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、神经元胞吐作用等无数细胞生物学机制提供了深入的见解。虽然参与这些过程的许多分子参与者已经被阐明，但定义这些过程的更多机械和结构方面却更加困难。重要的是，这些机制见解为我们理解疾病提供了细胞基础，并作为开发治疗方法的主要指南。发展机制洞察力的一个系统性问题是难以获得有关许多细胞复合物中成分的组织和化学计量的定量数据。例如，已记录了十多种蛋白质定位于突触活性区的细胞基质，这是一种突触前结构特征，对于大脑中神经元的囊泡神经递质释放位点至关重要。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超过一百种。定义这种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，那么它是至关重要的。在这里，我们建议开发定量评估体内亚细胞组装体各种成分组成的方法。先前已经描述了用于执行此类定量测量的多种方法，但没有一种方法易于实施来分析复杂的多细胞生物体中的结构。在此，我们建议创建体内内部校准标准品，该标准品可与荧光蛋白融合物结合使用，以量化体内特定位点存在的特定蛋白质的水平。具体来说，我们建议使用与以已知拷贝数整合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装中发现的分子数量。我们建议在酵母中开发该系统，将该技术转移到秀丽隐杆线虫中，然后应用该技术来表征突触前活性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可以使用转基因技术操纵的分子遗传模型系统，包括小鼠和斑马鱼以及人类细胞培养。公共健康相关性：这笔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装的成分。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发现了几种生物过程，其中每一个最近都获得了诺贝尔奖。第一个发现是程序性细胞死亡，它在细胞对中风的反应中发挥着关键作用。第二个是RNA干扰过程的发现，该过程被广泛认为是治疗人类疾病（例如由基因错误表达引起的癌症）非常有前途的方法。