用于研究人类疾病的基本细胞过程。了解细胞机制是设计治疗的基本要求。该模型系统对理解疾病的贡献包括诺贝尔奖最近认可的几种生物过程的发现。第一个发现是程序性细胞死亡，它在细胞对中风的反应中起关键作用。第二个是发现RNA干扰过程的发现，该过程被广泛认为是治疗基因误表达引起的人类疾病（例如癌症）的非常有前途的方法。