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

Research

JoVE Journal

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

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Production d'ensembles moteurs d'adn et de kinésine sur des nanostructures d'origami d'ADN pour l'observation d'une seule molécule

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

Recherche

Biochimie

Assembling Molecular Shuttles Powered by Reversibly Attached Kinesins

This protocol describes how to create kinesin-powered molecular shuttles with a weak and reversible attachment of the kinesins to the surface. In contrast to previous protocols, in this system, microtubules recruit kinesin motor proteins from solution and place them on a surface. The kinesins will, in turn, facilitate the gliding of the microtubules along the surface before desorbing back into the bulk solution, thus being available to be recruited again. This continuous assembly and disassembly leads to striking dynamic behavior in the system, such as the formation of temporary kinesin trails by gliding microtubules.
Several experimental methods will be described throughout this experiment: UV-Vis spectrophotometry will be used to determine the concentration of stock solutions of reagents, coverslips will first be ozone and ultraviolet (UV) treated and then silanized before being mounted into flow cells, and total internal reflection fluorescence (TIRF) microscopy will be used to simultaneously image kinesin motors and microtubule filaments.

We present a protocol to build molecular shuttles, where surface-adhered kinesin motor proteins propel dye-labelled microtubules. Weak interactions of the kinesins with the surface enables their reversible attachment to it. This creates a nanoscale system which exhibits dynamic assembly and disassembly of its components while retaining its functionality.

Assemblage des navettes moléculaires propulsés par réversiblement attaché kinésines

This protocol describes how to create kinesin-powered molecular shuttles with a weak and reversible attachment of the kinesins to the surface. In contrast ...

Ingénierie

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.

Motilité de molécules uniques et d’amas de kinésine-5 Cin8 bidirectionnelle purifiée à partir de cellules de S. cerevisiae

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of ...

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate (&#8220;map&#8221;) the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. &#8220;Cargo mapping&#8221; consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to &#8220;map&#8221; them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.

Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

Caractériser la composition de moteurs moléculaires sur les moyens axonale Cargo Utilisation de l&#39;analyse &quot;Cargo Mapping&quot;

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

Neurosciences

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

Analyse à molécule unique de moteurs de la famille Sf9 superprocessive kinésine-3 purifiée

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

Molécules simples à l&#39;imagerie du transport nucléaire

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Biologie

Measuring Axonal Cargo Transport in Mouse Primary Cortical Cultured Neurons

Neuronal cells are highly polarized cells that stereotypically harbor several dendrites and an axon. The length of an axon necessitates efficient bidirectional transport by motor proteins. Various reports have suggested that defects in axonal transport are associated with neurodegenerative diseases. Also, the mechanism of the coordination of multiple motor proteins has been an attractive topic. Since the axon has uni-directional microtubules, it is easier to determine which motor proteins are involved in the movement. Therefore, understanding the mechanisms underlying the transport of axonal cargo is crucial for uncovering the molecular mechanism of neurodegenerative diseases and the regulation of motor proteins. Here, we introduce the entire process of axonal transport analysis, including the culturing of mouse primary cortical neurons, transfection of plasmids encoding cargo proteins, and directional and velocity analyses without the effect of pauses. Furthermore, the open-access software "KYMOMAKER" is introduced, which enables the generation of a kymograph to highlight transport traces according to their direction and allow easier visualization of axonal transport.

The present protocol describes the whole procedure of analysis for axonal transport. In particular, the calculation of transport velocity, not including the pausing, and the visualization method using open-access software "KYMOMAKER" are shown here.

Neuronal cells are highly polarized cells that stereotypically harbor several dendrites and an axon. The length of an axon necessitates efficient ...

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 ...

Microtubule Associated Motor Proteins

Eukaryotic cells have different motor proteins for transporting various cargo within the cell. These motor proteins differ based on the filament they associate with, the direction they move within the cell, and the type of cargo they transport. Motor proteins that associate with microtubules are known as microtubule-associated motor proteins. There are two families of microtubule-associated motor proteins &#8212;Kinesins and Dyneins. Both these proteins assist in the transport of cellular cargos within the cell by hydrolyzing ATP molecules. The wide spectrum of cellular cargos transported includes organelles, vesicles, protein complexes, chromosomes, RNA-protein complexes, etc.
Kinesins
Kinesins are found in all eukaryotes.&#160; The human genome has forty-five genes encoding kinesin proteins, Arabidopsis thaliana has sixty, while the budding yeast Saccharomyces cerevisiae has six. Kinesin-1 (conventional kinesin) was the first molecular motor protein discovered in a squid neuron. It is involved in transporting cellular cargo via the microtubules. These motor proteins have a highly conserved motor domain of &#8764;340 amino-acid residues at its N-terminal, with some exceptions. The kinesin superfamily is broadly divided into 14 families with distinct members based on their conserved motor domains. These proteins are plus end-directed, i.e., the transport of the organelles and vesicles occurs from the center of the cell towards the cell periphery, showing the centrifugal movement of cellular cargos. This motor protein superfamily members are also involved in microtubule destabilization, DNA repair, transcription regulation, mitotic spindle assembly, and cell signaling during cell growth regulation.
Dyneins
Dynein is a family of minus-end directed motor proteins, i.e., it transports cellular cargos from the cell periphery towards the center of the cell, showing centripetal movement. They were initially identified from the core of eukaryotic cilia and flagella. The core, also termed axoneme, is where the dyneins make the microtubules slide, resulting in the characteristic whip-like movement of cilia and flagella. These motor proteins are widely present in eukaryotes except for most flowering plants, which lack dynein motor proteins.
Dyneins are classified as axonemal and cytoplasmic based on their location and functions. Cytoplasmic dynein is responsible for intraflagellar transport, while axonemal dynein is involved in the locomotory function of cilia and flagella. During interphase, these motor proteins transport vesicles, organelles,&#160; proteins, and mRNA particles, while in dividing cells, these proteins are responsible for spindle assembly.

Protéines motrices associées aux microtubules

Eukaryotic cells have different motor proteins for transporting various cargo within the cell. These motor proteins differ based on the filament they ...

Enseignement

JoVE Core

Biologie Cellulaire

Unité 1

The Cytoskeleton II: Microtubules and Intermediate Filaments

TERMES ET CONCEPTS CLÉS

The Movement of Organelles and Vesicles

In eukaryotic cells,&#160; cytoskeletal filaments such as actin, microtubules, and intermediate filaments form a mesh-like cytoskeletal network. These filaments serve as tracks for transporting cellular cargo. Specialized motor proteins use the chemical energy stored in adenosine triphosphate (ATP) for this transport. During interphase, microtubules are polarized, with the plus-end towards the cell periphery and the minus-end towards the cell center. Two microtubule-associated motor proteins, kinesin and cytoplasmic dynein, transport organelles and vesicles.
Transport via kinesins
Kinesin is a plus-end-directed, microtubule-associated motor protein made up of two heavy chains and two light chains. Kinesin motors are highly efficient as they undergo hundreds of ATP hydrolysis cycles without dissociating from the microtubules while transporting cellular cargos. During interphase, kinesins transport organelles and vesicles across the cell. The kinesins attach to specific vesicles or organelles with the help of the receptor domain present in its light chain. Some kinesin motor proteins have also shown preferential binding for post-translationally modified microtubules. For example,&#160; acetylation and detyrosination of microtubules alter the binding and mobility of kinesin-1. This selective binding plays a crucial role in the axonal cargo transport within the neurons.
Transport via dyneins
Dyneins are minus-end-directed motor proteins. These motors are responsible for carrying out crucial functions within the cell, such as the arrangement of axonal complex microtubule assemblies, signal transduction within the primary cilium, and positioning of Golgi apparatus near the cell center. Among the two dyneins present in the cell, cytoplasmic dynein is primarily responsible for transporting organelles and vesicles. Dynein has two globular heads attached to two stalks and intermediate and light chains that form the upper domain. ATP hydrolysis takes place within the globular heads. The movement of cytoplasmic dynein resembles walking, where one stalk moves ahead, followed by the other. The large cytoplasmic dynein molecule cannot bind directly to the organelles or vesicles; it requires an additional protein called dynactin. Dynactin has a short actin-like filament, Arp1. The Arp-1 domain recognizes and attaches to the receptor domain of the organelles or vesicles. The cytoplasmic dynein and dynactin are activated only after binding with a specific organelle or vesicle, forming a tripartite complex. The tripartite complex then transports the organelles and vesicles.

Le mouvement des organites et des vésicules

In eukaryotic cells,&#160; cytoskeletal filaments such as actin, microtubules, and intermediate filaments form a mesh-like cytoskeletal network. These ...

Chargement du fret sur la kinésine Powered navettes moléculaires

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