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. Preparação de células <ol><li> Crescer culturas de uma noite da estirpe desejada carregando a fliC pegajoso alelo 15,22 em Caldo de triptona (TB, 1% de peptona, 0,5% de NaCl), seguida de inoculação a diluição 1: 100 em 10 mL de TB fresco. Crescer a cultura a 33 ° C numa incubadora agitadora até DO600 = 0,5. </li><li> Agregar as células a 1500 xg durante 5-7 minutos e re-dispersar vigorosamente o sedimento em 10 mL de tampão de filtro esterilizado motilidade (MB; 10 mM de tampão de fosfato: 0,05-0,06 M de NaCl, 10 -4 M de EDTA, 1 | iM de metionina, pH 7,0). </li><li> Repita o passo 1.2 mais duas vezes e re-dispersar o sedimento final em 1 ml MB. </li><li> Cisalhamento da suspensão, passando para trás e para a frente ~ 75 vezes entre duas seringas com 21 a 23 adaptadores calibre ligados por tubos de polietileno (7 - 12 cm de comprimento, diâmetro 0,58 mm interno). Limitar o tempo total para corte de 30 - 45 s. </li><li> Centrifugar as células cortadas a 1.500 g durante 5-7 minutos e voltar a dispersar apelete em 100 - 500 mL de MB. </li></ol> 2. Deslize Preparação <ol><li> Preparar uma câmara de imagem imprensando duas fitas adesivas de dupla face entre um cover-derrapante e uma lâmina de microscópio. Para ensaios de quimiotaxia, empregar qualquer câmara de microfluidos que permite a troca de MB e estimulantes químicos. </li><li> Adicionar solução de 0,01% de poli-L-lisina na câmara e depois de 5 min lave as superfícies com MB (80-100 mL). </li><li> Adicionar 40 ul de suspensão de células para dentro da câmara e permitir tempo suficiente para a ligação à superfície do vidro (7-8 min). Fluxo de Saída células unstuck pela adição de 100 uL MB de um lado da câmara, enquanto wicking a solução com um papel de filtro a partir do outro lado. </li><li> Adicionam-se 10 - 15? L de esferas de látex para dentro da câmara e permitir que as esferas de tempo suficiente para se estabelecer e anexar as células (7-8 min). Lave com 100 ul de MB, tal como descrito na etapa 2.3, para remover descolar grânulos. Use uma gama de talão-sizes para as experiências tanto tempo como um bom contraste está disponível. </li></ol> 3. Acompanhamento Bead <ol><li> Colocar a amostra em um estágio do microscópio e digitalizar a superfície para grânulos ligados a motores. Utilize uma objectiva de 40x fase para fazer observações, embora microscopia de fase não é necessário. Alternativamente, empregam imagem de campo brilhante, desde que contraste suficiente é mantida a distinguir claramente um grânulo brilhante sobre um fundo escuro. </li><li> Uma vez que um grânulo tenha sido seleccionado, mover a fase lateralmente para posicionar o grânulo em um canto pré-determinado, como mostrado na Figura 1B. grânulos posição na mesma curva para garantir que o sentido de rotação do grânulo é correctamente conhecido. A trajetória talão ideal é aproximadamente circular, mas trajetórias elípticas são admissíveis. </li><li> Manter a frequência de amostragem mais elevada do que duas vezes a frequência de rotação do motor para evitar erros associados com serrilhado. Neste trabalho, utilizar um motor que foi rotativo a50 Hz e a amostra a frequências que eram 10 vezes mais elevada (500 Hz), para obter um sinal bom. </li></ol> Análise 4. Dados <ol><li> Centro e escalar as tensões de saída PMT e elipticidade correta nas trajetórias com transformações afins, se necessário 23. Use uma análise de poder de espectro para determinar as taxas de rotação 17. </li><li> Determinar ângulos polares, θ (t) = atan (y (t) / x (t)). Determinar as variações de velocidades do motor e mudar ao longo do tempo por meio do cálculo ω <img alt="equação 1" src="/files/ftp_upload/55240/55240eq1.jpg" /> 14. </li><li> Empregar um filtro de mediana para alisar os dados de velocidade do motor. Uma janela de filtro de mais de duas rotações completas é recomendado 23,24. </li></ol>

<ol>
	<li>Emody, L., Kerenyi, M., Nagy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+factors+of+uropathogenic+Echerichia+coli.">Virulence factors of uropathogenic Echerichia coli.</a> Int J Antimicrob. Ag. 22, 29-33 (2003).</li><li>Lane, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+motility+in+the+colonization+of+uropathogenic+Escherichia+coli+in+the+urinary+tract.">Role of motility in the colonization of uropathogenic Escherichia coli in the urinary tract.</a> Infect Immun. 73 (11), 7644-7656 (2005).</li><li>Kao, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complex+interplay+among+bacterial+motility+and+virulence+factors+in+different+Escherichia+coli+infections.">The complex interplay among bacterial motility and virulence factors in different Escherichia coli infections.</a> Eur J Clin Microbiol Infect Dis. 33 (12), 2157-2162 (2014).</li><li>Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rotary+motor+of+bacterial+flagella.">The rotary motor of bacterial flagella.</a> Annu Rev Biochem. 72, 19-54 (2003).</li><li>McCarter, L., Hilmen, M., Silverman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flagellar+Dynamometer+Controls+Swarmer+Cell+Differentiation+of+V.+parahaemolyticus.">Flagellar Dynamometer Controls Swarmer Cell Differentiation of V. parahaemolyticus.</a> Cell. 54 (3), 345-351 (1988).</li><li>Lele, P. P., Hosu, B. G., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+mechanosensing+in+the+bacterial+flagellar+motor.">Dynamics of mechanosensing in the bacterial flagellar motor.</a> Proc Natl Acad Sci U S A. 110 (29), 11839-11844 (2013).</li><li>Gode-Potratz, C. J., Kustusch, R. J., Breheny, P. J., Weiss, D. S., McCarter, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+sensing+in+Vibrio+parahaemolyticus+triggers+a+programme+of+gene+expression+that+promotes+colonization+and+virulence.">Surface sensing in Vibrio parahaemolyticus triggers a programme of gene expression that promotes colonization and virulence.</a> Mol Microbiol. 79 (1), 240-263 (2011).</li><li>Kearns, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+field+guide+to+bacterial+swarming+motility.">A field guide to bacterial swarming motility.</a> Nat Rev Microbiol. 8 (9), 634-644 (2010).</li><li>Belas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms,+flagella,+and+mechanosensing+of+surfaces+by+bacteria.">Biofilms, flagella, and mechanosensing of surfaces by bacteria.</a> Trends Microbiol. 22 (9), 517-527 (2014).</li><li>Silverman, M., Simon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flagellar+rotation+and+the+mechanism+of+bacterial+motility.">Flagellar rotation and the mechanism of bacterial motility.</a> Nature. 249, 73-74 (1974).</li><li>Block, S. M., Segall, J. E., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+Kinetics+in+Bacterial+Chemotaxis.">Adaptation Kinetics in Bacterial Chemotaxis.</a> J Bacteriol. 154 (1), 312-323 (1983).</li><li>Segall, J. E., Block, S. M., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+comparisons+in+bacterial+chemotaxis.">Temporal comparisons in bacterial chemotaxis.</a> Proc Natl Acad Sci U S A. 83, 8987-8991 (1986).</li><li>Blair, D. F., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+torque+in+defective+flagellar+motors.">Restoration of torque in defective flagellar motors.</a> Science. 242 (4886), 1678-1681 (1988).</li><li>Ryu, W. S., Berry, R. M., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Torque-generating+units+of+the+flagellar+motor+of+Escherchia+coli+have+a+high+duty+ratio.">Torque-generating units of the flagellar motor of Escherchia coli have a high duty ratio.</a> Nature. 403, 444-447 (2000).</li><li>Yuan, J., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resurrection+of+the+flagellar+rotary+motor+near+zero+load.">Resurrection of the flagellar rotary motor near zero load.</a> Proc Natl Acad Sci U S A. 105 (4), 1182-1185 (2008).</li><li>Yuan, J., Fahrner, K. A., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switching+of+the+bacterial+flagellar+motor+near+zero+load.">Switching of the bacterial flagellar motor near zero load.</a> J Mol Biol. 390 (3), 394-400 (2009).</li><li>Sowa, Y., Hotta, H., Homma, M., Ishijima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Torque-speed+Relationship+of+the+Na+-driven+Flagellar+Motor+of+Vibrio+alginolyticus.">Torque-speed Relationship of the Na+-driven Flagellar Motor of Vibrio alginolyticus.</a> J Mol Biol. 327 (5), 1043-1051 (2003).</li><li>Xing, J., Bai, F., Berry, R., Oster, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Torque-speed+relationship+of+the+bacterial+flagellar+motor.">Torque-speed relationship of the bacterial flagellar motor.</a> Proc Natl Acad Sci U S A. 103 (5), 1260-1265 (2006).</li><li>Meacci, G., Tu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+bacterial+flagellar+motor+with+multiple+stators.">Dynamics of the bacterial flagellar motor with multiple stators.</a> Proc Natl Acad Sci U S A. 106 (10), 3746-3751 (2009).</li><li>Lele, P. P., Roland, T., Shrivastava, A., Chen, Y. H., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+flagellar+motor+of+Caulobacter+crescentus+generates+more+torque+when+a+cell+swims+backwards.">The flagellar motor of Caulobacter crescentus generates more torque when a cell swims backwards.</a> Nat Phys. 12 (2), 175-178 (2016).</li><li>Lele, P. P., Shrivastava, A., Roland, T., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+thresholds+in+bacterial+chemotaxis.">Response thresholds in bacterial chemotaxis.</a> Sci Adv. 1 (9), e1500299 (2015).</li><li>Berg, H. C., Turner, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Torque+Generated+by+the+Flagellar+Motor+of+Escherichia+coli.">Torque Generated by the Flagellar Motor of Escherichia coli.</a> Biophys J. 65, 2201-2216 (1993).</li><li>Bai, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+Spread+as+a+Mechanism+for+Cooperativity+in+the+Bacterial+Flagellar+Switch.">Conformational Spread as a Mechanism for Cooperativity in the Bacterial Flagellar Switch.</a> Science. 327, 685-689 (2010).</li><li>Reid, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+maximum+number+of+torque-generating+units+in+the+flagellar+motor+of+Escherichia+coli+is+at+least+11.">The maximum number of torque-generating units in the flagellar motor of Escherichia coli is at least 11.</a> Proc Natl Acad Sci U S A. 103, 8066-8071 (2006).</li><li>Chen, X., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Torque-Speed+Relationship+of+the+Flagellar+Rotary+Motor+of+Escherichia+coli.">Torque-Speed Relationship of the Flagellar Rotary Motor of Escherichia coli.</a> Biophys J. 78, 1036-1041 (2000).</li><li>Turner, L., Caplan, S. R., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-induced+switching+of+the+bacterial+flagellar+motor.">Temperature-induced switching of the bacterial flagellar motor.</a> Biophys J. 71, 2227-2233 (1996).</li></ol>

The authors have nothing to disclose.

A fim de facilitar tethered talão de rastreamento e estimativa correcta do motor-torques, as seguintes informações devem ser revistos. Ao realizar essas medições com células flageladas, corte é um passo crítico. Cisalhamento reduz o filamento flagelar a um mero topo, assegurando desse modo que a carga no motor viscoso é predominantemente devido ao cordão e pode ser estimado dentro de erro de 10% 16. Shearing também melhora as chances de encontrar trajetórias circulares com excentricidades bem distribuídos (&lt;diâmetro talão 14). resultados de corte indevidos em trajetórias rebeldes, compostos que erros no rastreamento e no cálculo das drags viscosos, bem como resultando em relação sinal-ruído pobres. O uso de um osciloscópio permite uma rápida eliminação de tais dados. Desde são esperadas as propriedades biomecânicas de filamentos de flagelar a variar com as espécies, métodos de tosquia, provavelmente, precisam ser adaptados para garantir corte adequada em tele bactérias de interesse. Uma forma eficaz de reduzir os erros associados com cisalhamento é trabalhar com as células que não possuem os genes que codificam as proteínas de filamento. grânulos sonda pode então ser ligado directamente ao gancho por meio de anticorpos anti-gancho. Encontrar um talão que é apropriadamente tethered pode ser um desafio. Isto é porque a maioria dos grânulos no campo de visão ou será preso aos corpos celulares ou a superfície de vidro. Essas contas podem ser facilmente postos em destaque. Outras esferas aparece para vibrar ou girar visivelmente com grandes amplitudes ou grandes excentricidades (&gt; 1.5 - diâmetro 2x talão). Estes são tipicamente presa a flagelar filamentos que não foram totalmente cortados ou rodam num plano que é inclinado em relação ao plano focal. Amostragem de tais grânulos, normalmente resultam em maior ruído, e o tempo variações nas cargas viscosos podem resultar em uma subestimação de motores binários para uma dada pérola-tamanho. Uma pequena fração dos grânulos amarrados vai sofrer aleatóriomovimento; Estas são apenas submetidos a rotação browniano. Uma fracção dos grânulos aparecerá desfocada e não pode ser colocado em foco facilmente. Estas são mais susceptíveis de serem motores que foram amarradas de forma adequada e que se encontram os motores de interesse. Entre as limitações de um único motor de rastreamento, como a descrita aqui, é a incapacidade de realizar as experiências de alto rendimento. Uma câmara de alta velocidade com que as imagens de uma região maior de interesse pode ser vantajoso a este respeito. Outras limitações incluem os erros associados com sinais múltiplos resultantes de grânulos de rotação espaçados no campo de visão dos PMTs. Finalmente, os erros na determinação da posição correcta do grânulo gravado com respeito aos dois fotomultiplicadores fendas irá resultar em estimativas imprecisas da dinâmica de comutação. Vantagens da configuração aqui descrita incluem a capacidade para controlar a rotação dos grânulos durante longos períodos e em tempo real. estepermite a rápida eliminação das trajetórias propensas a erros, algo que talvez difícil de conseguir com câmeras ultra-rápidos. Além disso, com algumas modificações, esta configuração pode ser integrado com ensaios concebidos para submeter as células a uma variedade de estímulos. Combinado com termoeléctrica de arrefecimento 26, a técnica pode ser utilizada para medir as respostas de motores individuais aos estímulos térmicos. Integração com pinças ópticas pode permitir que as medições de remodelação de motores individuais em resposta a estímulos mecânicos, como tem sido feito recentemente 6. Finalmente, a adaptação do motor de estimulantes químicos pode ser medida com a utilização de uma câmara de perfusão adequada e as bombas 11. A maioria das espécies bacterianas conhecidas são móveis e motilidade mediada por flagelar é predominante na natureza. Os métodos demonstrados aqui são esperados para continuar a ajudar no desenvolvimento de insights sobre estrutural-remodelação e a adaptabilidade of o motor flagelar.

motores flagelares permitir que as células para nadar girando filamentos extracelulares helicoidais. A quantidade de torque do motor pode gerar para um dado comprimento do flagelo (isto é, a carga viscosa) determina as velocidades de natação. Por outro lado, a sua capacidade de mudar o sentido de rotação controla a migração celular em resposta a produtos químicos, um processo conhecido como quimiotaxia. Quimiotaxia e motilidade sendo fatores de virulência 1-3, motores flagelares foram bem caracterizado ao longo dos anos 4. Evidências crescentes agora sugere que o motor actua como um mechanosensor - lo mecanicamente detecta a presença de substratos sólidos 5,6. Esta capacidade provável ajuda no desencadeamento de colonização superfície e infecções 5,7. Como resultado, os mecanismos pelos quais o motor sentidos superfícies e iniciados de sinalização são de importância 8,9. O motor flagelar pode ser facilmente estudado por amarrar o flagellhum a um substrato e observando a rotação celular. Esta operação foi primeiramente alcançada por Silverman e Simon, que trabalhou com um mutante polyhook em E. coli e ganchos ligados com sucesso para substratos de vidro com anticorpos anti-gancho 10. O ensaio de célula tethered permitiu aos investigadores a estudar as respostas de o interruptor do motor a uma variedade de estímulos químicos. Por exemplo, Segall e co-trabalhadores quimicamente estimulado células conectada com o auxílio de pipetas iontoforéticos. As alterações correspondentes no viés CW (a fração dos motores tempo de rotação no sentido horário, CW) lhes permitiu medir a cinética de adaptação na rede quimiotaxia 11,12. Enquanto o ensaio de células tethered foi eficaz em estudar respostas de switch, ele só foi capaz de oferecer insights sobre a mecânica do motor através de uma gama limitada de cargas viscosos 13. Para ultrapassar este problema, Ryu e colaboradores tethered esféricas, esferas de látex de filamento topos sobre as células presas às superfícies. Os grânulos eramentão rastreados usando interferometria de back-focal com fracos armadilhas ópticas 14. Ao trabalhar com contas de diferentes tamanhos, os pesquisadores poderiam estudar o motor ao longo de uma gama muito maior de cargas. Este ensaio foi posteriormente melhorado por Yuan e Berg, que desenvolveu uma técnica de talão-de rastreamento baseado photomultiplier combinada com iluminação a laser de campo escuro. Seu método de rastreamento habilitado de nanobeads tethered de ouro que foram tão pequena (~ 60 nm) que as resistências viscosos externos foram menores em comparação com as resistências viscosos internos para rotação 15,16. Isto conduziu às medições das velocidades máxima alcançável em E. coli (~ 300 Hz). Em V. alginolyticus, ensaios talão semelhantes permitiu medições das taxas de fiação em cargas intermédias viscosos (~ 700 Hz) 17. Ao permitir que as medições de respostas motoras ao longo de toda a gama possível de cargas viscoso (a partir de zero a carga de quase-box), os ensaios de Bead-forneceu uma ferramenta importante para compreender a biofísica tprocesso orque geração 18,19. Recentemente, nós modificamos o ensaio Yuan-Berg para incluir pinças ópticas que nos permitiram aplicar estímulos mecânicos precisos para motores individuais 6. Usando esta técnica, nós mostramos que a força-geradores que giram o motor são mechanosensors dinâmicos - eles remodelar em resposta às mudanças nas cargas viscosos. É possível que tal sensor de carga provoca a diferenciação de células em bactérias enxame, embora os mecanismos permanecem obscuros. Também é provável que os motores flagelares em outras espécies também são mechanosensitive 20, apesar de evidência directa que falta. Aqui, discutimos a abordagem baseada em fotomultiplicador (PMT) para acompanhar a rotação de esferas de látex amarrados a filamentos de flagelar 15. Em comparação com o acompanhamento com câmeras ultra-rápidos, o fotomultiplicador-setup é vantajoso porque é relativamente simples para rastrear esferas individuais em tempo real e mais longa durações. É particularmente útil quando se estuda a longo tempo remodelação em complexos motor flagelar devido a estímulos do meio ambiente 21. Embora nós detalhe protocolos específicos para E. coli, que pode ser facilmente adaptado para estudar motores flagelares em outras espécies.

<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.

A configuração do fotomultiplicador é mostrado na Figura 1A. É importante que os PMTs ter alta sensibilidade ao longo do intervalo de comprimentos de onda dispersa pelas esferas de interesse. Os PMTs empregadas aqui operam nas faixas visíveis e do infravermelho próximo, e foram capazes de detectar a luz dispersa pelas esferas iluminadas por uma fonte de luz halógena. As condições de iluminação ideais e tensões de alimentação irá variar de uma instalação para outra. Para a configuração utilizada neste trabalho, um ganho de PMT ~ 10 abril - 10 maio mostrou-se adequada. Cada fotomultiplicador foi coberto com excepção de uma fenda 3 x 1mm posicionado em frente do fotomultiplicador. As fendas limitar a região na célula-amostra a partir da qual a luz pode entrar nos fotomultiplicadores, e as duas fendas são ortogonais entre si. Quando um cordão de rotação está posicionado na localização correcta (Figura 1B), a quantidade de luz que entra no fotomultiplicadores aumenta à medida que o cordão vema vista e diminui como o seu percurso circular leva-o longe da vista. As frequências das saídas de tensão sinusoidais PMT indicar a velocidade de rotação e as diferenças de fase entre os dois sinais de indicar o sentido de rotação. O uso de um osciloscópio para exibir as saídas PMT permite a visualização de trajectórias grânulo em tempo real. Os sinais PMT variáveis no tempo, Y (t) e x (t), a partir de um motor representativo são apresentados na Figura 2A. A ortogonalidade das duas fendas introduz um atraso de fase entre os dois sinais. As amplitudes do sinal dependerá da relação sinal-ruído, bem como a excentricidade de rotação. As trajectórias correspondentes do talão são indicados na Figura 2B. Um histograma das velocidades medidas a partir de um motor representativo de uma Chey - estirpe removido é mostrado na Figura 3A </strong&gt;. A eliminação garante que os motores flagelares não pode mudar e gire exclusivamente anti-horário (CCW). A velocidade média de este motor particular, foi de 60 Hz e as velocidades medidas são consistentes com relatos anteriores para motores amarrados com 1 mm grânulos 25. O grânulo foi posicionada em primeiro lugar no canto inferior direito, como se vê no esquema na Figura 1B. A velocidade angular correspondente é mostrada na Figura 3B (painel superior). Posicionando o talão para o canto esquerdo inferior adjacente resultou na inversão do sinal nas velocidades do motor (painel inferior). Deste modo, movendo-se o grânulo para uma zona adjacente irá alterar o sentido de rotação do motor observado. A este respeito, os cantos diagonalmente opostos são idênticos. É, portanto, crucial para saber a localização do cordão durante as medições para determinar corretamente a dinâmica de comutação. A Figura 3C mostra transições repetidas de um motor de tipo selvagem entre os dois sentidos de rotação. <html> Códigos personalizados para software de aquisição de dados foram adaptados de trabalho antes de gravar os dados em um computador 15. A saída PMT foi AC-coupled e low-pass filtrada com uma frequência de corte de 100 Hz. rastreamento em tempo real foi habilitado por ligar as saídas filtradas para um osciloscópio. <img alt="figura 1" src="/files/ftp_upload/55240/55240fig1.jpg" /> Figura 1: Configuração Bead-tracker. A) esquemático da configuração do controlo baseados em PMT. B) A posição ideal do grânulo (esfera negra) em relação às duas ranhuras ortogonais. A trajectória está indicado pelas linhas pontilhadas. A excentricidade é o raio do círculo pontilhado. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55240/55240fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> ove_content &quot;fo: manter-together.within-page =&quot; 1 &quot;&gt; <img alt="Figura 2" src="/files/ftp_upload/55240/55240fig2.jpg" /> Figura 2: PMT saídas. A) de baixa passagem filtrada saídas dos dois fotomultiplicadores, após a centragem / descamação. B) As trajetórias talão obtidos a partir dos dados PMT, amostrados ao longo de 3 s. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55240/55240fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/55240/55240fig3.jpg" /> Figura 3: Bead Trajetórias. A) Histograma de velocidades somente CCW de um motor representativo. B) velocidades de rotação de um motor só-CCW fotografada no canto inferior direito (painel superior). velocidades de rotação do mesmo motor, quando posicionado no canto inferior esquerdo (painel inferior). C) </strong&gt; Comutação em um motor do tipo selvagem representativa. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55240/55240fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

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.

Caracterização biofísica das funções Flagellar motor

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 San Sebastian

Research

JoVE Journal

Biology

8.2K visualizações.  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.

Vídeo: 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.

Produção de conjuntos de motor de dynein e de Kinesin em nanostructures do ADN origami para a única observação da molécula

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

Bioquímica

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.

Adaptações específicas de miose de ensaios de motilidade baseados em microscopia de fluorescência in vitro

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.

Motilidade de Moléculas Únicas e Aglomerados de Kinesin-5 Cin8 Bidiso

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.

Caracterizar a composição de motores moleculares sobre Movimentação axonal Carga Utilizando Análise &quot;Mapeamento Cargo&quot;

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

Neurociência

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.

Análise de molécula única de motores da família Sf9 superprocessiva superprocessiva cinesina-3

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.

Sondando a mecânica do conjunto de miosina em feixes de filamentos de actina usando pinças ópticas

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 ...

Bioengenharia

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.

Geração de Mutantes Nulos para Elucidar o Papel das Glicosyltransferases Bacterianas na Motilidade Bacteriana

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

Imunologia e Infecção

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.

Investigando a Motilidade Impulsionada por Flagella em Escherichia coli aplicando três técnicas estabelecidas em uma série

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

Biologia

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.

Dissecando as propriedades mecanoenzimáticas de miosinas processivas com espectroscopia de força-braçadeira ultrarrápida

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.

Visualizando a motilidade bacteriana com base em uma reação de cor

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