Le plasmide GFP-β-actine a été généreusement fourni par le Dr Wayne Orr, LSUHSC-S Département de pathologie, et a été amplifié dans le laboratoire du Dr Becky Worthylake, LSUHSC-NO Département de pharmacologie. Ce travail a été soutenu par des subventions des National Institutes of Health (P20 RR-018 766) et l&#39;American Heart Association (05835386N).

<ol>
	<li>Spindler, V., Schlegel, N., Waschke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+GTPases+in+control+of+microvascular+permeability.">Role of GTPases in control of microvascular permeability.</a> Cardiovasc. Res. 87, 243-253 (2010).</li><li>Wojciak-Stothard, B., Ridley, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+GTPases+and+the+regulation+of+endothelial+permeability.">Rho GTPases and the regulation of endothelial permeability.</a> Vascul. Pharmacol. 39, 187-199 (2002).</li><li>Yuan, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+pathways+in+enhanced+microvascular+permeability.">Signal transduction pathways in enhanced microvascular permeability.</a> Microcirculation. 7, 395-403 (2000).</li><li>Birukov, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+GTPases+in+mechanosensitive+regulation+of+endothelial+barrier.">Small GTPases in mechanosensitive regulation of endothelial barrier.</a> Microvasc. Res. 77, 46-52 (2009).</li><li>Duran, W. N., Sanchez, F. A., Breslin, J. W., Tuma, R. F., Duran, W. N., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcirculatory+Exchange+Function.">Microcirculatory Exchange Function.</a> Handbook of Physiology: Microcirculation. , 81-124 (2008).</li><li>Hu, Y. L., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+motion+of+paxillin+on+actin+filaments+in+living+endothelial+cells.">Dynamic motion of paxillin on actin filaments in living endothelial cells.</a> Biochem. Biophys. Res. Commun. 357, 871-876 (2007).</li><li>Borm, B., Requardt, R. P., Herzog, V., Kirfel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+ruffles+in+cell+migration:+indicators+of+inefficient+lamellipodia+adhesion+and+compartments+of+actin+filament+reorganization.">Membrane ruffles in cell migration: indicators of inefficient lamellipodia adhesion and compartments of actin filament reorganization.</a> Exp. Cell. Res. 302, 83-95 (2005).</li><li>Hotulainen, P., Lappalainen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+fibers+are+generated+by+two+distinct+actin+assembly+mechanisms+in+motile+cells.">Stress fibers are generated by two distinct actin assembly mechanisms in motile cells.</a> J. Cell. Biol. 173, 383-394 (2006).</li><li>Pellegrin, S., Mellor, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+stress+fibres.">Actin stress fibres.</a> J. Cell. Sci. 120, 3491-3499 (2007).</li><li>Ballestrem, C., Wehrle-Haller, B., Imhof, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+dynamics+in+living+mammalian+cells.">Actin dynamics in living mammalian cells.</a> J. Cell. Sci. 111, 1649-1658 (1998).</li><li>Helmke, B. P., Rosen, A. B., Davies, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+mechanical+strain+of+an+endogenous+cytoskeletal+network+in+living+endothelial+cells.">Mapping mechanical strain of an endogenous cytoskeletal network in living endothelial cells.</a> Biophys. J. 84, 2691-2699 (2003).</li><li>Birukova, A. A., Smurova, K., Birukov, K. G., Kaibuchi, K., Garcia, J. G., Verin, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Rho+GTPases+in+thrombin-induced+lung+vascular+endothelial+cells+barrier+dysfunction.">Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction.</a> Microvasc. Res. 67, 64-77 (2004).</li><li>van Nieuw Amerongen, G. P., van Delft, S., Vermeer, M. A., Collard, J. G., van Hinsbergh, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+RhoA+by+thrombin+in+endothelial+hyperpermeability:+role+of+Rho+kinase+and+protein+tyrosine+kinases.">Activation of RhoA by thrombin in endothelial hyperpermeability: role of Rho kinase and protein tyrosine kinases.</a> Circ. Res. 87, 335-340 (2000).</li><li>Moy, A. B., Blackwell, K., Kamath, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+histamine+and+thrombin+on+endothelial+barrier+function+through+actin-myosin+tension.">Differential effects of histamine and thrombin on endothelial barrier function through actin-myosin tension.</a> Am J Physiol Heart Circ Physiol. 282, H21-H29 (2002).</li><li>Wittman, T., Littlefield, R., Waterman-Storer, C., Goldman, R. D., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+speckle+microscopy+of+cytoskeletal+dynamics+in+living+cells.">Fluorescent speckle microscopy of cytoskeletal dynamics in living cells.</a> Live Cell Imaging: A Laboratory Manual. , 184-204 (2005).</li><li>Rabut, G., Ellenberg, J., Goldman, R. D., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobleaching+techniques+to+study+mobility+and+molecular+dynamics+of+proteins+in+live+cells:+FRAP,+iFRAP,+and+FLIP.">Photobleaching techniques to study mobility and molecular dynamics of proteins in live cells: FRAP, iFRAP, and FLIP.</a> Live Cell Imaging: A Laboratory Manual. , 101-126 (2005).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

L&#39;imagerie de la GFP-actine dans les cellules endothéliales en direct permet une analyse détaillée de la dynamique du cytosquelette d&#39;actine en réponse aux stimuli inflammatoires. Cette méthode peut également être utile de s&#39;appuyer sur les conclusions antérieures montrant remodelage du cytosquelette en réponse aux forces physiques comme la contrainte de cisaillement 11. En outre, cette méthode permet une évaluation détaillée de la contribution de la dynamique de l&#39;actine du cytosquelette à diverses activités de la cellule endothéliale, y compris la migration, la mitose, la formation des jonctions intercellulaires et la maturation jonctionnelle, et le maintien de la fonction de barrière. Dans les données présentées, le comportement du cytosquelette d&#39;actine endothéliales peuvent être observées avant et après traitement par la thrombine. Lamellipodes locale tout au long des bords des cellules endothéliales ont été observées former et régresse au fil du temps dans les monocouches de cellules à la fois nonconfluent et confluentes. Le traitement par la thrombine brièvement interrompu la forme lamellipodesation et le chiffre d&#39;affaires. La thrombine a également causé des cellules de se contracter légèrement, en accord avec les précédents rapports que la thrombine provoque la formation de fibres d&#39;actine stress et les tensions centripètes développement accru dans les cellules endothéliales 12-14. Cependant, à partir des études d&#39;imagerie des cellules vivantes de ce genre, l&#39;origine des fibres de stress peuvent désormais être déterminé. En HUVEC, la plupart des fibres de stress proviennent à la périphérie des cellules et des fibres ressemblent à l&#39;arc dans la migration de cellules transversales 8, 9. Une autre force de cette méthode sur l&#39;utilisation de cellules fixes est que le nombre de fibres de stress peuvent être quantifiés dans des cellules individuelles avant et après traitement de thrombine, en éliminant les biais de sélection entre les groupes expérimentaux. Avec ce protocole, nous évaluons le mouvement dynamique de la bordure des cellules et des fibres de stress d&#39;actine. Pour comprendre la dynamique de monomère d&#39;actine dans les cellules endothéliales, des techniques plus avancées telles que le recouvrement de fluorescence après photoblanchiment (FRAP) ou de fluorescence speckle microscopie (FSM) peut être appliquée 15, 16. En outre, parce cellules endothéliales microvasculaires peuvent représenter un meilleur modèle de la fonction de barrière microvasculaire, l&#39;optimisation des protocoles de transfection pour exprimer efficacement GFP-actine dans les cellules endothéliales microvasculaires représente une direction future logique. En résumé, l&#39;imagerie des cellules vivantes endothéliales exprimant la GFP-actine représente un outil puissant pour déterminer comment le cytosquelette d&#39;actine des cellules endothéliales répond à différents types de stimuli. Des études utilisant des monocouches confluentes étroitement endothéliales aideront à déterminer les rôles des structures dynamiques telles que l&#39;actine riches lamellipodes et transversale des fibres de stress d&#39;arc en fonction de la barrière endothéliale. En outre, imagerie des cellules vivantes des cellules endothéliales exprimant la GFP-actine ou d&#39;autres protéines de fusion qui permettent de visualiser d&#39;autres structures subcellulaires fournira des informations détaillées spatio-temporelle nécessaire pour comprendre la signalisation et les mécanismes structurels qui dintégrité de la barrière éterminer.

<table border="1"><tbody><tr><td colspan="4"> 1. Ringer stock 5x </td></tr><tr><td> Chimique </td><td> Société </td><td> Nombre de catalogue </td><td> Montant </td></tr><tr><td> Chlorure de sodium </td><td> EMD </td><td> SX0420-3 </td><td> 35 g </td></tr><tr><td> Chlorure de Potassium </td><td> JT Baker </td><td> 3040 </td><td> 1,75 g </td></tr><tr><td> Chlorure de calcium </td><td> Sigma </td><td> C-3881 </td><td> 1,47 g </td></tr><tr><td> Sulfate de magnésium </td><td> Sigma </td><td> M-9397 </td><td> 1,44 g </td></tr><tr><td> Stérile eau filtrée </td><td> N / A </td><td> N / A </td><td> Porter à 1 L </td></tr><tr><td colspan="4"> Filtre stérile dans des flacons à l&#39;autoclave et les magasins à 4 ° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 2. Tampon MOPS </td></tr><tr><td> Chimique </td><td>Société </td><td> Nombre de catalogue </td><td> Montant </td></tr><tr><td> MOPS </td><td> Sigma </td><td> M3183 </td><td> 125,6 g </td></tr><tr><td> Stérile eau filtrée </td><td> N / A </td><td> N / A </td><td> Porter à 1 L </td></tr><tr><td colspan="4"> Filtre stérile dans des flacons à l&#39;autoclave et les magasins à 4 ° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 3. L&#39;albumine du sérum physiologique (APSS) </td></tr><tr><td> Chimique </td><td> Société </td><td> Nombre de catalogue </td><td> Montant </td></tr><tr><td> Ringer stock (5x) </td><td> N / A </td><td> N / A </td><td> 200 ml </td></tr><tr><td> Tampon MOPS </td><td> N / A </td><td> N / A </td><td> 5 ml </td></tr><tr><td> Phosphate de sodium </td><td> Sigma </td><td> S-9638 </td><td> 0,168 g </td></tr><tr><td> Pyruvate de sodium &lt;/ Td&gt; <td> Sigma </td><td> P5280 </td><td> 0,22 g </td></tr><tr><td> Sel de sodium EDTA </td><td> Sigma </td><td> ED2SS </td><td> 0,0074 g </td></tr><tr><td> Le glucose </td><td> Sigma </td><td> G7528 </td><td> 0,901 g </td></tr><tr><td> L&#39;albumine, bovine </td><td> USB </td><td> 10856 </td><td> 10 g </td></tr><tr><td> Stérile eau filtrée </td><td> N / A </td><td> N / A </td><td> Porter à 1 L </td></tr><tr><td colspan="4"> Ajuster le pH à 7,4 à 37 ° C, puis filtre stérile dans des flacons à l&#39;autoclave et conserver à 4 ° C. </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 4. Saline à 0,9% </td></tr><tr><td> Chimique </td><td> Société </td><td> Nombre de catalogue </td><td> Montant </td></tr><tr><td> Chlorure de sodium </td><td> EMD </td><td> SX0420-3 </td><td> 9 g </td></tr><tr><td> Stérile eau filtrée </td><td> N / A </td><td> N / A </td><td> Porter à 1 L </td></tr><tr><td colspan="4"> Filtre stérile dans des flacons à l&#39;autoclave et les magasins à 4 ° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 5. 1,5% une solution de gélatine </td></tr><tr><td> Gélatine de la peau de porc </td><td> Sigma </td><td> G2500 </td><td> 15 g </td></tr><tr><td> Saline à 0,9% </td><td> N / A </td><td> N / A </td><td> Porter à 1 L </td></tr><tr><td colspan="4"> Chauffer la solution à 37 ° C pour dissoudre la gélatine suffisamment. Alors qu&#39;il était encore chaud, filtre stérile dans des flacons à l&#39;autoclave et conserver à 4 ° C </td></tr></tbody></table>

study of the actin cytoskeleton in live endothelial cells expressing gfp-actin

L&#39;endothélium microvasculaire joue un rôle important comme une barrière sélective perméable aux fluides et des solutés. Les jonctions d&#39;adhérence entre les cellules endothéliales régulation de la perméabilité de l&#39;endothélium, et de nombreuses études ont montré la contribution importante du cytosquelette d&#39;actine à la détermination de l&#39;intégrité jonctionnelle 1-5. Une ceinture d&#39;actine corticale est pensé pour être important pour le maintien de la stabilité des jonctions 1, 2, 4, 5. En revanche, les fibres de stress d&#39;actine sont censés générer des tensions centripètes dans les cellules endothéliales qui affaiblit les jonctions 2-5. Une grande partie de cette théorie a été basée sur des études dans lesquelles les cellules endothéliales sont traités avec des médiateurs inflammatoires connus pour augmenter la perméabilité endothéliale, puis fixer les cellules et l&#39;étiquetage de F-actine à l&#39;observation microscopique. Cependant, ces études fournissent une compréhension très limitée du rôle du cytosquelette d&#39;actine, car des images de cellules fixes fournissent des instantanés seulement danstemps avec aucune information sur la dynamique des structures d&#39;actine 5. Imagerie des cellules vivantes permet l&#39;incorporation de la nature dynamique du cytosquelette d&#39;actine dans les études des mécanismes de détermination intégrité de la barrière endothéliale. Un avantage majeur de cette méthode est que l&#39;impact de divers stimuli inflammatoires sur les structures d&#39;actine dans les cellules endothéliales peuvent être évalués dans le même ensemble de cellules vivantes avant et après traitement, en supprimant le biais potentiel pouvant survenir lors de l&#39;observation des échantillons fixés. Les cellules humaines endothéliales de veine ombilicale (HUVEC) sont transfectées avec GFP-β une actine plasmide et cultivées jusqu&#39;à confluence sur lamelles de verre. Time-lapse images de GFP-actine dans confluentes HUVEC sont capturés avant et après l&#39;ajout de médiateurs inflammatoires qui provoquent des changements dépendant du temps dans l&#39;intégrité de barrière endothéliale. Ces études permettent une observation visuelle de la séquence fluide de changements dans le cytosquelette d&#39;actine qui contribuent à des endothéliumsrupture de la barrière l et de restauration. Nos résultats montrent constamment locale, riche en actine formation de lamellipodes et le roulement dans les cellules endothéliales. La formation et le mouvement des fibres de stress d&#39;actine peuvent également être observés. Une analyse de la fréquence de la formation et la rotation de la section locale lamellipodes, avant et après traitement avec des stimuli inflammatoires peuvent être documentés par des analyses kymographe. Ces études fournissent des informations importantes sur la nature dynamique du cytosquelette d&#39;actine dans les cellules endothéliales qui peuvent servir à découvrir les mécanismes moléculaires non identifiés auparavant important pour le maintien de l&#39;intégrité barrière endothéliale.

Watch this Scientific Journal Video about Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin at JoVE.com

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

Imagerie microscopique des cellules endothéliales direct exprimant la GFP-actine permet la caractérisation des changements dynamiques dans les structures du cytosquelette. Contrairement aux techniques qui utilisent des échantillons fixés, cette méthode fournit une évaluation détaillée des changements temporels dans le cytosquelette d&#39;actine dans les mêmes cellules, avant, pendant, et après divers stimuli physiques, pharmacologiques ou inflammatoires.

Étude du cytosquelette d&#39;actine dans les cellules endothéliales en direct exprimant la GFP-actine

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

study-actin-cytoskeleton-live-endothelial-cells-expressing-gfp

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Unit 1

The Cytoskeleton I: Actin and Microfilaments

SCIENTISTS IN ACTION

27.0K vues.  Louisiana State University Health Sciences Center. Imagerie microscopique des cellules endothéliales direct exprimant la GFP-actine permet la caractérisation des changements dynamiques dans les structures du cytosquelette. Contrairement aux techniques qui utilisent des échantillons fixés, cette méthode fournit une évaluation détaillée des changements temporels dans le cytosquelette d'actine dans les mêmes cellules, avant, pendant, et après divers stimuli physiques, pharmacologiques ou inflammatoires.

Vidéo: Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test sensibilité visuelle à la vitesse et la direction du mouvement chez les lézards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Recherche

Biologie

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Computer-Generated Stimuli modèle animal

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Co-actine sédimentation Assay; pour l&#39;analyse des Binding Protein à F-actine

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

Imagerie de cellules vivantes de la F-actine fluorescente Dynamics via chatoiement de microscopie (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

Imagerie en temps réel de la motilité cellulaire et cytosquelette d&#39;actine des neurones individuels et de la crête neurale dans les embryons de poissons zèbres

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

La transplantation de blastomères exprimant la GFP pour l&#39;imagerie en direct sur le développement de la rétine et du cerveau dans les embryons de poissons zèbres chimériques

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself dispositif de récupération d&#39;échantillons cryoconservés tombé accidentellement dans les réservoirs de stockage cryogéniques

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Vivre imagerie de dédouanement cellulaire par apoptose au cours<em&gt; Drosophila</em&gt; Embryogenèse

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Microscopie intravitale pour l&#39;imagerie Subcellular ouvrages chez des souris vivantes exprimant des protéines fluorescentes

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Des cellules endothéliales Tube Formation test pour la<em&gt; In Vitro</em&gt; Etude de l&#39;angiogenèse

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...