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，对新药尤其是抗生素的开发也是至关重要的。出于这些原因，在过去的几年里，几个小组将他们的研究重点放在了肌动蛋白样蛋白MreB上，它是细菌细胞骨架的一部分，在细胞壁合成中发挥着关键作用。在与马尔堡大学Graumann教授的合作下，使用快速超分辨荧光显微镜(TIRF-SIM)以及生物物理分析，我们可以证明MreB细丝表现出与长度有关的速度。此外，这些细丝的运输方向经常发生变化，在细胞壁合成肽链的过程中似乎起到了机械偶联的作用。这些观察是在枯草芽孢杆菌类型的细菌中进行的，这将作为拟议研究的模型系统。在这个项目中，现有的将全内反射与结构照明相结合的超分辨率显微镜将增加额外的功能，并在采集速度方面显著提高。利用这个装置，我们想要观察MreB和其他与细胞壁相关的蛋白质的动力学，光学分辨率几乎为100 nm。此外，我们还希望实现用于荧光激发的第二个可见光激光器。这将使我们能够在共定位实验中观察到MreB和MreB相互作用的细胞壁合成机器的酶。此外，我们希望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光激光器。根据我们最近提出的机制模型，MreB组织了几个细胞壁链的合成。这将在更复杂的条件下进行实验测试。这些结果将被用来扩展我们的数学模型，该模型基于耦合分子马达，目的是描述在MreB动力学和细胞壁合成中观察到的所有现象。理论和实验之间的比较将提高对细胞壁合成的定性和定量理解。我们还想调查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，一个样本的所有细菌都可以用一定数量的生化反应物进行处理。通过荧光显微镜观察MreB和其他相关蛋白质，结果应该会变得明显。在第二步，我们希望通过光学和机械力的手段来局部扰乱单个细菌，这可能会导致蛋白质动力学的局部变化。这些操作应该通过光学镊子或使用紫外线激光来完成，紫外线激光可以局部破坏细胞材料并使蛋白质失活。因此，我们想解决的问题是，细胞壁合成和MreB动力学是局部自我调节的，还是更确切地说是由细胞全局控制的。

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.