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

使用超分辨率显微镜和光学机械操作技术研究枯草芽孢杆菌中的 MreB 动力学和细胞壁合成

• 批准号: 262837402
• 负责人: Professor Dr. Alexander Rohrbach
• 金额: --
• 依托单位: Institut für Mikrosystemtechnik (IMTEK)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/262837402?language=en
• 关键词: Investigation; MreB; dynamics; cell; wall

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.

了解细菌细胞壁的结构和合成对于基础研究非常重要，而且对于新药特别是抗生素的开发也至关重要。由于这些原因，在过去几年中，几个研究小组将研究重点放在肌动蛋白样蛋白 MreB 上，它是细菌细胞骨架的一部分，在细胞壁合成中发挥着至关重要的作用。在与 Graumann 教授（马尔堡大学）实验室的合作中，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 丝表现出长度依赖性速度。此外，这些丝经历运输方向的频繁变化，并且似乎在细胞壁中肽聚糖链的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，该细菌将作为拟议研究的模型系统。在该项目中，将全内反射与结构照明相结合的现有超分辨率显微镜将通过附加功能进行扩展，并在采集速度方面显着提高。使用此装置，我们希望以近 100 nm 的光学分辨率观察 MreB 和其他细胞壁相关蛋白的动态。此外，我们希望实现第二个可见激光用于荧光激发。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，我们希望实现用于光学镊子的红外激光器和用于显微解剖的紫外激光器。根据我们最近提出的机械模型，MreB 组织了多个细胞壁链的合成。这需要在更复杂的条件下进行实验测试。这些结果将用于扩展我们的基于耦合分子马达的数学模型，旨在描述 MreB 动力学和细胞壁合成中所有观察到的现象。理论与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，可以用规定量的生化反应物处理一个样品的所有细菌。通过荧光显微镜观察 MreB 和其他相关蛋白质，其后果应该会变得显而易见。第二步，我们希望通过光学和机械力局部扰动单个细菌，从而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可以局部破坏细胞材料并使蛋白质失活的紫外激光来完成。因此，我们想要解决细胞壁合成和 MreB 动力学是否是局部自我调节的问题，或者更确切地说是由细胞全局控制的问题。