Nous sommes très reconnaissants à Jonathan Howard, dont le groupe a développé le protocole de base pour un test de mobilité par glissement qui a ensuite été adapté par nos soins. Le soutien financier de la NSF subvention DMR0645023 est grandement appréciée.

1.) Tampons et réactifs Ces solutions doivent être préparées à l&#39;avance et stocké en aliquotes de taille pratique. Une aliquote doit contenir suffisamment de solution pour une expérience typique et une autre partie aliquote doit être utilisé pour chaque test de motilité. Les conditions de stockage et de tailles typiques aliquotes sont également mentionnées dans les protocoles suivants. 1. BRB80 tampon (80 mM PIPES, 1 mM MgCl2, 1 mM EGTA dans désionisée distillée (dd) d&#39;eau, le pH ajusté à 6,9 par KOH) <ul><li> Préparez une solution de 100 ml de bouillon de 0,5 M EGTA dans l&#39;eau dd. Ajuster le pH à 7,0 en utilisant une solution de NaOH 2 M. </li><li> Préparez une solution de 100 ml de bouillon de 1 M MgCl 2 dans l&#39;eau dd. Autoclaver la solution. </li><li> Ajouter 24,2 g de PIPES et 3,1 g de pastilles de KOH à environ 800 ml d&#39;eau dd et mélanger pour dissoudre. Ajuster le pH à 6,9 avec 1 M de solution de KOH. Ajouter 2 ml d&#39;une solution 0,5 M de stock EGTA et 1 ml de 1 M MgCl 2 SOLUTIsur. Montez le volume à 1000 ml avec de l&#39;eau dd. </li><li> Aliquoter dans des tubes de 50 ml falcon et congeler à -20 ° C pour une utilisation future. Le tube BRB80 actuellement utilisé peut être conservé à 4 ° C ou à température ambiante. </li></ul> 2. Chlorure de magnésium MgCl 2 (100 mM dans l&#39;eau dd) <ul><li> Diluer dans de l&#39;eau dd pour obtenir une concentration finale de 100 mM. </li><li> Aliquote (10 uL de volume) dans 0,5 ml microtubes et conserver à -20 ° C pour une utilisation ultérieure </li></ul> 3. Guanosine-5&#39;-triphosphate, sel disodique, le GTP (25 mM dans de l&#39;eau dd, pH ajusté à 7 par NaOH) <ul><li> Peser et dissoudre dans l&#39;eau dd et ajuster le pH à 7 par une solution de NaOH 2 M. </li><li> Vérifiez la concentration en mesurant l&#39;absorbance UV à 260 nm. (Utilisez un coefficient d&#39;extinction de 11,7 x 103 M -1 cm -1). </li><li> Aliquote (10 uL de volume) dans 0,5 ml microtubes et conserver à -20 ° C pour une utilisation ultérieure. </li&gt; </ul> 4. Le diméthylsulfoxyde, DMSO <ul><li> Aliquote (10 uL de volume) dans 0,5 ml microtubes et conserver à -20 ° C pour une utilisation ultérieure. </li></ul> 5. Taxol (1 mM dans du DMSO) <ul><li> Peser et dissoudre dans du DMSO sous une hotte pour obtenir une concentration finale de 1 mM. </li><li> Aliquote (20 uL de volume) dans 0,5 ml microtubes et conserver à -20 ° C pour une utilisation ultérieure. </li></ul> 6. D-(+)-glucose, (2 M dans l&#39;eau dd) <ul><li> Peser et dissoudre dans l&#39;eau dd pour obtenir une concentration finale de 2 M. </li><li> Aliquote (20 uL de volume) dans 0,5 ml microtubes et conserver à -20 ° C pour une utilisation ultérieure. </li></ul> 7. Glucose oxydase, (2 mg / ml dans BRB80) <ul><li> Dissoudre dans BRB80 pour atteindre une concentration finale de 2 mg / mL. </li><li> Aliquote (20 uL de volume) dans 0,5 ml microtubes et conserver à -20 ° C pour une utilisation ultérieure. </li></ul><pclass = &quot;jove_step&quot;&gt; 8. Dithiothréitol, TNT (1 M dans l&#39;eau dd) <ul><li> Dissoudre dans l&#39;eau dd sous une hotte pour obtenir une concentration finale de 1 M. </li><li> Aliquote (20 uL de volume) dans 0,5 ml microtubes et conserver à -20 ° C pour une utilisation ultérieure. </li></ul> 9. Catalase, (0,8 mg / ml dans BRB80) <ul><li> Dissoudre dans BRB80 en au moins 2 étapes pour atteindre une concentration finale de 0,8 mg / ml. Déterminer la concentration à chaque étape par mesure de l&#39;absorbance UV à 276 nm et 406 nm (Utilisez un coefficient d&#39;extinction de 3,1 x 10 5 M -1 cm -1 à 276 nm et 2,2 x 10 5 M -1 cm -1 à 406 nm et l&#39;équation A = Cl). </li><li> Aliquote (20 uL de volume) dans 0,5 ml microtubes et conserver à -20 ° C pour une utilisation ultérieure. </li></ul> 10. Adénosine-5&#39;-triphosphate, ATP (100 mM dans 100 mM de MgCl 2) <ul><li> Préparer une solution mère de 100 mM de MgCl2 jj water. Peser la poudre sèche et se dissolvent dans cette solution mère pour obtenir une concentration finale de 100 mM. </li><li> Vérifiez la concentration en mesurant l&#39;absorbance UV à 260 nm. (Utilisez un coefficient d&#39;extinction de 15,4 x 10 3 M -1 cm -1). </li><li> Aliquote (20 uL de volume) dans 0,5 ml microtubes et conserver à -20 ° C pour une utilisation ultérieure. </li></ul> 11. Caséine solution (20 mg / ml dans de la caséine BRB80) <ul><li> Ajouter environ 3 g de caséine à 30 ml d&#39;eau dd dans un tube Falcon de 50 ml. Vortex pendant environ 1 heure jusqu&#39;à ce que la solution se développe consistance épaisse. </li><li> Centrifuger à environ 15000 g pendant 30 minutes. Filtrer le surnageant à 0,5 um et 0,2 um filtres seringues. </li><li> Déterminer la concentration du surnageant en mesurant l&#39;absorbance UV à 280 nm (Utilisez un coefficient d&#39;extinction de 0,67 mg mL -1 -1 cm). Diluez à 20 mg / mL dans BRB80. </li><li> Aliquote (20 uL de volume) dans<html> à 0,5 ml microtubes et conserver à -20 ° C pour une utilisation ultérieure. </li></ul> 2.) Solutions standard Ceux-ci sont préparés le jour même de l&#39;expérience et doivent être jetés après l&#39;expérience est terminée. Préparer 1 ml de chaque. 1. BRB80CS0.5 <ul><li> Diluer la solution de caséine dans BRB80 à une concentration finale de 0,5 mg / ml et de stocker plus de glace. Cette solution est introduite dans la cellule d&#39;écoulement avant et permet de conserver la kinésine activité de kinésine, après l&#39;adsorption en surface. </li></ul> 2. BRB80CA <ul><li> Préparer 0,2 mg / ml de caséine et 1 mM d&#39;ATP dans BRB80 et de stocker plus de glace. Kinésine est encore diluée à l&#39;aide de cette solution avant l&#39;introduction dans la cellule d&#39;écoulement. </li></ul> 3. BRB80T <ul><li> Diluer la solution taxol dans BRB80 à une concentration finale de 10 uM et conserver à température ambiante. Cette solution est utilisée pour stabiliser les microtubules. </li></ul>jove_step &quot;&gt; 4. BRB80CT <ul><li> Préparer 10 uM de taxol et de 0,2 mg / ml de caséine dans BRB80 et stocker à température ambiante. Il est utilisé pour continuer à préparer le antifade et des solutions de microtubules. </li></ul> 5. BRB80AF <ul><li> Préparer 20 mM de D-glucose, 20 glucose oxydase pg / ml, 8 pg / ml de catalase, DTT 10 mM, et 20 uM d&#39;ATP dans BRB80CT et stocker à température ambiante. Cette solution est utilisée pour diluer la streptavidine et des nanosphères et &quot;laver&quot; la streptavidine en excès dans la cellule d&#39;écoulement. La vitesse de kinésine peut être contrôlée en ajustant la concentration d&#39;ATP dans cette solution. </li></ul> 3). Préparation kinésine <ul><li> Exprimer une kinésine construire composé du type sauvage, pleine longueur de la chaîne Drosophila melanogaster kinésine lourd et un C-terminal His-tag dans Escherichia coli et purifier l&#39;aide d&#39;une colonne de Ni-NTA comme décrit en 6. </li><li> Assurez aliquotes (10 ul chacun) dans 0,5 ml microtubes et magasins à -80 ° C pour une utilisation ultérieure. La concentration de la kinésine actif dans ces portions est d&#39;environ 200 nM. 7 </li><li> Pour une expérience typique, diluer la solution kinésine 20 fois BRB80CA. Étiquetez le KIN20 solution et de stocker plus de glace. </li></ul> Préparation des microtubules 4.) <ul><li> Dans un tube à centrifuger de 0,5 mL, préparer 25 pi de solution de croissance: 4 mM de MgCl2, 1 mM de GTP et 5% de DMSO (v / v) dans un tampon BRB80. </li><li> Ajoutez 6,25 ul de cette solution à une aliquote de 20 ug de tubuline biotinylé lyophilisée. </li><li> Vortex, puis mettre dans un bain thermique à 37 ° C pendant 30 minutes à polymériser. Diluer 100 fois dans BRB80T et vortexer doucement. Étiquetez le MT100 solution et conserver à température ambiante. </li><li> Faire une dilution de 10 fois dans BRB80AF MT100. Étiquetez le MT1000 solution. </li></ul> 5.) Solution streptavidine et Nanosphère Préparer AlexaFluor568 la streptavidine marquée à une concentration de 100 nM dans BRB80AF. Étiqueter et stocker STV100 sur la glace. De même, diluer nanosphères 5000 fois en solution BRB80AF. Étiqueter NS5000 et de stocker plus de glace. 6.) Construction Cellule à circulation Construire une cuve à circulation à l&#39;aide de deux lamelles de verre séparées par un adhésif double face. Cette cellule d&#39;écoulement est d&#39;environ 2 cm de long, 1 cm de large et 100 um de hauteur et a un volume d&#39;environ 20 mL. Solutions sont introduites dans la cellule d&#39;écoulement d&#39;un côté à l&#39;aide d&#39;une pipette et sur méchant de l&#39;autre à l&#39;aide de papier filtre. 7.) Assemblée dosage inversé Surface de verre est d&#39;abord revêtu avec de la caséine qui permet de conserver la kinésine sa fonctionnalité à adsorption. Après la kinésine est adsorbé, les microtubules sont introduites, qui sont détenus par la kinésine. Les microtubules sont ensuite revêtues de streptavidine fluorescente. Après lavage de la streptavidine excès, biotinylé polystyrène fluorescéine nanosphères (40 dia nmter) sont introduits. Surface adsorbés nanosphères fixes entrent en collision avec les microtubules en mouvement et se charger sur eux (figure 1). L&#39;ordre du débit des solutions et des temps d&#39;attente avant l&#39;introduction de la solution suivante sont listés ci-dessous. <ul><li> BRB80CS0.5, 5 minutes </li><li> KIN20, 5 minutes </li><li> MT1000, 5 minutes </li><li> STV100, 5 minutes </li><li> BRB80AF, 3x </li><li> NS5000 </li></ul> 8.) Microscopie Montez la cellule d&#39;écoulement sur la platine du microscope immédiatement après l&#39;introduction de nanosphères. Dans cette expérience, une Eclipse TE2000-U microscope à fluorescence (Nikon, Melville, NY) équipé d&#39;un objectif huile 100X (NA 1,45), un X-Cite 120 lampe (EXFO, Ontario, Canada) et un iXon EMCCD caméra (ANDOR, South Windsor, CT) a été utilisé. Un cube filtre FITC (# 48001) et un cube TRITC filtre (n ° 48002, Technologies Chroma, Rockingham, VT) ont été utilisés à des nanosphères d&#39;image et microtubules respectivement sur la surface de fond de cellules d&#39;écoulement. Le temps d&#39;exposition est 0.2s, alors que le temps entre l&#39;exposition est de 2 s. <img src="/files/ftp_upload/2006/2006fig1.jpg" alt="Figure 1" /> Figure 1. Schéma des navettes moléculaires.

<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>

Avec des modifications mineures, ce protocole a été utilisé avec succès par une variété de groupes d&#39;assembler kinésine-microtubules tests de motilité base. 10 mM de DTT dans la solution finale motilité peut être remplacé par 0,5% β-mercaptoéthanol. Solutions standard (BRB80AF, KIN20 et MT1000) plus de 2 heures ne doit pas utilisé. Toute solution contenant du taxol microtubules et surtout ne doit jamais être placé sur la glace. Une exposition excessive de la cellule d&#39;écoulement aux UV des résu...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"><html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en"><div><table border="1"><thead><tr><th> Nom du produit </th><th> Type </th><th> Entreprise </th><th> Numéro de catalogue </th><th> Commenter </th></tr></thead><tbody><tr><td> Adénosine-5&#39;-triphosphate (ATP) </td><td></td><td> Invitrogen </td><td> A1049 </td><td></td><tr><td> Tubuline biotine </td><td></td><td> Cytosquelette Inc </td><td> T333 </td><td></td><tr><td> Caséine </td><td></td><td> Sigma-Aldrich </td><td> C-0376 </td><td></td><tr><td> Catalase </td><td></td><td> Sigma-Aldrich </td><td> C-9322 </td><td></td><tr><td> D-(+)-glucose </td><td></td><td> Sigma-Aldrich </td><td> G-7528 </td><td></td><tr><td> Diméthylsulfoxyde (DMSO) </td><td></td><td> Sigma-Aldrich </td><td> D-8779 </td><td> &amp;nbsp; </td><tr><td> Dithiotréitol (TNT) </td><td></td><td> Bio-Rad </td><td> 161-0610 </td><td></td><tr><td> L&#39;acide N, N, N &#39;, N&#39;-tétraacétique (EGTA) - éthylène glycol-bis (2-aminoéthyléther) </td><td></td><td> Sigma-Aldrich </td><td> E-4378 </td><td></td><tr><td> FluoSpheres biotinylé microsphères, 40 nm, jaune-vert fluorescent (505/515) </td><td></td><td> Invitrogen </td><td> F-8766 </td><td></td><tr><td> La glucose oxydase </td><td></td><td> Sigma-Aldrich </td><td> G-7016 </td><td></td><tr><td> Guanosine-5&#39;-triphosphate (GTP) </td><td></td><td> Roche Diagnostic </td><td> 106399 </td><td></td><tr><td> Du chlorure de magnésium (MgCl 2) </td><td></td><td> Sigma-Aldrich </td><td> 63069 </td><td></td><tr><td> Le paclitaxel (Taxol) </td><td></td><td> Sigma-Aldrich </td><td> T1912 </td><td></td><tr><td> 1,4-Piperazinediethanesulfonic acide pipérazine-1 ,4-bis (2-éthanesulfonique), Ppipérazine-N, N&#39;-bis (2-éthanesulfonique) (PIPES) </td><td></td><td> Sigma-Aldrich </td><td> P-6757 </td><td></td><tr><td> L&#39;hydroxyde de potassium (KOH) </td><td></td><td> Sigma-Aldrich </td><td> P-6310 </td><td></td><tr><td> L&#39;hydroxyde de sodium (NaOH) </td><td></td><td> Sigma-Aldrich </td><td> 480878 </td><td></td><tr><td> Streptavidine conjugué Alexa Fluor 568 </td><td></td><td> Invitrogen </td><td> S11226 </td><td></td></tbody></table></div>

cargo loading onto kinesin powered molecular shuttles

Les cellules ont évolué sophistiqué machinerie moléculaire, tels que les protéines motrices kinésine microtubules et de filaments, pour soutenir le transport intracellulaire actif de la cargaison. Alors que kinésines queue domaine se lie à une variété de cargaisons, domaines kinésines tête d&#39;utiliser l&#39;énergie chimique stockée dans les molécules d&#39;ATP à l&#39;étape le long du réseau de microtubules. Les longs microtubules raides servir de pistes pour le transport longue distance intracellulaire. Ces moteurs et des filaments peut également être utilisé dans des environnements synthétiques microfabriqués en tant que composants de navettes moléculaires 1. Dans un motif fréquemment utilisés, les moteurs kinésine sont ancrées à la surface de la piste par le biais de leur queue, et microtubules fonctionnalisés servir éléments de transport de marchandise, qui sont propulsés par ces moteurs. Ces navettes peuvent être chargés de marchandises en utilisant la liaison forte et sélective entre la biotine et la streptavidine. Les composants clés (tubuline biotinylé, streptavidine et biotinylated fret) sont disponibles dans le commerce. En s&#39;appuyant sur ​​le test de motilité inversé classique 2, la construction de navettes moléculaires est détaillée ici. Kinésine protéines motrices sont adsorbés sur une surface préalablement enduite avec de la caséine; microtubules sont polymérisés à partir de tubuline biotinylé, adhéré à la kinésine et ensuite revêtus avec de la rhodamine-streptavidine marquée. La concentration d&#39;ATP est maintenue à une concentration subsaturating pour obtenir une vitesse de glissement des microtubules optimale pour le chargement 3. Enfin, biotinylé marqué à la fluorescéine nanosphères sont ajoutés en tant que cargaison. Nanosphères joindre aux microtubules du fait de collisions entre des microtubules de glisse et des nanosphères adhérant à la surface. Le protocole peut être facilement modifié pour charger une variété de marchandises telles que l&#39;ADN biotinylé 4, 5 ou points quantiques d&#39;une grande variété d&#39;antigènes au moyen d&#39;anticorps biotinylés 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

Navettes moléculaires constitués de microtubules fonctionnalisés glissant sur la surface collée protéines motrices kinésine peut servir de système de transport nanométrique. Ici, le montage d&#39;un système de navette typique est décrit.

Chargement du fret sur la kinésine Powered navettes moléculaires

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

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Biology

10.4K vues.  University of Florida. Navettes moléculaires constitués de microtubules fonctionnalisés glissant sur la surface collée protéines motrices kinésine peut servir de système de transport nanométrique. Ici, le montage d'un système de navette typique est décrit.

Vidéo: 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 Chargement mécanique du 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 ...

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Biologie

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.

Terminaux de chargement nerveuse chez la drosophile avec des indicateurs de calcium

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.

Molecular Evolution de la recombinase 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.

De FAE au Matrigel 2: Fractionnement des CSEh MEFs sur 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.

De FAE au Matrigel 3: Le repiquage du Matrigel CSEh sur 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.

Assemblée, de chargement, et l&#39;alignement d&#39;une cellule échantillon ultracentrifugation analytique

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.

Isolement et purification de la kinésine de Drosophila Embryons

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.

Imagerie directe du calcium ER avec Targeted-estérase colorant induit Chargement (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 &quot;Humanized&quot; 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.

Génération d&#39;un «humanisé»<em&gt; Drosophila</em&gt; S2 Cell Line Sensible à pharmacologique inhibition de 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.

Identification des Kinesin-1 Cargos utilisant microscopie à fluorescence

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