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. Preparazione delle cellule <ol><li> Grow culture durante la notte del ceppo desiderato portando il appiccicoso flic allele 15,22 a triptone Broth (TB, 1% Peptone, 0.5% NaCl) seguita da inoculo in diluizione 1: 100 in 10 ml TB fresco. Crescere la coltura a 33 ° C in un incubatore shaker fino OD 600 = 0.5. </li><li> Agglomerare le cellule a 1500 xg per 5 - 7 min e ri-disperdere il pellet vigorosamente in 10 mL di tampone di filtro sterilizzato motilità (MB; 10 mM tampone fosfato: 0,05-0,06 M NaCl, 10 -4 M EDTA, 1 mM metionina, pH 7,0). </li><li> Ripetere il punto 1.2 altre due volte e ri-disperdere il pellet finale in 1 ml MB. </li><li> Tosare la sospensione passando avanti e indietro ~ 75 volte tra due siringhe con 21 a 23 adattatori calibro connesso con un tubo in polietilene (7 - 12 cm di lunghezza, diametro 0,58 mm interno). Limitare il tempo totale di taglio a 30 - 45 s. </li><li> Centrifugare le cellule tranciate a 1.500 xg per 5-7 minuti e ri-disperdere lapellet in 100 - 500 ml di MB. </li></ol> 2. Far scorrere Preparazione <ol><li> Preparare una camera di imaging da sandwich due nastri adesivi doppia faccia tra una copertura antiscivolo e un vetrino da microscopio. Per i saggi di chemiotassi, impiegare qualsiasi camera di microfluidica che consente lo scambio di MB e stimolanti chimici. </li><li> Aggiungere 0,01% soluzione di poli-L-lisina nella camera e dopo 5 min risciacquare delicatamente le superfici con MB (80 - 100 l). </li><li> Aggiungere 40 ml di sospensione cellulare nella camera e consentire un tempo sufficiente per il fissaggio alla superficie di vetro (7 - 8 min). Defluire cellule scollate aggiungendo 100 microlitri MB su un lato della camera, mentre traspirante la soluzione con un filtro di carta dal lato opposto. </li><li> Aggiungere 10 - 15 ml di perle di lattice nella camera e consentire le perle tempo sufficiente per risolvere e allegare alle cellule (7-8 min). Lavare delicatamente con 100 ml di MB, come descritto al punto 2.3, per rimuovere scollarsi perline. Utilizzare un intervallo di bead-SIzes per gli esperimenti così a lungo come un buon contrasto è disponibile. </li></ol> 3. Monitoraggio Bead <ol><li> Posizionare il campione su un palco microscopio e la scansione della superficie per perline attaccate ai motori. Utilizzare un obiettivo 40X fase per fare osservazioni anche se la microscopia di fase non è necessario. In alternativa, utilizzare l&#39;imaging in campo chiaro fino a quando un sufficiente contrasto è mantenuto per distinguere chiaramente una perlina luminoso su uno sfondo scuro. </li><li> Una volta selezionato un tallone, spostare la fase lateralmente per posizionare il tallone in un angolo predeterminato, come mostrato in Figura 1B. perline posizione alla stessa corner per garantire che il senso di rotazione del tallone è correttamente noto. Il tallone traiettoria ideale è approssimativamente circolare, ma ellittica traiettorie sono ricevibili. </li><li> Mantenere la frequenza di campionamento superiore al doppio della frequenza di rotazione del motore per evitare errori associati aliasing. In questo lavoro, utilizzare un motore che ruotava a50 Hz e il campione a frequenze che erano 10 volte superiore (500 Hz), per ottenere un segnale liscia. </li></ol> Analisi 4. I dati <ol><li> Centro e scalare le tensioni di uscita PMT e corretta ellitticità nelle traiettorie con trasformazioni affini, se necessario 23. Utilizzare l&#39;analisi di potenza spettro per determinare i tassi di rotazione 17. </li><li> Determinare angoli polari, θ (t) = atan (y (t) / x (t)). Determinare le variazioni di velocità del motore e la commutazione nel tempo calcolando ω <img alt="Equazione 1" src="/files/ftp_upload/55240/55240eq1.jpg" /> 14. </li><li> Impiegare un filtro mediano per lisciare i dati di velocità del motore. Una finestra di filtro più di due giri completi si raccomanda 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.

Al fine di facilitare tethered tallone-tracking e stima corretta del motore coppie, le seguenti informazioni dovrebbe essere rivisto. Quando si eseguono queste misure con cellule flagellate, tosatura è un passaggio fondamentale. Shearing riduce la flagellar filamento ad un semplice stub, garantendo così che il carico sul motore viscosa è prevalentemente dovuta al tallone e può essere stimato entro l&#39;errore 10% 16. Shearing migliora anche la possibilità di trovare le traiettorie circolari con eccentricità strettamente distribuiti (&lt;diametro tallone 14). Risultati tranciatura improprie in traiettorie ostinati, quali composti errori nel monitoraggio e nel calcolo delle boccate viscosi, nonché con conseguente scarsa rapporto segnale-rumore. L&#39;uso di un oscilloscopio permette una rapida eliminazione di tali dati. Dal momento che le proprietà biomeccaniche di filamenti flagellari potranno variare con specie, sarà probabilmente necessario adattare i metodi di taglio al fine di garantire un adeguato taglio in tegli batteri di interesse. Un modo efficace per ridurre gli errori associati alla tosatura è quello di lavorare con le cellule che mancano i geni che codificano le proteine ​​del filamento. perline sonda può poi essere collegato direttamente al gancio tramite anticorpi anti-gancio. Trovare un cordolo che viene opportunamente legato può essere impegnativo. Questo è perché la maggior parte perline nel campo visivo sarà o essere bloccato ai corpi cellulari o la superficie del vetro. Tali sfere possono essere facilmente portati a fuoco. Altre perle appariranno a vibrare o ruotare visibilmente con grandi ampiezze o grandi eccentricità (&gt; 1,5 - diametro 2x tallone). Questi sono tipicamente legato a flagellare filamenti che non sono stati completamente tranciati o ruotare in un piano inclinato rispetto al piano focale. Il campionamento di tali perle in genere causare rumore più elevato, e di tempo variazioni di carichi viscosi può comportare una sottostima di motori coppie per una data tallone-size. Una piccola frazione di perline frenati subirà casualemovimento; Questi sono solo in fase di rotazione browniano. Una frazione dei branelli apparirà sfocato e non possono essere portati a fuoco facilmente. Questi sono più probabilità di essere i motori che sono stati impastoiati in modo appropriato e sono i motori di interesse. Tra i limiti del monomotore monitoraggio come quello descritto qui è l&#39;incapacità di condurre esperimenti high throughput. Una telecamera ad alta velocità che immagini una regione più grande di interesse può essere vantaggioso in questo senso. Altre limitazioni sono errori associati con più segnali derivanti da perline rotanti strettamente distanziati nel campo di vista dei PMT. Infine, errori nella determinazione della corretta posizione del tallone registrato rispetto ai due fenditure fotomoltiplicatori comporterà stima imprecisa della dinamica di commutazione. Vantaggi della configurazione descritta qui includono la possibilità di monitorare la rotazione delle sfere su lunghe durate e in tempo reale. Questopermette la rapida eliminazione delle traiettorie soggette a errori, cosa che forse difficile da raggiungere con le fotocamere ultraveloci. Inoltre, con alcune modifiche questa configurazione può essere integrato con saggi intese a sottoporre le cellule ad una varietà di stimoli. Combinato con raffreddamento termoelettrico 26, la tecnica può essere utilizzata per misurare le risposte dei singoli motori a stimoli termici. Integrazione con pinzette ottiche può attivare le misurazioni di rimodellamento dei singoli motori in risposta a stimoli meccanici, come è stato fatto recentemente 6. Infine, l&#39;adattamento del motore stimolanti chimici può essere misurata con l&#39;uso di una camera di perfusione adeguata e pompe 11. La maggior parte delle specie batteriche conosciute sono mobili e flagellare mediata motilità è predominante in natura. I metodi dimostrato qui si prevede di continuare a contribuire allo sviluppo di intuizioni strutturali-rimodellamento e l&#39;adattabilità of il motore flagellare.

I motori flagellare consentono alle cellule di nuotare ruotando filamenti extracellulari elicoidali. La quantità di coppia del motore può generare per una data lunghezza del flagello (cioè, il carico viscoso) determina le velocità di nuoto. D&#39;altro canto, la capacità di cambiare la direzione di rotazione controlla migrazione delle cellule in risposta ad agenti chimici, un processo noto come chemiotassi. Chemiotassi e la motilità di essere fattori di virulenza 1-3, motori flagellare sono stati ben caratterizzati nel corso degli anni 4. Prove di montaggio ora suggerisce che il motore funge mechanosensor - rileva meccanicamente la presenza di substrati solidi 5,6. Questa capacità aiuta probabilmente a scatenare la colonizzazione della superficie e le infezioni 5,7. Di conseguenza, i meccanismi per cui il motore individua automaticamente superfici e iniziati segnalazione sono di importanza 8,9. Il motore flagellare può essere facilmente studiato da legare il flagellum ad un substrato e osservando la rotazione cellulare. Tale tethering è stato raggiunto prima da Silverman e Simon, che lavorava con un mutante polyhook in E. coli e ganci fissati con successo per substrati di vetro con anticorpi anti-gancio 10. Il test legato celle consentito ai ricercatori di studiare le risposte del motore interruttore ad una varietà di stimoli chimici. Ad esempio, Segall e collaboratori stimolati chimicamente le cellule in collegamento, con l&#39;ausilio di pipette iontoforesi. I cambiamenti corrispondenti pregiudizi CW (la frazione dei motori tempo di rotazione in senso orario, CW) ha permesso loro di misurare le cinetiche di adattamento della rete chemiotassi 11,12. Mentre il test delle cellule legato è stato efficace nello studio delle risposte degli interruttori, è stato solo in grado di offrire intuizioni meccanica del motore su una gamma limitata di carichi viscosi 13. Per superare questo problema, Ryu e collaboratori impastoiati sferiche, sfere di lattice a filamento stub su cellule attaccate alle superfici. Le perle sono statipoi monitorati tramite interferometria back-focale con trappole ottiche deboli 14. Lavorando con perline di diverse dimensioni, i ricercatori possono studiare il motore su una gamma più ampia di carichi. Questo test è stato poi migliorato da Yuan e Berg, che ha sviluppato una tecnica di tallone-tracking fotomoltiplicatore a base combinata con illuminazione laser in campo scuro. Il loro metodo attivato il monitoraggio di nanobeads frenati d&#39;oro che erano così piccola (~ 60 nm) che le resistenze viscose esterni sono stati inferiori rispetto alle resistenze viscose interne a rotazione 15,16. Ciò ha portato alle misure delle velocità massime ottenibili in E. coli (~ 300 Hz). In V. alginolyticus, simili saggi di perline attivate misure dei tassi di filatura a carichi intermedi viscosi (~ 700 Hz) 17. Consentendo misurazioni di risposte motorie sull&#39;intera gamma possibile di carichi viscosi (da zero-carico quasi stallo), le perline-saggi fornito un importante strumento biofisico per comprendere il tprocesso orque generazione 18,19. Recentemente, abbiamo modificato il dosaggio Yuan-Berg per includere pinzette ottiche che ci hanno permesso di applicare stimoli meccanici precisi per singoli motori 6. Usando questa tecnica, abbiamo dimostrato che la forza-generatori che ruotano il motore sono meccanosensori dinamici - si rimodellare in risposta ai cambiamenti di carichi viscosi. È possibile che tale load sensing innesca differenziazione cellulare nei batteri brulicanti, sebbene i meccanismi rimangono poco chiari. E &#39;anche probabile che i motori flagellare in altre specie sono anche meccanosensibili 20, anche se la prova diretta è carente. Qui, si discute l&#39;approccio fotomoltiplicatore-based (PMT) per il monitoraggio della rotazione delle sfere di lattice impastoiati ai filamenti flagellari 15. Rispetto al monitoraggio con telecamere ultraveloci, il fotomoltiplicatore-setup è vantaggioso perché è relativamente semplice per tenere traccia delle singole perle in tempo reale e più a lungo durazioni. E &#39;particolarmente utile quando si studia a lungo tempo rimodellamento in complessi a motore flagellare a causa di stimoli ambientali 21. Anche se abbiamo protocolli dettaglio specifico per E. coli, che possono essere facilmente adattati per lo studio di motori flagellare in altre specie.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#38;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&#38;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,&#160; 0.78 ns response time&#160; 
			http://pdf1.alldatasheet.com/datasheet-pdf/view/212308/HAMAMATSU/R7400U-20.html</td>
		</tr>
		<tr>
			<td>3x1 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&#160; 
			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.&#160;&#160;&#160; 
			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. 
			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.

La configurazione fotomoltiplicatore è mostrato nella Figura 1A. È importante che i PMT hanno alte sensibilità nel range di lunghezze d&#39;onda sparse dalle perline di interesse. Le PMT impiegati qui operano nelle gamme visibile e vicino infrarosso, e sono stati in grado di rilevare la luce diffusa dalla perle illuminate da una sorgente di luce alogena. Le condizioni di luce ottimali e tensioni di alimentazione variano da una configurazione all&#39;altra. Per la configurazione utilizzata in questo lavoro, un guadagno di PMT ~ 10 aprile - 10 maggio si è rivelata adeguata. Ogni fotomoltiplicatore è stato coperto tranne una fessura 3 x 1 mm posizionato davanti al fotomoltiplicatore. Le fessure limitano regione nella cella campione da cui la luce può entrare fotomoltiplicatori, e le due fenditure sono ortogonali tra loro. Quando una perlina rotante è posizionato nella posizione corretta (Figura 1B), la quantità di luce che entra aumenti fotomoltiplicatori come il tallone vienenella vista e diminuisce come percorso circolare prende lontano dalla vista. Le frequenze delle uscite sinusoidali di tensione PMT indicano la velocità di rotazione e le differenze di fase tra i due segnali indicano il senso di rotazione. L&#39;uso di un oscilloscopio per visualizzare i risultati PMT consente la visualizzazione delle traiettorie tallone in tempo reale. I segnali PMT tempo-varianti, y (t) e x (t), da un motore rappresentante sono mostrati nella Figura 2A. L&#39;ortogonalità dei due fenditure introduce un ritardo di fase tra i due segnali. Le ampiezze dei segnali dipende dal rapporto segnale-rumore e l&#39;eccentricità di rotazione. Le corrispondenti traiettorie del tallone sono indicati nella Figura 2B. Un istogramma delle velocità misurate da un motore rappresentativo in uno Chey - ceppo soppresso è mostrato in Figura 3A </strong&gt;. La cancellazione assicura che i motori flagellare non possono cambiare e ruotare esclusivamente in senso antiorario (CCW). La velocità media di questo particolare motore era di 60 Hz e le velocità misurate sono coerenti con precedenti relazioni per motori in collegamento, con 1 micron perline 25. Il tallone è stato posizionato nell&#39;angolo inferiore destro, come visto nello schema in Figura 1B. La corrispondente velocità angolare è mostrato nella Figura 3B (pannello superiore). Posizionando il tallone verso l&#39;angolo inferiore sinistro adiacente ha provocato l&#39;inversione del segno sulle velocità del motore (pannello inferiore). Quindi, muovendo il tallone a un angolo adiacente cambierà la direzione osservata di rotazione del motore. A questo proposito, angoli diagonalmente opposti sono identici. È pertanto fondamentale conoscere la posizione del tallone durante le misurazioni per determinare correttamente le dinamiche di commutazione. Figura 3C mostra ripetuti passaggi di un motore wild-type tra i due sensi di rotazione. <html> Codici personalizzati per il software di acquisizione dati sono stati adattati dal lavoro prima di registrare i dati su un computer 15. L&#39;uscita PMT è accoppiato in AC e filtro passa-basso con frequenza di taglio di 100 Hz. il monitoraggio in tempo reale è stato attivato collegando le uscite filtrate a un oscilloscopio. <img alt="Figura 1" src="/files/ftp_upload/55240/55240fig1.jpg" /> Figura 1: Impostazione Bead-tracker. A) Schema di impostazione del monitoraggio PMT-based. B) La posizione ideale del tallone (sfera nera) rispetto ai due fessure ortogonali. La traiettoria è indicata dalle linee tratteggiate. L&#39;eccentricità e è il raggio del cerchio tratteggiato. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55240/55240fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> ove_content &quot;fo: keep-together.within-page =&quot; 1 &quot;&gt; <img alt="figura 2" src="/files/ftp_upload/55240/55240fig2.jpg" /> Figura 2: PMT uscite. A) passa-basso uscite filtrato dai due fotomoltiplicatori, dopo il centraggio / ridimensionamento. B) Le traiettorie tallone ottenuti dai dati PMT, campionati più di 3 s. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55240/55240fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/55240/55240fig3.jpg" /> Figura 3: Traiettorie tallone. A) Istogramma di velocità CCW solo di un motore rappresentativo. B) velocità di rotazione di un motore CCW solo ripreso in basso a destra (pannello superiore). velocità di rotazione dello stesso motore quando posizionati in basso a sinistra (pannello inferiore). C) </strong&gt; di commutazione in un motore rappresentante wild-type. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55240/55240fig3large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa 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.

Caratterizzazione biofisica delle funzioni flagellare a motore

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

Nanyang Technological University

Research

JoVE Journal

Biology

8.2K Views.  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.

Video: Biophysical Characterization of Flagellar Motor Functions

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Exploring Cognitive Functions in Babies, Children & Adults with Near Infrared Spectroscopy

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

Esplorare le funzioni cognitive in neonati, bambini e adulti con Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Ricerca

Biologia

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Preparazione del Aplysia Senso-motorio colture cellulari neuronali

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

Sezioni incluse in paraffina e surgelati di Drosophila Adulti Muscoli

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.1 A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer 'epicuticular' layer as well as the blend consisting of the cutin biopolymer and 'intracuticular' waxes.2 Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Sch&auml;ffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) 13C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS 13C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH2O) chemical moieties.
Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state 13C NMR reveals differences in the relative numbers of CHO and CH2O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-&mu;m length scale.

Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.

Isolamento e studio della biofisica cuticole di frutta

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

Cattura selettiva di 5 hydroxymethylcytosine dal DNA genomico

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

Singola molecola di imaging del regolamento Gene<em&gt; In vivo</em&gt; Utilizzo di attivazione Cotranslational da Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

Super-risoluzione Imaging della Divisione Macchine batterica

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

Introduzione alla membrana supportata Solid Sulla Elettrofisiologia

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Produzione efficiente e purificazione di ricombinante murino kindlina-3 da cellule di insetto per Biofisica Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.