Assembly of multifunctional domains at bacterial cell poles

描述（由申请人提供）：这项工作的目标是了解控制蛋白质组装到有组织的细胞结构域的分子机制。实验将检查结构域在细菌细胞中的组装，但结构域的建立是发生在所有细胞类型中的普遍现象。考虑到这个项目的原理证明，同样的实验方法可以转移到真核系统，并用于人类生物学的新发现。这项工作将集中在杆状新月形茎杆菌的细胞极组装上，它在连续细胞质的两端产生两个功能不同的极。这些极点是复杂的结构域，具有多种功能，包括鞭毛组装、染色体定位和转录因子活性的调节。极性组装因子将在两种基因筛选中被发现：一种是直接识别已知极性蛋白正确定位所需的基因的视觉筛选，另一种是新的极性蛋白的生物信息学筛选。对于在筛选中识别的每个基因，组装过程中的上游和下游因素将使用遗传，微观和生化方法进行识别。生物信息学筛选已经产生了一种新的蛋白质，这种蛋白质似乎利用染色体复制作为细胞极点组装的线索。分析的一个关键方面将是使用蛋白质特异性标记技术进行冷冻电子显微镜断层扫描的蛋白质定位的精确测定。这将揭示结构域超微结构背景下蛋白质和功能复合物的排列，从而表征遗传和生化方法无法达到的组装方面。该项目将定义组装步骤的时间顺序，并显示单个组件如何放置在单元极的整体架构中，提供该领域构建的详细四维视图。蛋白质是作为单独的亚基制造的，必须找到一种方法与其他蛋白质结合在一起，形成具有生物功能的更大的复合物。为了让细胞生长和分裂，这种组装必须正确进行，而硬币的另一面是，我们可能会阻止病毒和细菌的这一过程，以抑制它们的生长。我正在研究细菌中的蛋白质组装，这可能会直接导致新抗生素的开发。这项工作还将对我们了解复杂的组装如何影响所有生命形式的细胞功能产生更广泛的影响。

DESCRIPTION (provided by applicant): The goal of this work is to understand the molecular mechanisms that govern the assembly of proteins into an organized cellular domain. The experiments will examine domain assembly in bacterial cells, but the establishment of domains is a general phenomenon that occurs in all cell types. Given this project as proof- of-principle, the same experimental approach could be transferred to eukaryotic systems and used to make new discoveries in human biology. The work will focus on cell pole assembly in the rod shaped Caulobacter crescentus, which creates two functionally distinct poles at opposite ends of a contiguous cytoplasm. These poles are complex domains that carry out multiple functions, including flagellar assembly, chromosome positioning, and the regulation of transcription factor activity. Polar assembly factors will be discovered in two genetic screens: one a visual screen to directly identify genes that are required for the proper localization of known polar proteins, the other a bioinformatic screen for new polar proteins. For every gene identified in the screens, upstream and downstream factors in the assembly process will be identified using genetic, microscopic, and biochemical methods. The bioinformatic screen has already yielded a novel protein that seems to use chromosome replication as a cue for assembly at the cell pole. A key aspect of the analysis will be a precise determination of protein localization using protein-specific labeling techniques for cryo-EM tomography. This will reveal the arrangement of proteins and functional complexes in the context of domain ultrastructure, thereby characterizing an aspect of assembly that is inaccessible by genetic and biochemical methods. The project will define a temporal order of assembly steps and show how individual components are placed in the overall architecture of the cell pole, providing a detailed four dimensional view of the construction of this domain. Proteins are made as individual subunits, and must find a way to fit together with other proteins to create larger complexes with biological function. Such assembly must occur properly in order for our cells to grow and divide, and the flip side of the coin is that we may block this process in viruses and bacteria to inhibit their growth. I am studying protein assembly in a bacterium, which could lead directly to the development of new antibiotics. The work will also have broader impact on our knowledge of how complex assembly contributes to cellular function in all life forms.