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

这项研究项目的目的是发现嵌入细菌坚硬外壳中的机械安全阀，即所谓的“机械敏感通道”是如何发挥作用的，以及它们如何帮助保护细菌，使其在环境突然变化时不会因过高的内部压力而破裂。对这一基本细菌功能的了解可能会影响人类健康和细菌疾病的控制。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克服这些保护的新的药理学方法。此外，细菌在商业农业、环境修复和替代能源生产中发挥着关键作用。在所有这些情况下，了解生长规律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位于生物学、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在这个交叉点工作的培训。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。来自少数族裔群体的本科生将参加加州大学洛杉矶分校的暑期研究体验，重点是计算建模。来自不同背景的初中生和高中生将参加杜克大学的课外和夏令营体验，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。细菌被一个复杂的多层细胞膜包裹着，使它们能够在一个或多个大气压下保持较高的内部膨胀压力。当体外渗透压显著下降时，过度膨胀会导致细胞破裂。为了防止这种情况，嵌入内脂膜中的机械敏感通道(MSCs)起到安全阀的作用，释放溶质以减少膨胀。目前尚不清楚活细胞中的MSCs是仅在细胞内膜的侧向张力增加时才开放，还是也对通过其复杂的机械微环境传递的其他机械刺激产生反应。生化调节如何影响导致通道门控的力传递也尚不清楚。该项目将利用一种新的方法来观察活细菌中单个间充质干细胞在机械压缩下的开口，基本上是在原子力显微镜(AFM)的平板之间。这种方法可以精确地量化充气压力，同时解决单个MSCs门控产生的小至0.01毫升的细胞体积变化。该项目将研究革兰氏阴性大肠杆菌和革兰氏阳性枯草杆菌，并比较野生型菌株和仅表达特定MSCs的菌株的行为。实验将与解析和数值粗粒度建模相结合，以了解通过复杂的细胞壁结构向MSCs传递的力，包括脂膜(S)、蛋白聚糖层和周质聚电解质层，重点是细胞壁缺陷的作用。MSCs体内特性的新方法将有助于解决有关MSC在其原始生理环境中的功能的根本难题。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.