Collaborative Research: Single-molecule in vivo analysis of mechanosensitive channels in bacteria using force spectroscopy

该研究项目的目的是发现机械安全阀，即所谓的“机械敏感通道”，如何嵌入细菌坚韧的外壳中发挥作用，以及它们如何帮助保护细菌在环境突然变化时因内部压力过大而破裂。了解这种基本的细菌功能可能会影响人类健康和细菌性疾病的控制。多重耐药性是一个巨大的健康威胁。对细菌保护功能的详细了解可能会导致新的药理学方法来克服这些保护。此外，细菌在商业农业、环境修复和替代能源生产中发挥着至关重要的作用。在所有这些情况下，了解生长调节和对不断变化的环境条件的反应是至关重要的。这项合作研究计划的工作在于生物学，实验生物物理学和机械工程的交叉点，团队中的研究生和博士后研究人员将接受培训，在这个交叉点工作。杜克大学和加州大学洛杉矶分校的本科生也将参与这一研究项目。来自代表性不足的少数群体的本科生将参加加州大学洛杉矶分校的夏季研究经验，重点是计算建模。来自不同背景的初中和高中学生将参加杜克的课后和夏令营体验，向参与者介绍最先进的细胞成像技术。通过教育外展，这项工作将增加对STEM职业感兴趣的本科生群体并使其多样化，包括来自社区学院和少数族裔服务机构的本科生群体。 细菌被复杂的多层细胞膜包围，使它们能够保持一个或多个大气压的高内部膨压。当外部渗透压显著下降时，过度的膨压会导致细胞破裂。为了防止这种情况，嵌入内脂膜中的机械敏感通道（MSC）充当安全阀并释放溶质以减少膨压。目前尚不清楚活细胞中的MSC是否仅在细胞内膜的横向张力增加时才打开，或者它们是否也对通过其复杂的机械微环境传递的其他机械刺激做出反应。它也仍然是未知的生化调节如何影响力传递导致通道门控。该项目将利用一种新的方法来观察活细菌中单个MSC对机械压缩的反应，基本上是在原子力显微镜（AFM）中的平板之间。这种方法可以精确地量化膨压，同时解决单个MSC门控产生的小至0.01毫微微升的细胞体积变化。本项目将研究革兰氏阴性大肠杆菌。大肠杆菌和革兰氏阳性B.枯草杆菌中，并比较野生型菌株与仅表达特异性MSC的菌株的行为。实验将结合分析和数值粗粒度建模，以了解通过复杂的细胞壁结构（包括脂质膜，蛋白聚糖层和周质膜层）向MSC传递力，重点关注细胞壁缺陷的作用。这种新的方法来在体内表征间充质干细胞将有助于解决基本的困惑MSC功能在其原生的生理environment.This奖项反映了NSF的法定使命，并已被认为是值得的支持，通过评估使用基金会的智力价值和更广泛的影响审查标准。

The objective of this research project is to discover how the mechanical safety valves, so called “mechanosensitive channels”, embedded in the tough outer shells of bacteria function and how they help to protect bacteria against rupture due to excessive internal pressure during sudden changes in their environment. An understanding of this essential bacterial function could impact human health and the control of bacterial disease. Multiple drug resistance is an immense health threat. A detailed understanding of bacterial protective functions may lead to new pharmacological approaches to overcoming these protections. Furthermore, bacteria play crucial roles in commercial agriculture, environmental remediation, and alternative energy production. In all these situations, understanding of growth regulation and reactions to changing environmental conditions is critical. The work of this collaborative research program lies at the intersection of biology, experimental biophysics, and mechanical engineering, and graduate students and postdoctoral researchers in the team will be trained to work at this intersection. Undergraduate students at Duke and UCLA will also participate in this research project. Undergraduates from underrepresented minority groups will participate in summer research experiences at UCLA that focus on computational modeling. Middle and high school students from diverse backgrounds will participate in afterschool and camp experiences at Duke that introduce participants to state-of-the-art cell imaging technologies. Through the educational outreach, this work will increase and diversify the group of undergraduates interested in STEM-based careers, including those from community colleges and Minority Serving Institutions. Bacteria are enclosed by a complex multi-layered cell envelope that enables them to maintain a high internal turgor pressure of one or more atmospheres. When external osmolarity drops significantly, excessive turgor can cause cells to burst. To prevent this, mechano-sensitive channels (MSCs) embedded in the inner lipid membrane act as safety valves and release solutes to decrease turgor. It remains unknown if MSCs in the living cell open only when the lateral tension in the inner cell membrane increases, or if they also react to other mechanical stimuli transmitted through their complex mechanical microenvironments. It also remains unknown how biochemical regulation affects force transmission leading to channel gating. This project will utilize a new approach to observe the opening of single MSCs in live bacteria in response to mechanical compression, essentially between flat plates in an atomic force microscope (AFM). This method can precisely quantify turgor pressure and, at the same time, resolve cell volume changes as small as 0.01 femtoliters, produced by the gating of individual MSCs. The project will study gram-negative E. coli and gram-positive B. subtilis bacteria and compare the behavior of wildtype strains with strains expressing only specific MSCs. Experiments will be combined with analytical and numerical coarse-grained modeling to understand force transmission to MSCs through the complex cell wall structures, including the lipid membrane(s), the proteoglycan layer, and the periplasmic polyelectrolyte layer, with a focus on the role of cell wall defects. The new approach to in vivo characterization of MSCs will help to solve fundamental puzzles about MSC function in their native physiological environment.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.