The authors acknowledge Howard Berg for the gift of the bead-tracking microscope/photomultipliers and the Texas A&amp;M Engineering Experiment Station for funds.

1.细胞的制备<ol><li>生长所需的应变在胰蛋白胨肉汤（TB，1％胨，0.5％氯化钠），随后接种于1等位基因15,22携带粘的fliC的过夜培养物:100稀释在10mL新鲜结核病。生长的培养物在33℃下在摇床培养箱直到OD 600 = 0.5。 </li><li>沉淀细胞在1500×g离心5 - 7分钟和重新分散沉淀大力在10mL过滤灭菌蠕动缓冲器（MB; 10mM磷酸盐缓冲液:0.05-0.06 M氯化钠，10 -4 M EDTA，1μM的蛋氨酸， pH为7.0）。 </li><li>重复步骤1.2两次并重新分散于1mL MB为最终的沉淀。 </li><li> （ - 19厘米长，0.58内径7）由两个注射器之间来回传递〜75倍，由聚乙烯管连接21至23号的适配器剪切停牌。限制的总时间剪切到30 - 45秒。 </li><li>离心剪切细胞以1500 xg离心5-7分钟，并再分散的颗粒在100 - 500μLMB的。 </li></ol> 2.将准备<ol><li>由盖防滑和显微镜载玻片之间夹在2张双面粘合带制备的成像室中。对于趋化性测定法，使用任何微流体室，使MB的和化学刺激物的交换。 </li><li>添加0.01％聚-L-赖氨酸溶液在腔室中，5分钟后轻轻冲洗以MB为表面（80 - 100微升）。 </li><li>添加细胞悬浮液的40微升到室中，并允许有足够的时间用于附着到玻璃表面（7 - 8分钟）。通过在腔室的一侧加入100微升MB的，而芯吸距离的另一侧用滤纸将溶液流出粘住细胞。 </li><li>添加10 - 的胶乳珠15微升到室中，并允许珠粒足够的时间来解决，并连接到细胞中（7 - 8分钟）。用100微升MB的轻轻冲洗，如在步骤2.3中所述，以除去未粘住珠。使用范围珠硅ZES的实验，只要一个良好的对比度是可用的。 </li></ol> 3.珠跟踪<ol><li>放置在显微镜载物台上的样本和扫描表面为连接至马达珠。使用40X阶段目标进行观察，虽然相差显微镜是没有必要的。另外，采用亮场成像，只要足够的对比度保持明确区分一个黑暗的背景上用亮珠。 </li><li>一旦珠已被选择，移动台横向以预定的角胎圈定位为如图1B所示。在相同的角位置珠，以确保胎圈的旋转方向正确知的。理想的珠轨迹近似圆形的椭圆形，但轨迹是受理。 </li><li>保持采样频率的两倍的马达，以避免与混叠相关联的错误的旋转频率高。在这项工作中，使用的是在旋转的电机在该均高10倍（500赫兹）的频率50 Hz和样品，以获得平滑的信号。 </li></ol> 4.数据分析<ol><li>中心，如果需要23缩放轨迹的PMT输出电压和正确的椭圆与仿射变换。使用功率谱分析，以确定旋转速率17。 </li><li>确定的极角，θ（T）= ATAN（Y（T）/ X（t））。确定变化的电动机速度和通过计算ω切换时间<img alt="式（1）" src="/files/ftp_upload/55240/55240eq1.jpg" /> 14。 </li><li>采用中值滤波器平滑电动机速度数据。在两整圈的过滤器窗口，推荐23,24。 </li></ol>

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The authors have nothing to disclose.

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（&lt;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?...

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。

<table><tbody><tr><td>Poly-L-lysine Solution (0.1%)</td><td>Sigma-Aldrich</td><td>P8920</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/p8920?lang=en&amp;amp;region=US</td></tr><tr><td>Polybead Microspheres</td><td>Polysciences, Inc.</td><td>7307</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/p8920?lang=en&amp;amp;region=US</td></tr><tr><td>1 mL Luer Slip Tip Syringe</td><td>Exel Int.</td><td>26048</td><td>http://www.exelint.com/tuberculin_syringes.php</td></tr><tr><td>Clay Adams Intramedic Luer-Stub Adapter 23-gauge</td><td>Becton, Dickinson and Company</td><td>427565</td><td>http://www.bd.com/ds/productCenter/ES-LuerStubAdaptors.asp</td></tr><tr><td>Polyethylene tubing</td><td>Harvard Apparatus</td><td>59-8325</td><td>http://www.harvardapparatus.com/laboratory-polye-polyethylene-non-sterile-tubing.html</td></tr><tr><td>Photomultiplier Tubes</td><td>Hamamatsu</td><td>R7400U-20</td><td>Spectral response range of 300 to 920 nm, Peak wavelength 630 nm,&amp;nbsp; 0.78 ns response time&amp;nbsp;&lt;br/&gt; http://pdf1.alldatasheet.com/datasheet-pdf/view/212308/HAMAMATSU/R7400U-20.html</td></tr><tr><td>3 x 1 mm precision slits</td><td>Edmund Optics</td><td>NT39-908</td><td>2 slits mounted at right angles to one another on photomultiplier tubes</td></tr><tr><td>Oscilloscope</td><td>Tektronix</td><td>TBS 1032B</td><td>Alternative brands are acceptable. Digital Oscilloscope, TBS 1000B Series, 2 Analogue, 30 MHz, 500 MSPS, 2.5 kpts&amp;nbsp;&lt;br/&gt; http://www.tek.com/oscilloscope/tbs1000b-digital-storage-oscilloscope</td></tr><tr><td>8 Pole LP/HP Filter</td><td>Krohn-Hite</td><td>3384</td><td>Alternative brands are acceptable. A frequency range from 0.1 Hz to 200 kHz is recommended.&amp;nbsp;&amp;nbsp;&amp;nbsp;&lt;br/&gt; http://www.krohn-hite.com/htm/filters/PDF/3384Data.pdf</td></tr><tr><td>Optiphot microscope</td><td>Nikon</td><td>NA</td><td>Any upright or inverted phase microscope can be used.&lt;br/&gt; https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=754</td></tr><tr><td>50:50 (R:T) Cube Beamsplitter</td><td>ThorLabs</td><td>BS013</td><td></td></tr></tbody></table>

biophysical characterization of flagellar motor functions

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.

的光电倍增管设置示于图1A。重要的是，所述光电倍增管具有在由所感兴趣的珠散落的波长范围内的高灵敏度。这里使用的光电倍增管在可见光和近红外范围内工作，并能够检测由被卤素光源照射珠散射光。最佳的照明条件和电源电压的变化会从一个设置到另一个。对于在这项工作中所使用的安装，一个PMT增益〜4月10日至5月10日被证明足够。?...

Watch this Scientific Journal Video about Biophysical Characterization of Flagellar Motor Functions at JoVE.com

Biophysical Characterization of Flagellar Motor Functions

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.

鞭毛马达功能的生物物理表征

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel ...

biophysical-characterization-of-flagellar-motor-functions

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.2K 次观看.  Texas A&M University. 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.

视频: Biophysical Characterization of Flagellar Motor Functions

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

科研

生物化学

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic properties are adapted for the particular function they execute in cells. Myosin 5a processively walks on F-actin to transport melanosomes and vesicles in cells. Conversely, nonmuscle myosin 2b operates as a bipolar filament containing approximately 30 molecules. It moves F-actin of opposite polarity toward the center of the filament, where the myosin molecules work asynchronously to bind actin, impart a power stroke, and dissociate before repeating the cycle. Nonmuscle myosin 2b, along with its other nonmuscle myosin 2 isoforms, has roles that include&#160;cell adhesion, cytokinesis, and tension maintenance. The mechanochemistry of myosins can be studied by performing in vitro motility assays using purified proteins. In the gliding actin filament assay, the myosins are bound to a microscope coverslip surface and translocate fluorescently labeled F-actin, which can be tracked. In the single molecule/ensemble motility assay, however, F-actin is bound to a coverslip and the movement of fluorescently labeled myosin molecules on the F-actin is observed. In this report, the purification of recombinant myosin 5a from Sf9 cells using affinity chromatography is outlined. Following this, we outline two fluorescence microscopy-based assays: the gliding actin filament assay and the inverted motility assay. From these assays, parameters such as actin translocation velocities and single molecule run lengths and velocities can be extracted using the image analysis software. These techniques can also be applied to study the movement of single filaments of the nonmuscle myosin 2 isoforms, discussed herein in the context of nonmuscle myosin 2b. This workflow represents a protocol and a set of quantitative tools that can be used to study the single molecule and ensemble dynamics of nonmuscle myosins.

Presented here is a procedure to express and purify myosin 5a followed by a discussion of its characterization, using both ensemble and single molecule in vitro fluorescence microscopy-based assays, and how these methods can be modified for the characterization of nonmuscle myosin 2b.

体外荧光显微镜基于动感检测的肌素特异性适应

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic ...

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.

从 酿酒酵母 细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of ...

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate (&#8220;map&#8221;) the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. &#8220;Cargo mapping&#8221; consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to &#8220;map&#8221; them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.

Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

表征分子马达的组成对运动轴突货物用"货物映射"分析

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative ...

神经科学

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Probing Myosin Ensemble Mechanics in Actin Filament Bundles Using Optical Tweezers

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle contraction. To understand their force-generating mechanisms, myosin II has been investigated both at the single-molecule (SM) level and as teams of motors in vitro using biophysical methods such as optical trapping.
These studies showed that myosin force-generating behavior can differ greatly when moving from the single-molecule level in a three-bead arrangement to groups of motors working together on a rigid bead or coverslip surface in a gliding arrangement. However, these assay constructions do not permit evaluating the group dynamics of myosin within viscoelastic structural hierarchy as they would within a cell. We have developed a method using optical tweezers to investigate the mechanics of force generation by myosin ensembles interacting with multiple actin filaments.
These actomyosin bundles facilitate investigation in a hierarchical and compliant environment that captures motor communication and ensemble force output. The customizable nature of the assay allows for altering experimental conditions to understand how modifications to the myosin ensemble, actin filament bundle, or the surrounding environment result in differing force outputs.

Formation of actomyosin bundles in vitro and measuring myosin ensemble force generation using optical tweezers is presented and discussed.

使用光学镊子探测肌动蛋白丝束中的肌球蛋白系综力学

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle ...

生物工程

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans constitute a strain-specific barcode. The associated glycans show higher diversity in sugar composition and structure than those of eukaryotes and are important in bacterial-host recognition processes and interaction with the environment. In pathogenic bacteria, glycoproteins have been involved in different stages of the infectious process, and glycan modifications can interfere with specific functions of glycoproteins. However, despite the advances made in the understanding of glycan composition, structure, and biosynthesis pathways, understanding of the role of glycoproteins in pathogenicity or interaction with the environment remains very limited. Furthermore, in some bacteria, the enzymes required for protein glycosylation are shared with other polysaccharide biosynthetic pathways, such as lipopolysaccharide and capsule biosynthetic pathways. The functional importance of glycosylation has been elucidated in several bacteria through mutation of specific genes thought to be involved in the glycosylation process and the study of its impact on the expression of the target glycoprotein and the modifying glycan. Mesophilic Aeromonas have a single and O-glycosylated polar flagellum. Flagellar glycans show diversity in carbohydrate composition and chain length between Aeromonas strains. However, all strains analyzed to date show a pseudaminic acid derivative as the linking sugar that modifies serine or threonine residues. The pseudaminic acid derivative is required for polar flagella assembly, and its loss has an impact on adhesion, biofilm formation, and colonization. The protocol detailed in this article describes how the construction of null mutants can be used to understand the involvement of genes or genome regions containing putative glycosyltransferases in the biosynthesis of a flagellar glycan. This includes the potential to understand the function of the glycosyltransferases involved and the role of the glycan. This will be achieved by comparing the glycan deficient mutant to the wild-type strain.

Here, we describe the construction of null mutants of Aeromonas in specific glycosyltransferases or regions containing glycosyltransferases, the motility assays, and flagella purification performed to establish the involvement and function of their encoded enzymes in the biosynthesis of a glycan, as well as the role of this glycan in bacterial pathogenesis.

产生空突变体以阐明细菌糖基转移酶在细菌运动中的作用

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans ...

免疫与感染

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, to elucidate the function and assembly of flagellar parts, and to examine and understand patterns of movement. Here we demonstrate a combination of three of these methodologies. Motility in soft agar is the oldest, offering a strong selection for isolating gain-of-function suppressor mutations in motility-impaired strains, where motility is restored through a second mutation. The cell-tethering technique, first employed to demonstrate the rotary nature of the flagellar motor, can be used to assess the impact of signaling effectors on the motor speed and its ability to switch rotational direction. The &#8220;border-crossing&#8221; assay is more recent, where swimming bacteria can be primed to transition into moving collectively as a swarm. In combination, these protocols represent a systematic and powerful approach to identifying components of the motility machinery, and to characterizing their role in different facets of swimming and swarming. They can be easily adapted to study motility in other bacterial species.

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Many bacteria use flagella-driven motility to navigate their environment and colonize favorable surroundings both individually and as a collective. Demonstrated here is the use of three established methods that exploit motility as a selection tool to identify components/pathways contributing to swimming and swarming motility.

通过在系列中应用三种既定技术，调查大 肠杆菌 的弗拉盖拉驱动的动性

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, ...

生物学

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of both conventional and unconventional myosins under load with unprecedented time resolution. In particular, the possibility to probe myosin motors under constant force right after the actin-myosin bond formation, together with the high rate of the force feedback (200 kHz), has shown UFFCS to be a valuable tool to study the load dependence of fast dynamics such as the myosin working stroke. Moreover, UFFCS enables the study of how processive and non-processive myosin-actin interactions are influenced by the intensity and direction of the applied force.
By following this protocol, it will be possible to perform ultrafast force-clamp experiments on processive myosin-5 motors and on a variety of unconventional myosins. By some adjustments, the protocol could also be easily extended to the study of other classes of processive motors such as kinesins and dyneins. The protocol includes all the necessary steps, from the setup of the experimental apparatus to sample preparation, calibration procedures, data acquisition and analysis.

Presented here is a comprehensive protocol to perform ultrafast force-clamp experiments on processive myosin-5 motors, which could be easily extended to the study of other classes of processive motors. The protocol details all the necessary steps, from the setup of the experimental apparatus to sample preparation, data acquisition and analysis.

超快力钳谱分析合成性肌球蛋白的机械酶学特性

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of ...

Visualizing Bacterial Motility Based on a Color Reaction

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or dissemination of many pathogenic species. Therefore, it is important to detect bacterial motility. Bacterial growth conditions, such as oxygen, pH, and temperature, can affect bacterial growth and the expression of bacterial flagella. This can lead to reduced motility or even loss of motility, resulting in the inaccurate evaluation of bacterial motility. Based on the color reaction of 2,3,5-triphenyl tetrazolium chloride (TTC) by intracellular dehydrogenases of living bacteria, TTC was added to traditional semisolid agar for bacterial motility detection. The results showed that this TTC semisolid agar method for the detection of bacterial motility is simple, easy to operate, and does not involve large and expensive instruments. The results also showed that the highest motility was observed in semisolid medium prepared with 0.3% agar. Compared with the traditional semisolid medium, the results are easier to evaluate and more accurate.

Here, we present a protocol to detect bacterial motility based on a color reaction. Key advantages of this method are that it is easy to evaluate and more accurate, and does not require specialized equipment.

基于颜色反应可视化细菌运动

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or ...

鞭毛马达功能的生物物理表征

摘要

探索更多视频

此视频中的章节

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

体外荧光显微镜基于动感检测的肌素特异性适应

从酿酒酵母细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

表征分子马达的组成对运动轴突货物用"货物映射"分析

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

使用光学镊子探测肌动蛋白丝束中的肌球蛋白系综力学

产生空突变体以阐明细菌糖基转移酶在细菌运动中的作用

通过在系列中应用三种既定技术，调查大肠杆菌的弗拉盖拉驱动的动性

超快力钳谱分析合成性肌球蛋白的机械酶学特性

基于颜色反应可视化细菌运动

鞭毛马达功能的生物物理表征

摘要

探索更多视频

此视频中的章节

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

体外荧光显微镜基于动感检测的肌素特异性适应

从 酿酒酵母 细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

表征分子马达的组成对运动轴突货物用"货物映射"分析

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

使用光学镊子探测肌动蛋白丝束中的肌球蛋白系综力学

产生空突变体以阐明细菌糖基转移酶在细菌运动中的作用

通过在系列中应用三种既定技术，调查大 肠杆菌 的弗拉盖拉驱动的动性

超快力钳谱分析合成性肌球蛋白的机械酶学特性

基于颜色反应可视化细菌运动

从酿酒酵母细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

通过在系列中应用三种既定技术，调查大肠杆菌的弗拉盖拉驱动的动性