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: 8401468
• 负责人: William E Moerner
• 金额: $ 51.43万
• 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/8401468
• 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. PUBLIC HEALTH RELEVANCE: By combining new methods for three-dimensional high resolution optical imaging in living cells with expertise in bacterial cell biology, this research ill define the structural organization of the bacterial cell pole, a site of critical regulatory functin, in unprecedented detail. Such precise information about how bacteria actually work will bear directly upon biotechnological and biomedical problems where microbes are either essential symbionts or pathogens. The ability to specifically and noninvasively measure positions of key proteins and their superstructures at high resolution in live cells without requiring ionizing radiation or low temperatures will have strong implications for biomedical imaging and analysis of eukaryotic cells whose cellular structures and behavior are altered in the progress of disease.

描述（由申请人提供）：细菌极点在微观成像中使用单个荧光分子的最新进展的调节蛋白复合物的亚细胞体系结构导致了超分辨率信息，从而可以在可见的可见度中观察到具有超出标准光学衍射极限的对象的能力。在 同时，细菌组织的复杂性变得越来越明显，并且鉴于人体所包含的原始细胞比真核细胞更多，因此必须了解我们的微生物伴侣，以进行科学利益和预防病理学。组织中的大部分组织发生在细胞极中，不仅是鞭毛的锚点，而且还出现在染色体起源，趋化设备以及关键调节和信号传导子系统，以协调细胞周期的进展。虽然可获得有关细胞极的近似信息，但仍然存在许多谜团，并且需要有关极性蛋白质的身份和确切相对位置的高分辨率信息 了解并最终影响细菌生物学。 该应用程序提出了一项新的研究系列，以了解以空前的分辨率在Caulobacter细胞极处的调节蛋白的亚细胞组织。这样的努力要求将生化遗传学与高级三维（3D）超分辨率荧光成像超出光学衍射极限之外，以充分量化细菌细胞极点处的关键蛋白的位置和空间相互作用，以便在x，y和z中的精度为〜20-30 nm。 Caulobacter Crescentus是通过其不对称的细胞分裂周期的细胞分化的强大模型，其中一个PI是专家。 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. 三个推力定义了以下程序：目标1：使用DH-PSF显微镜开发高级两色，3D成像：将精确延伸至X，Y和Z中〜20nm的极性蛋白的相对位置的方法进行开发和验证。 AIM 2：基准蛋白组件的超分辨率3D成像，以定义极点的坐标系。极性参考坐标系将通过执行TIPN，MCPA，CRES和POPZ的精确3D成像（关键极性标记）来定义。 AIM 3：定义细菌细胞极点关键调节蛋白组件的3D结构组织和动力学。通过将一系列突变菌株与两色3D超分辨率成像相结合，我们将在该空间组织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从细胞周期的不同时间中的成像中提取，从而提供了对结构的前所未有的视图以及控制细菌细胞组织和功能的动力学。 公共卫生相关性：通过结合具有细菌细胞生物学专业知识的活细胞中三维高分辨率光学成像的新方法，这项研究不正确地定义了细菌细胞极的结构性组织，这是一个固定的细节。关于细菌如何实际发挥作用的精确信息将直接依靠生物技术和生物医学问题，在这些问题中，微生物是必不可少的共生体或病原体。在无需电离辐射或低温的无需电离辐射或低温的无需电离辐射或低温的情况下，主要和非侵入性测量关键蛋白及其上层建筑的位置将对生物医学成像和真核细胞的分析具有很大的影响，而真核细胞的细胞结构和行为在疾病的进展中会改变。