Super-Resolution Imaging of the Nucleoid-Associated Protein HU in Caulobacter crescentus
Super-Resolution Imaging of the Nucleoid-Associated Protein HU in Caulobacter crescentus
复制标题
DOI：
10.1016/j.bpj.2011.02.022
复制
发表时间：
2011-04-06
影响因子：
3.4
通讯作者：
Moerner, W. E.
中科院分区：
文献类型：
--
作者：
Lee, Steven F.;Thompson, Michael A.;Moerner, W. E.
细菌中大多数核相关蛋白（nap）的结构和功能尚不清楚。其中一个原因是，在标准的宽视场和共聚焦荧光成像中，蛋白质的分布和结构被衍射极限所模糊。特别是，作为NAP最丰富的HU的分布很少受到关注。在这项研究中，我们利用超分辨率荧光成像和空间点统计相结合的方法研究了HU在新月形茎杆菌中的分布。通过简单地增加激光功率，可以使荧光蛋白融合HU2-eYFP的单分子闪烁，从而在单一激发源下实现超分辨率成像。通过Ripley’s k检验的量化和与蒙特卡罗模拟的比较，我们发现蛋白质在细胞周期的群集和跟踪阶段略微聚集在一个基本均匀的分布中，但在分裂前细胞中聚集得更多。这封信中提出的方法应该在未来的原核nap研究中具有广泛的适用性。
Little is known about the structure and function of most nucleoid-associated proteins (NAPs) in bacteria. One reason for this is that the distribution and structure of the proteins is obfuscated by the diffraction limit in standard wide-field and confocal fluorescence imaging. In particular, the distribution of HU, which is the most abundant NAP, has received little attention. In this study, we investigate the distribution of HU in Caulobacter crescentus using a combination of super-resolution fluorescence imaging and spatial point statistics. By simply increasing the laser power, single molecules of the fluorescent protein fusion HU2-eYFP can be made to blink on and off to achieve super-resolution imaging with a single excitation source. Through quantification by Ripley's K-test and comparison with Monte Carlo simulations, we find the protein is slightly clustered within a mostly uniform distribution throughout the swarmer and stalked stages of the cell cycle but more highly clustered in pre-divisional cells. The methods presented in this letter should be of broad applicability in the future study of prokaryotic NAPs.