Department of Biochemistry and Biomedical Engineering, University of Wisconsin, Madison, WI 53706, USA.
Proc Natl Acad Sci U S A. 2011 Apr 12;108(15):6264-9. doi: 10.1073/pnas.1015757108. Epub 2011 Mar 28.
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.
许多蛋白质位于杆状细菌的细胞两极。有几个假说将蛋白质定位与两极处的大细胞壁曲率联系起来。一个假说是围绕着脂质心磷脂 (CL) 的微域形成，其定位到高膜曲率区域，以及其与膜相关蛋白的相互作用。缺乏实验技术使得这个假说没有得到解答。本文描述了一种基于微技术的技术，用于操纵细菌膜曲率并定量测量其对 CL 和细胞内蛋白质定位的影响。我们将限定形状的大肠杆菌原生质体限制在微室中，微室的形状被压印到聚合物层中，并且观察到膜的形状可预测地变形以适应微室的壁。将这种技术与荧光显微镜和定量图像分析相结合，我们研究了 CL 微域在响应大肠杆菌膜曲率时的定位。CL 微域定位于微室施加的高固有负曲率区域。我们在原生质体中表达了黄色荧光蛋白与 MinD 的 N 端区域融合的嵌合体 - 大肠杆菌分裂平面组装的空间决定因素 - 并观察到其与 CL 共定位到大的负膜曲率区域。有趣的是，MinD 的分布在源自 CL 合酶敲除株的原生质体中相似。这些研究表明了 CL 在膜中的曲率依赖性，并测试了这些结构是否参与 MinD 到细胞中负曲率区域的定位。
