鞭毛马达功能的生物物理表征
Summary
Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.
Abstract
The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.
Introduction
鞭毛马达使细胞通过旋转螺旋灯丝外游。转矩的电动机可产生用于鞭毛（ 即 ，粘性载荷）的给定长度的量决定了游泳速度。另一方面，其可切换旋转方向的能力控制响应于化学物质，被称为趋化处理细胞迁移。趋化和运动是毒力因子1-3，鞭毛马达已良好的特点，多年来4。越来越多的证据现在表明电动机充当mechanosensor -它机械地检测固体基材5,6的存在。这种能力可能有助于触发表面定植和感染5,7。其结果是，由此，电动机感应表面，并发起信令的机制意义8,9。
该鞭毛马达可通过圈养的flagell很容易研究微米到衬底上并观察细胞的旋转。这种束缚首先由西尔弗曼和Simon，谁在大肠杆菌 polyhook突变体，并成功地附着钩玻璃基板用抗钩抗体10的工作来实现。拴系细胞测定法使研究人员能够电动开关的响应研究的各种化学刺激。例如，西格尔和同事化学刺激的拴系细胞用离子电渗疗法移液器的助剂。在CW 偏置相应的变化（时间电机的部分按顺时针方向旋转，CW）使他们能够衡量网络的趋化性11,12适应的动力学。而拴系细胞测定是有效的学习开关响应，那也只是能提供分析上市马达力学上的粘性负载13的一个有限的范围内。为了克服这个问题，柳和同事拴球形，乳胶珠灯丝上粘到表面上的细胞存根。将珠然后使用后焦干涉与微弱光陷阱14跟踪。通过与不同大小的珠子时，研究人员可以研究电机在更宽的负载范围。此法后来被元和Berg，谁开发基于光电倍增管珠，跟踪技术与激光暗视场照明相结合的提高。他们的方法启用的系留黄金纳米珠说人话的小（〜60纳米），相比内部电阻粘稠旋转15,16外部电阻粘性较低的跟踪。这导致在大肠杆菌中可达到的最大速度（〜300赫兹）的测量结果。在溶藻弧菌 ，在中间的粘性负载（〜700赫兹）17启用类似珠检测纺纱率的测量。通过使电动机响应的测量结果在整个可能范围的粘性载荷（从零负载到近失速），珠的测定法提供了一个重要的生物物理工具来了解第torque生成过程18,19。
最近，我们修改了元伯格法包括使我们能够精确的机械刺激适用于单个电机6光学镊子。使用这种技术，我们发现，该旋转电机的力发电机是动态mechanosensors – 它们响应于粘性载荷变化重塑。这可能是这样的负荷感测触发细胞分化成蜂拥细菌，虽然机制尚不清楚。它也可能是在其他物种的鞭毛电机也机械敏感20，虽然直接的证据不足。在这里，我们讨论了跟踪拴鞭毛细丝15乳胶颗粒旋转基于光电倍增（PMT）的方法。相比于与超快摄像机跟踪，光电倍增管，设置是有利的，因为它是相对简单的实时和在长硬脑膜跟踪单个珠系统蒸发散。研究长期在鞭毛马达络合物重塑由于环境刺激21时，它是特别有用的。虽然专门为大肠杆菌我们详细介绍的协议，它们可容易地适用于其它物种研究鞭毛电机。
Protocol
Representative Results
Discussion
为了便于拴珠跟踪和电机扭矩的正确估计，下面的信息进行审查。当与有鞭毛的细胞执行这些测量，剪切是一个关键步骤。剪切降低了鞭毛丝到仅仅存根，从而确保电机的粘性载荷主要是由于胎圈和可在10％的误差16内进行估计。剪切还改善寻找具有紧密分布的偏心（<珠粒径14）的圆形轨迹的机会。不当的剪切导致任性轨迹，这在跟踪和粘阻力的计算，以及导致差信号噪声比化?…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors acknowledge Howard Berg for the gift of the bead-tracking microscope/photomultipliers and the Texas A&M Engineering Experiment Station for funds.
Materials
| Poly-L-lysine Solution (0.1%) | Sigma-Aldrich | P8920 | http://www.sigmaaldrich.com/catalog/product/sigma/p8920?lang=en®ion=US |
| Polybead Microspheres | Polysciences, Inc. | 7307 | http://www.sigmaaldrich.com/catalog/product/sigma/p8920?lang=en®ion=US |
| 1 ml Luer Slip Tip Syringe | Exel Int. | 26048 | http://www.exelint.com/tuberculin_syringes.php |
| Clay Adams Intramedic Luer-Stub Adapter 23-gauge | Becton, Dickinson and Company | 427565 | http://www.bd.com/ds/productCenter/ES-LuerStubAdaptors.asp |
| Polyethylene tubing | Harvard Apparatus | 59-8325 | http://www.harvardapparatus.com/laboratory-polye-polyethylene-non-sterile-tubing.html |
| Photomultiplier Tubes | Hamamatsu | R7400U-20 | Spectral response range of 300 to 920 nm, Peak wavelength 630 nm, 0.78 ns response time http://pdf1.alldatasheet.com/datasheet-pdf/view/212308/HAMAMATSU/R7400U-20.html |
| 3×1 mm precision slits | Edmund Optics | NT39-908 | 2 slits mounted at right angles to one another on photomultiplier tubes |
| Oscilloscope | Tektronix | TBS 1032B | Alternative brands are acceptable. Digital Oscilloscope, TBS 1000B Series, 2 Analogue, 30 MHz, 500 MSPS, 2.5 kpts http://www.tek.com/oscilloscope/tbs1000b-digital-storage-oscilloscope |
| 8 Pole LP/HP Filter | Krohn-Hite | 3384 | Alternative brands are acceptable. A frequency range from 0.1 Hz to 200 kHz is recommended. http://www.krohn-hite.com/htm/filters/PDF/3384Data.pdf |
| Optiphot microscope | Nikon | NA | Any upright or inverted phase microscope can be used. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=754 |
| 50:50 (R:T) Cube Beamsplitter | ThorLabs | BS013 |
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