Subcellular architecture of regulatory protein complexes at the bacterial pole

细菌极调节蛋白复合物的亚细胞结构

• 批准号: 8515456
• 负责人: William E Moerner
• 金额: $ 49.51万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8515456
• 关键词: Advanced Development; Architecture; Bacteria; Behavior; Benchmarking; Binding; Biochemical Genetics; Biology; Blinking; Caulobacter; Caulobacter crescentus; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell model; Cell surface; Cells; Cellular Structures; Cellular biology; Centromere; Chemotaxis; Chimeric Proteins; Chromosomes; Color; Cytoskeletal Proteins; Disease; Electron Microscopy; Elements; Engineering; Eukaryotic Cell; Flagella; Genetic; Grant; HU Protein; Human body; Image; Image Analysis; Individual; Ionizing radiation; Label; Life; Light; Location; Maps; Masks; Measures; Methodology; Methods; Microbe; Microscope; Microscopic; Microscopy; Optics; Pathology; Pattern; Peptide Hydrolases; Phase; Positioning Attribute; Prevention; Process; Prokaryotic Cells; Proteins; Proteobacteria; Relative (related person); Research; Resolution; SHPS-1 protein; Side; Signal Transduction; Signaling Protein; Site; Speed; Structure; Surface; System; Three-Dimensional Imaging; Time; Ursidae Family; Work; active control; base; bioimaging; cell type; cold temperature; density; design; design and construction; fluorescence imaging; fluorophore; genetic regulatory protein; interest; microbial; mutant; novel; optical imaging; pathogen; photoactivation; programs; protein complex; response; segregation; single molecule; small molecule; spatial relationship; system architecture; two-dimensional

DESCRIPTION (provided by applicant): Subcellular Architecture of Regulatory Protein Complexes at the Bacterial Pole Recent advances in microscopic imaging with single fluorescent molecules have led to super-resolution information providing the ability to observe objects with resolution beyond the standard optical diffraction limit of ~250 nm in the visible. At the same time, the complexity of bacterial organization has become more and more apparent, and given that the human body contains more prokaryotic cells than eukaryotic cells, it is essential to understand our microbial partners, for scientific benefit and for prevention of pathology. Much of the organization in ?-proteobacteria occurs in the cell pole, the anchor not only for the flagellum, but also for the chromosomal origin, the chemotactic apparatus and for critical regulatory and signaling subsystems that coordinate cell cycle progression. While approximate information is available about the cell pole, many mysteries remain, and high resolution information on the identity and precise relative locations of polar proteins is required to understand and ultimately influence bacterial biology. This application proposes a new line of research to understand the subcellular organization of regulatory proteins at the Caulobacter cell pole at unprecedented resolution. Such an effort requires the close integration of biochemical genetics with advanced three-dimensional (3D) super-resolution fluorescence imaging beyond the optical diffraction limit, in order to fully quantify the locations and spatial interactions of key proteins at the bacterial cell pole down to a precision of ~20-30 nm in x, y, and z. Caulobacter crescentus is a powerful model of cellular differentiation by virtue of its asymmetric cell division cycle, of which one of the PIs is expert. The new imaging methodology in which the other PI is expert relies on two components: (a) a two- color method for 3D imaging in cells with the double-helix point spread function (DH-PSF) microscope, which allows precise 3D imaging over a large depth of field, and (b) single-molecule active control microscopy, which provides super-resolution detail by sequentially imaging and localizing sparse subsets of individual emitters. Three thrusts define this program: Aim 1: Development of advanced two-color, 3D imaging with the DH- PSF microscope: Methods for localizing relative locations of pairs of polar proteins with precision extending down to ~20nm in x, y, and z will be developed and validated. Aim 2: Super-resolution 3D imaging of benchmark protein assemblies to define the coordinate system of the pole. The polar reference coordinate system will be defined by performing precise 3D imaging of TipN, McpA, CreS, and PopZ, key polar markers. Aim 3: Define 3D structural organization and dynamics of key regulatory protein assemblies at the bacterial cell pole. By combining an array of mutant strains with two-color 3D super-resolution imaging, we will establish the spatial organization of multiple pairs of regulatory proteins at the bacterial cell pole. Dynamical information in live cells will be extracted from imaging at differen times of the cell cycle, thus providing an unprecedented view of the structure as well as the dynamics controlling bacterial cell organization and function.

描述（由申请人提供）：细菌极点处调节蛋白复合物的亚细胞结构使用单个荧光分子的显微成像的最新进展已经导致超分辨率信息，提供了观察物体的能力，其分辨率超过可见光中约250 nm的标准光学衍射极限。在 与此同时，细菌组织的复杂性已变得越来越明显，鉴于人体含有的原核细胞比真核细胞多，为了科学利益和预防病理，了解我们的微生物伙伴至关重要。大部分组织在？变形菌出现在细胞极，不仅是鞭毛的锚，而且是染色体起源的锚，是趋化性装置和协调细胞周期进程的关键调节和信号子系统的锚。虽然关于细胞极的近似信息是可用的，但许多谜团仍然存在，并且需要关于极性蛋白质的身份和精确相对位置的高分辨率信息 来理解并最终影响细菌生物学。 该申请提出了一种新的研究路线，以前所未有的分辨率了解柄杆菌细胞极调节蛋白的亚细胞组织。这样的努力需要将生化遗传学与超越光学衍射极限的先进三维（3D）超分辨率荧光成像紧密结合，以便完全量化细菌细胞极处关键蛋白质的位置和空间相互作用，精确到x，y和z的约20-30 nm。新月柄杆菌由于其不对称的细胞分裂周期而成为细胞分化的强大模型，其中一个PI是专家。新的成像方法，其中另一个PI是专家依赖于两个组件：（a）一个双色方法的细胞中的三维成像与双螺旋点扩散函数（DH-PSF）显微镜，它允许精确的三维成像在一个大的景深，和（B）单分子主动控制显微镜，它提供了超分辨率的细节，通过顺序成像和定位稀疏子集的个别发射器。 目标1：开发使用DH-PSF显微镜的高级双色3D成像：将开发和验证用于定位极性蛋白质对的相对位置的方法，其精度在x，y和z方向上延伸至约20 nm。目标2：对基准蛋白质组装体进行超分辨率三维成像，以确定极点的坐标系。将通过对TipN、McpA、克雷斯和PopZ（关键极标）进行精确3D成像来定义极参考坐标系。目的3：确定细菌细胞极关键调控蛋白组装体的三维结构组织和动力学。通过将一系列突变菌株与双色3D超分辨率成像相结合，我们将在基因组中建立多对调节蛋白的空间组织。 细菌细胞极活细胞中的动态信息将在细胞周期的第三个时间从成像中提取，从而提供对结构以及控制细菌细胞组织和功能的动态的前所未有的视图。