Cardiolipin microdomains localize to negatively curved regions of Escherichia coli membranes
Cardiolipin microdomains localize to negatively curved regions of Escherichia coli membranes
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DOI：
10.1073/pnas.1015757108
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发表时间：
2011-04-12
影响因子：
11.1
通讯作者：
Weibel, Douglas B.
中科院分区：
文献类型：
--
作者：
Renner, Lars D.;Weibel, Douglas B.
许多蛋白质存在于杆状细菌的细胞两极。有几种假说将蛋白质定位与两极的大细胞壁曲率联系起来。一种假说集中在脂质心磷脂（CL）的微区的形成，其定位于高膜曲率的区域，以及其与膜相关蛋白的相互作用。由于缺乏实验技术，这一假设没有得到回答。本文介绍了一种基于显微技术的技术，用于操纵细菌膜曲率和定量测量其对CL和蛋白质在细胞中的定位的影响。我们将大肠杆菌原生质球限制在具有确定形状的微室中，这些微室被压印到聚合物层中，并观察到膜的形状可预测地变形以容纳微室的壁。结合这种技术与落射荧光显微镜和定量图像分析，我们表征了响应于E.大肠杆菌膜弯曲。CL微区定位于由微室施加的高固有负曲率的区域。我们表达了一个黄色荧光蛋白的嵌合体融合到MinD的N-末端区域，MinD是大肠杆菌的空间决定簇。大肠杆菌分裂平面组装在原生质球，并观察其与CL的大，负膜曲率的区域共定位。有趣的是，MinD的分布是相似的，在原生质球来自CL合酶敲除菌株。这些研究证明了CL在膜中的曲率依赖性，并测试这些结构是否参与MinD在细胞中负曲率区域的定位。
Many proteins reside at the cell poles in rod-shaped bacteria. Several hypotheses have drawn a connection between protein localization and the large cell-wall curvature at the poles. One hypothesis has centered on the formation of microdomains of the lipid cardiolipin (CL), its localization to regions of high membrane curvature, and its interaction with membrane-associated proteins. A lack of experimental techniques has left this hypothesis unanswered. This paper describes a microtechnology-based technique for manipulating bacterial membrane curvature and quantitatively measuring its effect on the localization of CL and proteins in cells. We confined Escherichia coli spheroplasts in microchambers with defined shapes that were embossed into a layer of polymer and observed that the shape of the membrane deformed predictably to accommodate the walls of the microchambers. Combining this technique with epifluorescence microscopy and quantitative image analyses, we characterized the localization of CL microdomains in response to E. coli membrane curvature. CL microdomains localized to regions of high intrinsic negative curvature imposed by microchambers. We expressed a chimera of yellow fluorescent protein fused to the N-terminal region of MinD-a spatial determinant of E. coli division plane assembly-in spheroplasts and observed its colocalization with CL to regions of large, negative membrane curvature. Interestingly, the distribution of MinD was similar in spheroplasts derived from a CL synthase knockout strain. These studies demonstrate the curvature dependence of CL in membranes and test whether these structures participate in the localization of MinD to regions of negative curvature in cells.