QUANTIFYING CELLULAR COMPLEX COMPOSITION IN VIVO

描述(申请人提供)：现代分子生物学方法提供了大量的细胞生物学机制，从极化细胞分裂到神经细胞迁移、自噬、神经元胞吐和无数其他机制。虽然许多参与这些过程的分子已经被阐明，但定义这些过程的更多机械性和结构性方面更加困难。重要的是，这些机制洞察力为我们理解疾病提供了细胞基础，并成为开发治疗方法的主要指南。发展机械洞察力的一个系统性问题是很难获得关于许多细胞复合体中成分的组织和化学计量的定量数据。例如，已有十几种蛋白质被证明定位于突触活动区的细胞线，这是突触前的一种结构性特征，对于大脑中神经元的囊泡性神经递质释放位置是必不可少的。此外，对突触活动区的蛋白质组学分析表明，与突触前释放部位相关的不同蛋白质数量超过100个。确定这种复杂的亚细胞集合体的结构和组织是一个艰巨的挑战，但如果我们想要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，我们建议开发一种方法，用于定量评估体内亚细胞组件的各种成分的组成。以前已经描述了执行这种定量测量的多种方法，但没有一种方法容易实施来分析复杂的多细胞生物体的结构。在此，我们建议创建体内内部校准标准，该标准可与荧光蛋白融合一起使用，以量化体内特定部位存在的特定蛋白的水平。具体地说，我们建议使用GFP-Laci与已知拷贝数整合到染色体上特定位置的LACO位点结合作为校准曲线，以定量在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，将该技术转移到线虫，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比。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。

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.