Super-resolution Imaging of the Bacterial Division Machinery
Super-resolution Imaging of the Bacterial Division Machinery
复制标题
DOI：
10.3791/50048
复制
发表时间：
2013-01-01
影响因子：
1.2
通讯作者：
Xiao, Jie
中科院分区：
文献类型：
--
作者：
Buss, Jackson;Coltharp, Carla;Xiao, Jie
细菌细胞分裂需要在中间细胞协调组装十种以上的必需蛋白质（1，2）。该过程的核心是FtsZ蛋白在分裂平面上形成环状超结构（Z环）（3，4）。Z环由多个单链FtsZ原丝组成，理解Z环内原丝的排列将有助于深入了解Z环组装的机制及其作为力发生器的功能（5，6）。由于传统的荧光显微镜和电子显微镜目前的限制，这些信息仍然难以捉摸。由于光的衍射极限（类似于200 nm），传统的荧光显微镜无法提供Z环的高分辨率图像。电子冷冻断层成像在小C. crescentus细胞（7），但难以应用于较大的细胞，如大肠杆菌。coli或B.枯草芽孢杆菌在这里，我们描述了应用超分辨率荧光显微镜方法，光激活定位显微镜（PALM），定量表征的结构组织的E。PALM成像提供了高空间分辨率（类似于35 nm）和特异性标记，以实现靶蛋白的明确鉴定。我们用光活化荧光蛋白mEos 2标记FtsZ，其在405 nm处活化后从绿色荧光（激发= 488 nm）转换为红色荧光（激发= 561 nm）（9）。在PALM实验期间，单个FtsZ-mEos 2分子被随机激活，并且以< 20 nm的精度确定单个分子的相应质心位置。通过叠加所有检测到的FtsZ-mEos 2分子的质心位置，我们重建了Z环的超分辨率图像。使用这种方法，我们发现Z环具有近似100 nm的固定宽度，由一束松散的FtsZ原丝组成，它们在三维空间中相互重叠。这些数据为进一步研究Z环的细胞周期依赖性变化提供了跳板（10），并可应用于其他感兴趣的蛋白质。
Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell(1,2). Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan(3,4). The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator(5,6). This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (similar to 200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells(7), but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring(8).PALM imaging offers both high spatial resolution (similar to 35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm(9). During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with < 20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.Using this method, we found that the Z-ring has a fixed width of similar to 100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring(10) and can be applied to other proteins of interest.