Subcellular architecture of regulatory protein complexes at the bacterial pole

描述(申请人提供)：细菌极地调节蛋白质复合体的亚细胞结构最近在单个荧光分子显微成像方面的进展导致了超分辨率信息的产生，提供了在可见光下观察分辨率超过标准光学衍射极限~250 nm的物体的能力。在… 与此同时，细菌组织的复杂性也变得越来越明显，鉴于人体中含有的原核细胞比真核细胞多，了解我们的微生物伙伴对于科学和预防病理都是至关重要的。蛋白细菌中的大部分组织存在于细胞极，不仅是鞭毛的锚，也是染色体起源、趋化装置以及协调细胞周期进程的关键调控和信号子系统的锚。虽然有关于细胞极点的大致信息，但许多谜团仍然存在，需要关于极地蛋白质的身份和精确相对位置的高分辨率信息。 了解并最终影响细菌生物学。这项申请提出了一条新的研究路线，以了解调节蛋白的亚细胞组织在Caulbacter细胞极点以前所未有的分辨率。这项工作需要生化遗传学与先进的超三维(3D)超分辨荧光成像的紧密结合，以充分量化细菌细胞极点上关键蛋白质的位置和空间相互作用，精确度在x，y和z方向上为~20-30 nm。新月弯杆菌是一个强大的细胞分化模型，凭借其不对称的细胞分裂周期，其中一种PI是专家。另一位PI是专家的新成像方法依赖于两个组件：(A)双螺旋点扩散函数(DH-PSF)显微镜的双色细胞3D成像方法，它允许在大景深上进行精确的3D成像，以及(B)单分子主动控制显微镜，它通过顺序成像和定位单个发射器的稀疏子集来提供超分辨率细节。这项计划有三个重点：目标1：发展先进的双色三维成像技术-目标1：发展先进的双色三维成像技术目的2：对基准蛋白质组件进行超分辨率3D成像，以确定极点的坐标系。将通过对TipN、MCPA、Cres和POPZ这四个关键的极地标记执行精确的3D成像来定义极地参考坐标系。目标3：确定细菌细胞极点关键调控蛋白组装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 细菌细胞杆。活细胞中的动态信息将从细胞周期的不同时间从成像中提取，从而提供一个前所未有的结构以及控制细菌细胞组织和功能的动力学视图。

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.