Siamo fortemente indebitati a Jonathon Howard, il cui gruppo ha sviluppato il protocollo di base per un test di volo a vela motilità che è stato successivamente adattato da noi. Il sostegno finanziario NSF concedere DMR0645023 Si ringrazia.

1.) Tamponi e reagenti Queste soluzioni dovrebbero essere preparate in anticipo e conservato in aliquote comodamente dimensioni. Una aliquota dovrebbe contenere soluzione sufficiente per un tipico esperimento e una nuova aliquota dovrebbe essere utilizzato per ogni dosaggio motilità. Le condizioni di conservazione e le dimensioni aliquota tipici sono menzionati anche i seguenti protocolli. 1. BRB80 tampone, (80 TUBI mM, 1 mM MgCl 2, 1 mM EGTA in deionizzata distillata (gg) l&#39;acqua, pH regolato a 6,9 con KOH) <ul><li> Portare a 100 mL di soluzione madre di 0,5 M EGTA in acqua gg. Regolare il pH a 7,0 con 2 M NaOH. </li><li> Portare a 100 mL di soluzione madre 1 M MgCl 2 in acqua gg. Autoclave la soluzione. </li><li> Aggiungere 24,2 g di TUBI e 3,1 g pellet KOH in circa 800 ml di acqua dd e mescolare per sciogliere. Regolare il pH a 6,9 con 1 M soluzione di KOH. Aggiungere 2 ml di soluzione 0,5 M EGTA magazzino e 1 ml di 1 M MgCl 2 soluzione madre. Portare il volume a 1000 ml con acqua dd. </li><li> Aliquota in 50 mL tubi falco e congelare a -20 ° C per un uso futuro. Il tubo BRB80 attualmente in uso può essere conservato a 4 ° C oa temperatura ambiente. </li></ul> 2. Cloruro di magnesio, MgCl 2 (100 mM in acqua gg) <ul><li> Diluire in acqua dd per ottenere una concentrazione finale di 100 mm. </li><li> Aliquota (10 volumi mL) in provette da 0,5 ml microcentrifuga e conservare a -20 ° C per un uso futuro </li></ul> 3. Guanosina-5&#39;-trifosfato, sale disodico, GTP (25 mM gg acqua, pH regolato a 7 da NaOH) <ul><li> Pesare e si sciolgono in acqua dd e regolare il pH a 7 per soluzione 2 M di NaOH. </li><li> Verificare la concentrazione di misurare l&#39;assorbanza UV a 260 nm. (Utilizzare un coefficiente di estinzione di 11,7 x 103 M -1 cm -1). </li><li> Aliquota (10 volumi mL) in provette da 0,5 ml microcentrifuga e conservare a -20 ° C per un uso futuro. </li></ul> 4. Dimetil solfossido, DMSO <ul><li> Aliquota (10 volumi mL) in provette da 0,5 ml microcentrifuga e conservare a -20 ° C per un uso futuro. </li></ul> 5. Taxol (1 mM in DMSO) <ul><li> Pesare e si dissolvono in DMSO sotto cappa d&#39;aspirazione a raggiungere una concentrazione finale di 1 mm. </li><li> Aliquota (20 volumi mL) in provette da 0,5 ml microcentrifuga e conservare a -20 ° C per un uso futuro. </li></ul> 6. D-(+)-glucosio, (2 M in acqua gg) <ul><li> Pesare e si sciolgono in acqua dd per ottenere una concentrazione finale di 2 M. </li><li> Aliquota (20 volumi mL) in provette da 0,5 ml microcentrifuga e conservare a -20 ° C per un uso futuro. </li></ul> 7. Glucosio ossidasi, (2 mg / ml in BRB80) <ul><li> Sciogliere in BRB80 per ottenere una concentrazione finale di 2 mg / ml. </li><li> Aliquota (20 volumi mL) in provette da 0,5 ml microcentrifuga e conservare a -20 ° C per un uso futuro. </li></ul> 8. Ditiotreitolo, DTT (1 M gg acqua) <ul><li> Sciogliere in acqua gg sotto cappa d&#39;aspirazione a raggiungere una concentrazione finale di 1 M. </li><li> Aliquota (20 volumi mL) in provette da 0,5 ml microcentrifuga e conservare a -20 ° C per un uso futuro. </li></ul> 9. Catalasi, (0,8 mg / ml in BRB80) <ul><li> Sciogliere in BRB80 in almeno 2 fasi per ottenere una concentrazione finale di 0,8 mg / ml. Determinare la concentrazione in ogni fase di misurare l&#39;assorbanza UV a 276 nm e 406 nm (Utilizzare un coefficiente di estinzione di 3,1 x 10 5 M -1 cm -1 a 276 nm e 2,2 x 10 5 M -1 cm -1 a 406 nm e l&#39;equazione A = cL). </li><li> Aliquota (20 volumi mL) in provette da 0,5 ml microcentrifuga e conservare a -20 ° C per un uso futuro. </li></ul> 10. Adenosina-5&#39;-trifosfato, ATP (100 mM a 100mM MgCl 2) <ul><li> Preparare una soluzione stock di mM MgCl 100 2 gg in acqua. Pesare polvere secca e si dissolvono in questa soluzione, per ottenere una concentrazione finale di 100 mm. </li><li> Verificare la concentrazione di misurare l&#39;assorbanza UV a 260 nm. (Utilizzare un coefficiente di estinzione di 15,4 x 10 3 M -1 cm -1). </li><li> Aliquota (20 volumi mL) in provette da 0,5 ml microcentrifuga e conservare a -20 ° C per un uso futuro. </li></ul> 11. Caseina soluzione (20 mg / mL caseina in BRB80) <ul><li> Aggiungere circa 3 g di caseina e 30 ml di acqua gg in un tubo da 50 ml falco. Vortex per circa 1 ora fino a quando la soluzione si sviluppa consistenza densa. </li><li> Centrifugare a 15000 g per circa 30 minuti. Filtrare il surnatante con 0,5 micron e 0,2 micron filtri per siringa. </li><li> Determinare la concentrazione del supernatante di misurare l&#39;assorbanza UV a 280 nm (Utilizzare un coefficiente di estinzione di 0,67 centimetri ml mg -1 -1). Diluirlo a 20 mg / ml in BRB80. </li><li> Aliquota (20 volumi mL) inper tubi da microcentrifuga da 0,5 ml e conservare a -20 ° C per un uso futuro. </li></ul> 2.) Soluzioni standard Questi sono preparati il ​​giorno dell&#39;esperimento e deve essere eliminata dopo l&#39;esperimento è finito. Preparare 1 ml di ciascuna. 1. BRB80CS0.5 <ul><li> Soluzione diluita di caseina in BRB80 ad una concentrazione finale di 0,5 mg / ml e memorizzare sul ghiaccio. Questa soluzione viene introdotto nella cella di flusso prima di chinesina e aiuta a mantenere l&#39;attività dopo chinesina superficie di adsorbimento. </li></ul> 2. BRB80CA <ul><li> Preparare 0,2 mg / ml caseina e 1 mM ATP in BRB80 e memorizzare sul ghiaccio. Chinesina è ulteriormente diluita utilizzando questa soluzione prima introduzione nella cella di flusso. </li></ul> 3. BRB80T <ul><li> Soluzione diluita di taxolo in BRB80 ad una concentrazione finale di 10 mM e conservare a temperatura ambiente. Questa soluzione viene utilizzata per stabilizzare i microtubuli. </li></ul> 4. BRB80CT <ul><li> Preparare 10 taxolo mM e 0,2 mg / ml di caseina in BRB80 e conservare a temperatura ambiente. Questo è utilizzato per preparare ulteriormente il Antifade e soluzioni microtubuli. </li></ul> 5. BRB80AF <ul><li> Preparare 20 mM D-glucosio, 20 mg / ml glucosio ossidasi, 8 mg / mL catalasi, 10 mM DTT, e 20 mM ATP in BRB80CT e conservare a temperatura ambiente. Questa soluzione viene utilizzata per diluire streptavidina e nanosfere e &quot;lavare via&quot; la streptavidina in eccesso nella cella di flusso. La velocità chinesina può essere controllata regolando la concentrazione di ATP in questa soluzione. </li></ul> 3.) Kinesin Preparazione <ul><li> Esprimere una chinesina costruire costituito dalla wild-type, full-length catena Drosophila melanogaster chinesina pesanti e un C-terminale His-tag in Escherichia coli e purificare con un Ni-NTA colonna, come descritto al punto 6. </li><li> Fai aliquote (10 L ciascuno) in tubi da microcentrifuga da 0,5 ml e conservare a -80 ° C per un uso futuro. La concentrazione di chinesina attivi in queste aliquote è di circa 200 nM 7. </li><li> Per un tipico esperimento, diluire la soluzione chinesina 20 volte in BRB80CA. Etichettare il KIN20 soluzione e memorizzare sul ghiaccio. </li></ul> 4.) Microtubuli Preparazione <ul><li> In un tubo da microcentrifuga 0,5 ml, preparare 25 ml di soluzione di crescita: 4 mM MgCl 2, 1 mM GTP e 5% DMSO (v / v) in BRB80 buffer. </li><li> Aggiungere 6,25 ml di questa soluzione per un&#39;aliquota di 20 mg di liofilizzato tubulina biotinilato. </li><li> Vortex, poi posto in un bagno di calore a 37 ° C per 30 minuti per polimerizzare. Diluire 100-volte in BRB80T vortice e delicatamente. Etichettare il MT100 soluzione e conservare a temperatura ambiente. </li><li> Fai una diluizione di 10 volte MT100 in BRB80AF. Etichettare il MT1000 soluzione. </li></ul> 5.) Streptavidina e Nanosphere Solution Preparare AlexaFluor568 streptavidina marcata ad una concentrazione di 100 nM in BRB80AF. Etichettarla STV100 e memorizzare sul ghiaccio. Allo stesso modo, diluire nanosfere 5000 volte in soluzione BRB80AF. Etichettarla NS5000 e memorizzare sul ghiaccio. 6.) Flusso costruzione cellulare Costruire una cella di flusso utilizzando due coprioggetto di vetro separate da nastro biadesivo. Questa cella di flusso è di circa 2 cm di lunghezza, 1 cm di larghezza e 100 micron alto, e ha un volume di circa 20 l. Le soluzioni sono introdotti nella cella di flusso da un lato con una pipetta e fuori malvagio dall&#39;altro utilizzando carta da filtro. 7.) Assemblea Saggio invertito Superficie del vetro viene prima rivestita con la caseina che consente chinesina di mantenere la sua funzionalità su adsorbimento. Dopo kinesina è assorbito, microtubuli sono introdotti, che sono detenuti da cinesina. I microtubuli sono poi rivestiti con streptavidina fluorescenti. Dopo aver lavato i streptavidina eccesso, biotinilato polistirolo fluoresceina nanosfere (40 nm di diametro) vengono introdotti. Superficie nanosfere adsorbito stazionario si scontrano con i microtubuli in movimento e vengono caricate su di loro (Figura 1). L&#39;ordine del flusso di soluzioni e di tempo concesso prima dell&#39;introduzione della soluzione successiva sono elencati di seguito. <ul><li> BRB80CS0.5, a 5 minuti </li><li> KIN20, a 5 minuti </li><li> MT1000, a 5 minuti </li><li> STV100, a 5 minuti </li><li> BRB80AF, 3x </li><li> NS5000 </li></ul> 8.) Microscopia Montare la cella di flusso sul palco subito dopo l&#39;introduzione del microscopio nanosfere. In questo esperimento, un TE2000-U Eclipse microscopio a fluorescenza (Nikon, Melville, NY) dotata di un obiettivo olio 100X (NA 1,45), una X-Cite 120 lampada (EXFO, Ontario, Canada) e un ixon EMCCD fotocamera (ANDOR, South Windsor, CT) è stato utilizzato. Un filtro FITC cubo (# 48001) e un cubo TRITC filtro (# 48002, Tecnologie Chroma, Rockingham, VT) sono stati utilizzati per nanosfere immagine e microtubuli respectivamente sulla superficie inferiore di celle di flusso. Il tempo di esposizione è stato 0.2s, mentre il tempo tra le esposizioni è stato 2 s. <img src="/files/ftp_upload/2006/2006fig1.jpg" alt="Figura 1" /> Figura 1. Schema del navette molecolari.

<ol>
	<li>Agarwal, A., Hess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomolecular+motors+at+the+intersection+of+nanotechnology+and+polymer+science.">Biomolecular motors at the intersection of nanotechnology and polymer science.</a> Progress in Polymer Science. 35 (1-2), 252-252 (2010).</li><li>Howard, J., Hunt, A. J., Baek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+microtubule+movement+driven+by+single+kinesin+molecules.">Assay of microtubule movement driven by single kinesin molecules.</a> Methods Cell Biol. 39, 137-137 (1993).</li><li>Agarwal, A., Katira, P., Hess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond+curing+time+of+a+molecular+adhesive+causes+velocity-dependent+cargo-loading+of+molecular+shuttles.">Millisecond curing time of a molecular adhesive causes velocity-dependent cargo-loading of molecular shuttles.</a> Nano Lett. 9 (3), 1170-1170 (2009).</li><li>Diez, S., Reuther, C., Dinu, C., Seidel, R., Mertig, M., Pompe, W., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretching+and+Transporting+DNA+Molecules+Using+Motor+Proteins.">Stretching and Transporting DNA Molecules Using Motor Proteins.</a> Nano Lett. 3 (9), 1251-1251 (2003).</li><li>Bachand, G. D., Rivera, S. B., Boal, A. K., Gaudioso, J., Liu, J., Bunker, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+transport+of+nanocrystal+CdSe+quantum+dot+nanocomposites+using+microtubules+and+kinesin+motor+proteins.">Assembly and transport of nanocrystal CdSe quantum dot nanocomposites using microtubules and kinesin motor proteins.</a> Nano Lett. 4 (5), 817-817 (2004).</li><li>Coy, D. L., Wagenbach, M., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin+takes+one+8-nm+step+for+each+ATP+that+it+hydrolyzes.">Kinesin takes one 8-nm step for each ATP that it hydrolyzes.</a> J. Biol. Chem. 274 (6), 3667-3667 (1999).</li><li>Katira, P., Agarwal, A., Fischer, T., Chen, H. -. Y., Jiang, X., Lahann, J., Hess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+performance+of+protein-resisting+surfaces+at+ultra-low+protein+coverages+using+kinesin+motor+proteins+as+probes.">Quantifying the performance of protein-resisting surfaces at ultra-low protein coverages using kinesin motor proteins as probes.</a> Advanced Materials. 19, 3171-3171 (2007).</li><li>Vigers, G. P. A., Coue, M., McIntosh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Microtubules+Break+Up+Under+Illumination.">Fluorescent Microtubules Break Up Under Illumination.</a> J. Cell Biol. 107, 1011-1011 (1988).</li><li>Brunner, C., Hess, H., Ernst, K. -. H., Vogel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifetime+of+biomolecules+in+hybrid+nanodevices.">Lifetime of biomolecules in hybrid nanodevices.</a> Nanotechnology. 15 (10), S540-S540 (2004).</li></ol>

Nessun conflitto di interessi dichiarati.

Con piccole modifiche, questo protocollo è stato usato con successo da una varietà di gruppi di assemblare chinesina-microtubuli saggi motilità base. DTT 10 mM nella soluzione motilità finale può essere sostituito con lo 0,5% β-mercaptoetanolo. Soluzioni standard (BRB80AF, KIN20 e MT1000) più di 2 ore di vita non deve utilizzare. Qualsiasi soluzione contenente microtubuli taxolo e soprattutto non devono mai essere posti su ghiaccio. L&#39;eccessiva esposizione della cella di flusso ai risultati UV luce di eccitaz...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Adenosine-5&#x2019;-triphosphate (ATP)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A1049</td>
<td>&nbsp;</td>
<tr>
<td>Biotin tubulin</td>
<td>&nbsp;</td>
<td>Cytoskeleton Inc.</td>
<td>T333</td>
<td>&nbsp;</td>
<tr>
<td>Casein</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C-0376</td>
<td>&nbsp;</td>
<tr>
<td>Catalase</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C-9322</td>
<td>&nbsp;</td>
<tr>
<td>D-(+)-Glucose</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G-7528</td>
<td>&nbsp;</td>
<tr>
<td>Dimethylsulfoxide (DMSO)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D-8779</td>
<td>&nbsp;</td>
<tr>
<td>Dithiotreitol (DTT)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>161-0610</td>
<td>&nbsp;</td>
<tr>
<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#x2032;,N&#x2032;-tetraacetic acid (EGTA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>E-4378</td>
<td>&nbsp;</td>
<tr>
<td>FluoSpheres Biotinylated microspheres, 40 nm, yellow-green fluorescent (505/515)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>F-8766</td>
<td>&nbsp;</td>
<tr>
<td>Glucose oxidase</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G-7016</td>
<td>&nbsp;</td>
<tr>
<td>Guanosine-5&#x2019;-triphosphate (GTP)</td>
<td>&nbsp;</td>
<td>Roche Diagnostic</td>
<td>106399</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium Chloride (MgCl2)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>63069</td>
<td>&nbsp;</td>
<tr>
<td>Paclitaxel (Taxol)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T1912</td>
<td>&nbsp;</td>
<tr>
<td>1,4-Piperazinediethanesulfonic acid, Piperazine-1,4-bis(2-ethanesulfonic acid), Piperazine-N,N&#x2032;-bis(2-ethanesulfonic acid) (PIPES)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P-6757</td>
<td>&nbsp;</td>
<tr>
<td>Potassium hydroxide (KOH)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P-6310</td>
<td>&nbsp;</td>
<tr>
<td>Sodium hydroxide (NaOH)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>480878</td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin Alexa Fluor 568 conjugate</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S11226</td>
<td>&nbsp;</td>		</tbody>
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cargo loading onto kinesin powered molecular shuttles

Le cellule hanno sviluppato sofisticate macchine molecolari, come chinesina proteine ​​motrici e filamenti di microtubuli, al sostegno attivo di trasporto intracellulare di carico. Mentre kinesins coda dominio si lega ad una varietà di carichi, domini testa kinesins utilizzare l&#39;energia chimica immagazzinata nelle molecole di ATP per passo lungo il reticolo microtubuli. Il lungo e rigido microtubuli fungono da piste per lunghe distanze di trasporto intracellulare. Questi motori e filamenti possono anche essere impiegati in microfabbricazione ambienti sintetici come componenti di navette molecolari 1. In un progetto di uso frequente, i motori chinesina sono ancorati alla superficie della pista con la coda, e microtubuli funzionalizzate servire come elementi costitutivi di trasporto merci, che sono spinti da questi motori. Queste navette possono essere caricati con il carico utilizzando il legame forte e selettiva tra biotina e streptavidina. I componenti chiave (tubulina biotinilato, streptavidina, e carico biotinilato) sono disponibili in commercio. Sulla base del classico saggio motilità invertita 2, la costruzione di navette molecolari è dettagliata qui. Chinesina proteine ​​motrici sono assorbiti da una superficie coperte con caseina; microtubuli sono polimerizzati da tubulina biotinilato, ha aderito al chinesina e successivamente rivestito con streptavidina marcata con rodamina. La concentrazione di ATP è mantenuto a concentrazione subsaturating di raggiungere una velocità ottimale microtubulo scorrimento per il carico merci 3. Infine, biotinilato marcato con fluoresceina nanosfere vengono aggiunti come merce. Nanosfere allegare ai microtubuli a causa di collisioni tra microtubuli volo a vela e nanosfere aderire alla superficie. Il protocollo può essere facilmente modificato per caricare una varietà di carichi come il DNA biotinilato 4, 5 o quantum dots una vasta gamma di antigeni tramite anticorpi biotinilati 4-6.

Watch this Scientific Journal Video about Cargo Loading onto Kinesin Powered Molecular Shuttles at JoVE.com

Cargo Loading onto Kinesin Powered Molecular Shuttles

Navette molecolari costituiti da microtubuli funzionalizzati scivolando sulla superficie aderito chinesina proteine ​​motore può servire come sistema di trasporto su scala nanometrica. Qui, l&#39;assemblaggio di un sistema di navetta tipico è descritto.

Caricamento di merci su Kinesin Navette Powered molecolare

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport ...

cargo-loading-onto-kinesin-powered-molecular-shuttles

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.4K Views.  University of Florida. Navette molecolari costituiti da microtubuli funzionalizzati scivolando sulla superficie aderito chinesina proteine ​​motore può servire come sistema di trasporto su scala nanometrica. Qui, l'assemblaggio di un sistema di navetta tipico è descritto.

Video: Cargo Loading onto Kinesin Powered Molecular Shuttles

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

Ex vivo Carico meccanico del tendine

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

Ricerca

Biologia

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Caricamento terminazioni nervose Drosophila con gli indicatori di calcio

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Molecular Evolution of the Tre Recombinase

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. 
We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of "molecular surgery" and molecular medicine.

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of molecular surgery and molecular medicine.

Evoluzione molecolare del ricombinasi Tre

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within ...

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.


This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

Dal MEF per Matrigel 2: hESC scissione dal MEF sul Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated.

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3.

Dal MEF per Matrigel 3: hESC Passaging da Matrigel su Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Assemblaggio, carico, allineamento e di una cella analitica del campione Ultracentrifuga

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Isolamento e purificazione di chinesina dal Drosophila Embrioni

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Imaging diretta di calcio ER con Targeted-esterasi Induced Loading Dye (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Generating a &#34;Humanized&#34; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined.

Generazione di un &quot;umanizzato&quot;<em&gt; Drosophila</em&gt; S2 Cell Line Sensibile alla farmacologica L&#39;inibizione di Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper ...

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

Identificazione Kinesin-1 Cargos Utilizzando la microscopia a fluorescenza

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...