Investigation of MreB dynamics and cell wall synthesis in B. subtilis using superresolution microscopy and optical-mechanical manipulation techniques

了解细菌细胞壁的结构和合成在基础研究中具有重要意义，对新药特别是抗生素的开发也至关重要。由于这些原因，在过去的几年里，几个小组集中研究了肌动蛋白样蛋白MreB，它是细菌细胞骨架的一部分，在细胞壁合成中起着至关重要的作用。在与格劳曼教授（马尔堡大学）的实验室合作中，使用快速超分辨率荧光显微镜（TIRF-SIM）以及生物物理分析，我们可以证明MreB细丝表现出长度依赖的速度。此外，这些细丝经历频繁的运输方向变化，似乎在细胞壁的肽聚糖链合成过程中充当机械耦合器。这些观察是在枯草芽孢杆菌类型的细菌中进行的，它将作为拟议调查的模型系统。在这个项目中，现有的全内反射和结构照明相结合的超分辨率显微镜需要增加额外的功能，并在采集速度上有明显的提高。使用这种装置，我们希望以接近100nm的光学分辨率观察MreB和其他细胞壁相关蛋白的动力学。此外，我们想要实现第二个可见激光荧光激发。这将使在共定位实验中观察细胞壁合成机制的MreB和MreB相互作用酶成为可能。此外，我们还想实现一个用于光学镊子的红外激光器和一个用于显微解剖的紫外激光器。根据我们最近提出的机制模型，MreB组织了几种细胞壁链的合成。这需要在更复杂的条件下进行实验测试。这些结果扩展了我们基于耦合分子马达的数学模型，旨在描述在MreB动力学和细胞壁合成中观察到的所有现象。理论与实验的比较将提高对细胞壁合成的定性和定量认识。我们还想研究细菌的全局和局部扰动如何影响细胞壁合成。对于全局操作，一个样品的所有细菌都可以用一定数量的生化反应物处理。通过荧光显微镜观察MreB和其他相关蛋白，结果应该是显而易见的。在第二步中，我们希望通过光学和机械力来局部扰动单个细菌，这可以导致蛋白质动力学的局部变化。这些操作应该通过光镊或使用紫外线激光来完成，紫外线激光可以局部破坏细胞物质并使蛋白质失活。因此，我们想要解决细胞壁合成和MreB动力学是局部自我调节还是由细胞全局控制的问题。

Understanding the structure and synthesis of the cell wall of bacteria is of great importance in fundamental research, but also crucial for the development of new drugs, especially antibiotics. For these reasons, during the last years several groups have focused their research on the actin-like protein MreB, which is part of the bacterial cytoskeleton and plays a crucial role in cell wall synthesis. In a collaboration with the lab of Prof. Graumann (University of Marburg) and using fast superresolution fluorescence microscopy (TIRF-SIM) as well as biophysical analysis we could show that MreB filaments exhibit a length-dependent velocity. Furthermore, these filaments undergo frequent changes of transport direction and seem to serve as a mechanical coupler during the synthesis of peptidoglycan strands in the cell wall. These observations were made in bacteria of the type Bacillus subtilis, which will serve as a model system for the proposed investigations.In this project an existing superresolution microscope that combines total internal reflection with structured illumination shall be extended by additional features and significantly improved in terms of acquisition speed. Using this set-up we want to observe the dynamics of MreB and other cell wall-related proteins with an optical resolution of almost 100nm. Furthermore, we want to implement a second visible laser for fluorescence excitation. This will enable the observation of MreB and MreB-interacting enzymes of the cell wall synthetic machinery in colocalization experiments. Additionally, we want to implement an IR-laser for optical tweezing and a UV-laser for micro-dissection.According to our recently presented, mechanistic model, MreB organzises the synthesis of several cell wall strands. This is to be tested experimentally under more complex conditions. These results shall be used to extend our mathematical model, which is based on coupled molecular motors, with the aim to describe all observed phenomena in MreB dynamics and cell wall synthesis. Comparisons between theory and experiment will improve the qualitative and quantitative understanding of cell wall synthesis.We also want to investigate how global and local perturbations of the bacteria influence cell wall synthesis. For a global manipulation all bacteria of one sample can be treated with a defined amount of biochemical reactant. The consequences should become apparent by observing MreB and other relevant proteins by fluorescence microscopy. In a second step, we want to perturb single bacteria locally by the means of optical and mechanical forces which can lead to local changes in protein dynamics. These manipulations should be done either by optical tweezers or by the use of a UV-laser that can locally destroy cell material and deactivate proteins. Hereby, we want to address the question if cell wall synthesis and MreB dynamics are locally self-regulated or rather globally controlled by the cell.