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. 셀 준비 <ol><li> 10 mL의 신선한 TB 100 희석 : 1에 접종 한 다음 트립 톤 국물 (TB, 1 % 펩톤, 0.5 % 염화나트륨)에 대립 유전자 15, 22 끈적 끈적한 플릭를 들고 원하는 균주의 야간 문화를 성장. OD 600 = 0.5까지 진탕 배양기에서 33 ° C에서 문화를 성장. </li><li> 1,500 XG에서 세포 펠렛 5-7 분, 재 분산 펠렛 적극적으로 필터 살균 운동 버퍼의 10 ㎖의 (MB를 10 mM의 인산 완충액 : 0.05-0.06 M 염화나트륨, 10-4 M EDTA, 1 μm의 메티오닌, pH가 7.0). </li><li> 단계를 반복 1.2 두 번 더 1 ML의 MB의 마지막 펠릿을 재 분산. </li><li> (- 12cm 길이, 0.58 mm 내경 7) 폴리에틸렌 튜브로 연결된 21 ~ 23 게이지 어댑터와 함께 두 주사기 사이에 앞뒤로 ~ 75 번 전달하여 정지 전단. 45 초 - (30) 전단의 총 시간을 제한합니다. </li><li> 5-7 분 동안 1,500 XG에서 전단 된 세포를 원심 분리하여 다시 분산MB의 500 μL - 100 펠렛. </li></ol> 2. 슬라이드 준비 <ol><li> 커버 슬립 현미경 슬라이드 사이에 두 개의 양면 접착 테이프를 개재하여 촬상 챔버를 준비한다. 화성 분석을 위해, MB, 화학적 자극의 교환을 가능하게하는 미세 유체 챔버를 사용합니다. </li><li> 챔버에서 0.01 % 폴리 -L- 라이신 솔루션을 추가하고 5 분 후 부드럽게 MB와 표면 세척 (80-100 μL)를. </li><li> 챔버 내로 세포 현탁액 40 μL의 추가, 유리 표면에 부착을위한 충분한 시간이 허용 (7-8 분). 다른 측면에서 여과지로 용액을 심지하면서 상기 챔버의 일측에 100 μL를 첨가하여 MB 떨어진 세포를 흐른다. </li><li> 챔버로 라텍스 구슬 15 μL와 비즈를 정착 세포에 부착 할 충분한 시간 수 - 10 추가 (7-8 분). 구슬 떨어진 제거하는 단계 2.3에 설명 된대로 조심스럽게, MB의 100 μL 씻어. 비드 실리콘의 범위를 사용하여좋은 대비를 사용할 정도로 오랫동안 실험 ZES. </li></ol> 3. 비드 추적 <ol><li> 현미경 스테이지에 샘플을 놓고 모터에 연결된 구슬 위해 표면을 검사합니다. 위상 현미경이 필요하지 않지만 관측을 만들기 위해 40X 위상 목적을 사용합니다. 또한, 너무 오래 충분한 대비가 명확하게 어두운 배경에 밝은 구슬을 구분하기 위해 유지되는 밝은 - 필드 이미징을 사용한다. </li><li> 비드가 선택되면,도 1b에 도시 된 바와 같이 소정의 모서리에 비드를 위치 시키도록 측 방향으로 상기 스테이지를 이동. 같은 코너에 위치 구슬은 구슬의 회전 방향이 제대로 알려져 있는지 확인합니다. 이상적인 비드 궤도는 대략 원형이지만, 타원형 궤도가 허용됩니다. </li><li> 에일리어싱과 연관된 에러를 방지하기 위해 모터의 회전 주파수의 두 배 이상의 샘플링 주파수를 높게 유지. 본 연구에서는으로 회전 된 모터를 사용10 시간 이상 (500 Hz에서)했다 주파수 50 Hz의 시료는 평활 한 신호를 획득한다. </li></ol> 4. 데이터 분석 <ol><li> 23 필요한 경우 센터는 아핀 변환과 궤적의 PMT 출력 전압과 정확한 타원의 크기를 조절합니다. 회전 속도 (17)를 결정하기 위해 전력 스펙트럼 분석을 사용한다. </li><li> 극 각도 θ (t) = ATAN (Y (t) / X (t))를 결정합니다. 모터 속도의 변화를 확인하고 ω를 계산하여 시간이 지남에 따라 전환 <img alt="식 (1)" src="/files/ftp_upload/55240/55240eq1.jpg" /> 14. </li><li> 모터 속도 데이터를 부드럽게 중앙값 필터를 사용한다. 이 전체 회전을 통해 필터 창 (23, 24)를 권장합니다. </li></ol>

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The authors have nothing to disclose.

테더 비드 추적 모터 토크의 정확한 추정을 용이하게하기 위해, 다음과 같은 정보가 검토되어야한다. flagellated 세포 이러한 측정을 수행 할 때, 전단은 중요한 단계입니다. 전단시켜 상기 모터의 점성 부하가 비드에 주로 기인하고 10 %의 오차 (16)에서 추정 될 수 있도록, 단순한 스터브로 편모 필라멘트를 감소시킨다. 양털 깎기도 밀접하게 분산 기행 (&lt;비드 직경 14)와 원형 궤적?...

편모 모터는 나선형 외 필라멘트를 회전하여 수영을 셀 수 있습니다. 모터가 편모 (즉, 점성 부하)의 소정 기간 동안 생성 할 수있는 토크의 양이 수영 속도를 결정한다. 한편, 회전 방향을 전환 할 수있는 능력은 화학 약품, 화학 주성으로 공지 된 과정에 응답하여 세포 이동을 제어한다. 화성과 운동성되는 독성은 1-3, 편모 모터는 년 4로 잘 특성화 된 요인. 증거 확보 해주기 모터가 mechanosensor 역할을 제안 - 그것은 기계적 고체 기판 -5,6-의 존재를 검출한다. 이 기능은 가능성이 표면 식민지 및 감염 5,7 트리거링에 도움이됩니다. 결과적으로, 모터의 표면과 동수 신호를 감지하여 그에 메커니즘의 중요성 8,9이다. 편모 모터는 쉽게 flagell를 테 더링으로 공부하실 수 있습니다UM 기판에 세포 회전을 관찰. 이러한 테 더링이 먼저 안티 - 후크 항체 (10)와 유리 기판에 polyhook의 대장균에서 돌연변이 성공적으로 연결된 후크와 협력 실버 사이먼에 의해 달성되었다. 속박 세포 분석은 화학 자극의 다양한 모터 스위치의 반응을 연구 연구 할 수 있었다. 예를 들어, Segall와 동료들은 화학적으로 이온 영동 피펫의 도움으로 닿는 세포를 자극. CW 바이어스에 해당 변경 화성 네트워크 (11, 12)에서 적응의 반응 속도를 측정하기 위해 그들을 사용 (시간 모터의 비율은 CW, 시계 방향으로 회전). 닿는 세포 분석은 스위치 응답을 공부에 효과적이지만, 단지 점성로드 (13)의 제한된 범위에서 모터 역학에 대한 통찰력을 제공 할 수 있었다. 이 문제를 극복하기 위해 류와 동료들은 표면에 붙어있는 세포 스텁 필라멘트 구면 라텍스 비즈 닿는. 비드이었다다음 약한 광 트랩 (14) 백 초점 간섭을 사용하여 추적. 서로 다른 크기의 구슬과 협력함으로써, 연구진은 부하의 훨씬 넓은 범위에 걸쳐 모터를 연구 할 수있다. 이 분석법 후에 레이저 암시 야 조명과 결합 된 광전자 계 비드 추적 기술을 개발 위안 베르그 향상했다. 외부 점성 저항이 회전 (15, 16)에 내부 점성 저항에 비해 낮았다 너무 작았 닿는 골드 나노 비드 (~ 60 ㎚)의 그들의 방법을 사용 추적. 이 대장균에서 달성 가능한 최대 속도 (~ 300 Hz에서)의 측정을했다. V.의 alginolyticus에서 유사한 비드 분석은 중간 점성 부하 (~ 700 Hz에서) (17)의 회전 속도를 측정 할 수 있었다. (제로로드에서 가까운 매점에) 점성 부하의 전체 가능한 범위에서 모터 반응의 측정을 가능하게함으로써, 비드 분석은 t을 이해하는 중요한 생물 리 학적 도구를 제공orque 생성 프로세스 (18, 19). 최근에, 우리는 각각의 모터 6 정밀 기계적인 자극을 적용 할 수있게 광 핀셋을 포함하도록 위안 - 버그 분석을 수정했습니다. 그들은 점성 부하의 변화에 ​​응답하여 개조 -이 기술을 이용하여, 모터 회전 힘 발전기 동적 mechanosensors이다 것으로 나타났다. 이 메커니즘 불분명 남아 있지만 이러한 하중 감지가 진을 치고 박테리아로 세포 분화를 유발하는 것이 가능하다. 직접적인 증거는 부족하지만 다른 종의 편모 모터는,도 20 mechanosensitive 것으로도 보인다. 여기, 우리는 편모 필라멘트 (15)에 닿는 라텍스 구슬의 회전을 추적하기위한 광전자 기반 (PMT) 접근 방식에 대해 설명합니다. 이 실시간으로 긴 경질 위에 단일 비드를 추적 비교적 간단하기 때문에 초고속 카메라 추적 비교에서, 광전자 - 설치 유리한TIONS. 때문에 환경 자극 (21)에 편모 모터 단지에 리모델링 긴 시간을 공부 할 때 특히 유용합니다. 구체적으로는 E. coli에 대한 우리 상세히 프로토콜이지만, 그들은 용이하게 다른 종 편모 모터 공부하도록 할 수있다.

<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.

광전자 증 배관 설치는 그림 1A에 표시됩니다. 이는 관심의 PMT가 비드에 의해 산란의 파장 범위에서 높은 감도를 갖는 것이 중요하다. 여기에서 사용되는 PMT가 가시 근적외선 범위에서 동작하고, 할로겐 광원에 의해 조명 비즈 산란광을 검출 할 수 있었다. 최적의 조명 조건 및 공급 전압을 하나의 설정마다 다를 것이다. 이 작업에 사용되는 설정하는 PMT 이득 ~...

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.

편모 모터 기능의 생물 물리학 적 특성

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

Research

JoVE Journal

Biology

8.2K 조회수.  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.

비디오: 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.

단일 분자 관찰을 위한 DNA 종이 접기 나노 구조에 다인 및 키네신 모터 앙상블 생산

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

연구

생화학

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.

체외 형광 현미경 검사법 기반 운동성 검사의 Myosin 특이적 적응

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.

S. cerevisiae 세포로부터 정제된 양방향 Kinesin-5 Cin8의 단일 분자 및 클러스터의 이동성

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.

사용 축삭화물을 이동하는 분자 모터의 구성을 특성화 &quot;화물 매핑&quot;분석

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

신경과학

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 정제 초공정 키네신-3 제품군 모터의 단일 분자 분석

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.

광학 핀셋을 사용하여 액틴 필라멘트 번들에서 미오신 앙상블 역학 조사

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 ...

생체공학

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.

박테리아 운동성에서 박테리아 글리코실트랜스퍼라제의 역할을 밝히기 위한 Null 돌연변이체의 생성

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

면역학 및 감염병학

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.

에슈리치아 대장균에서 모토렐라 구동 운동성 조사

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

생물학

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.

초고속 포스 클램프 분광법을 사용한 공정 미오신의 기계적 효소 특성 해부

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.

색상 반응을 기반으로 한 박테리아 운동성 시각화

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