Institute of Condensed Matter and Nanosciences, Université catholique de Louvain, Croix du Sud 2/18, B-1348 Louvain-la-Neuve, Belgium.
ACS Nano. 2010 Sep 28;4(9):5498-504. doi: 10.1021/nn101598v.
Traditionally, the structural details of microbial cell walls are studied by thin-section electron microscopy, a technique that is very demanding and requires vacuum conditions, thus precluding live cell experiments. Here, we present a method integrating single-molecule atomic force microscopy (AFM) and protein design to measure cell wall thickness in a living yeast cell. The basic idea relies on the expression of His-tagged membrane sensors of increasing lengths in yeast and their subsequent specific detection at the cell surface using a modified AFM tip. After establishing the method on a wild-type strain, we demonstrate its potential by measuring changes in cell wall thickness within a few nanometers range, which result from (bio)chemical treatments or from mutations affecting the cell wall structure. The single molecular ruler method presented here not only avoids cell fixation artifacts but also provides new opportunities for studying the dynamics of microbial cell walls during growth, drug action, or enzymatic modification.
传统上，微生物细胞壁的结构细节是通过薄截面电子显微镜技术来研究的，该技术要求很高，需要真空条件，因此排除了活细胞实验。在这里，我们提出了一种将单分子原子力显微镜（AFM）和蛋白质设计结合起来的方法，用于测量活酵母细胞的细胞壁厚度。基本思路依赖于在酵母中表达带有 His 标签的越来越长的膜传感器，并使用改良的 AFM 尖端在细胞表面上对其进行特异性检测。在对野生型菌株进行方法建立后，我们通过测量由于（生物）化学处理或影响细胞壁结构的突变而导致的几纳米范围内的细胞壁厚度变化，证明了该方法的潜力。本文提出的单分子标尺方法不仅避免了细胞固定假象，而且为研究微生物细胞壁在生长、药物作用或酶修饰过程中的动力学提供了新的机会。
