El plásmido GFP-β-actina fue generosamente proporcionado por el Dr. Wayne Orr, LSUHSC-S del Departamento de Patología, y se amplificó en el laboratorio del Dr. Becky Worthylake, LSUHSC-NO Departamento de Farmacología. Este trabajo fue apoyado por becas de los Institutos Nacionales de Salud (P20 RR-018766) y la Asociación Americana del Corazón (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>

No hay conflictos de interés declarado.

La imagen de la GFP-actina en las células endoteliales en vivo permite un análisis detallado de la dinámica del citoesqueleto de actina en respuesta a estímulos inflamatorios. Este método también puede ser útil para construir sobre los hallazgos previos que muestran que la remodelación del citoesqueleto en respuesta a las fuerzas físicas como la tensión de corte 11. Además, este método permite una evaluación detallada de la contribución de la dinámica del citoesqueleto de actina a las diversas actividades de las células endoteliales, como la migración, la mitosis, la formación de uniones intercelulares y la maduración de la unión, y el mantenimiento de la función de barrera. En los datos presentados, el comportamiento del citoesqueleto de actina endotelial se puede observar antes y después del tratamiento con trombina. Lamellipodia locales a lo largo de los bordes de las células endoteliales se observó la formación y la regresión en el tiempo en monocapas de células tanto nonconfluent y confluente. El tratamiento con trombina interrumpió brevemente la forma lamellipodiación y el volumen de ventas. La trombina también hizo que las células se contraen ligeramente, de acuerdo con los informes anteriores que la trombina hace que el estrés de actina formación de la fibra y el desarrollo de aumento de la tensión centrípeta en las células endoteliales 12-14. Sin embargo, a partir de estudios de células en vivo de imágenes de este tipo, el origen de las fibras de estrés se puede determinar. En HUVEC, la mayoría de las fibras de estrés se originan en la periferia de la célula y se asemejan a las fibras transversales del arco en la migración de las células 8, 9. Otro punto fuerte de este método sobre el uso de células fijas es que el número de fibras de estrés pueden ser cuantificados en las células antes y después del tratamiento de trombina, lo que elimina el sesgo de selección entre los grupos experimentales. Con este protocolo se evalúa el movimiento dinámico del borde de la celda y las fibras de actina estrés. Para entender la dinámica de actina monómeros en las células endoteliales, las técnicas más avanzadas como la recuperación de fluorescencia después de photobleaching (FRAP) o fluorescencia moteado microscopia (FSM) se puede aplicar 15, 16. Además, debido a las células endoteliales microvasculares pueden representar un mejor modelo de la función de barrera microvascular, la optimización de los protocolos de transfección para expresar eficazmente GFP-actina en las células endoteliales microvasculares representa una futura dirección lógica. En resumen, las imágenes en vivo de las células endoteliales que expresan GFP-actina constituye un poderoso instrumento para determinar cómo el citoesqueleto de actina de células endoteliales responden a diversos tipos de estímulos. Los estudios que utilizan monocapas endoteliales confluentes bien le ayudará a determinar las funciones de las estructuras dinámicas como la actina-ricos lamellipodia y transversal las fibras de estrés de arco en función de barrera endotelial. Además, imágenes de células vivas de las células endoteliales que expresan GFP-actina y otras proteínas de fusión que permiten la visualización de otras estructuras subcelulares proporcionará información detallada espacio-temporal necesaria para comprender la señalización y los mecanismos estructurales que determine integridad de la barrera.

<table border="1"><tbody><tr><td colspan="4"> 1. Ringer 5x de archivo </td></tr><tr><td> Químico </td><td> Empresa </td><td> Número de catálogo </td><td> Cantidad </td></tr><tr><td> Cloruro de sodio </td><td> EMD </td><td> SX0420-3 </td><td> 35 g </td></tr><tr><td> Cloruro de Potasio </td><td> JT Baker </td><td> 3040 </td><td> 1,75 g </td></tr><tr><td> Cloruro de calcio </td><td> Sigma </td><td> C-3881 </td><td> 1,47 g </td></tr><tr><td> Sulfato de magnesio </td><td> Sigma </td><td> M-9397 </td><td> 1,44 g </td></tr><tr><td> El agua filtrada estéril </td><td> N / A </td><td> N / A </td><td> Llevar a 1 L </td></tr><tr><td colspan="4"> Filtro estéril en botellas autoclave y se almacena a 4 º C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 2. MOPS buffer </td></tr><tr><td> Químico </td><td>Empresa </td><td> Número de catálogo </td><td> Cantidad </td></tr><tr><td> MOPS </td><td> Sigma </td><td> M3183 </td><td> 125,6 g </td></tr><tr><td> El agua filtrada estéril </td><td> N / A </td><td> N / A </td><td> Llevar a 1 L </td></tr><tr><td colspan="4"> Filtro estéril en botellas autoclave y se almacena a 4 º C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 3. Solución de albúmina salina fisiológica (APSS) </td></tr><tr><td> Químico </td><td> Empresa </td><td> Número de catálogo </td><td> Cantidad </td></tr><tr><td> Timbre de valores (5x) </td><td> N / A </td><td> N / A </td><td> 200 ml </td></tr><tr><td> Trapeadores Buffer </td><td> N / A </td><td> N / A </td><td> 5 ml </td></tr><tr><td> Fosfato de sodio </td><td> Sigma </td><td> S-9638 </td><td> 0,168 g </td></tr><tr><td> El piruvato de sodio &lt;/ Td&gt; <td> Sigma </td><td> P5280 </td><td> 0,22 g </td></tr><tr><td> EDTA sal sódica </td><td> Sigma </td><td> ED2SS </td><td> 0,0074 g </td></tr><tr><td> Glucosa </td><td> Sigma </td><td> G7528 </td><td> 0,901 g </td></tr><tr><td> Albúmina, bovina </td><td> USB </td><td> 10856 </td><td> 10 g </td></tr><tr><td> El agua filtrada estéril </td><td> N / A </td><td> N / A </td><td> Llevar a 1 L </td></tr><tr><td colspan="4"> Ajustar el pH de 7,4 a 37 ° C, luego de filtro estéril en botellas autoclave y se almacena a 4 º C. </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 4. 0,9% </td></tr><tr><td> Químico </td><td> Empresa </td><td> Número de catálogo </td><td> Cantidad </td></tr><tr><td> Cloruro de sodio </td><td> EMD </td><td> SX0420-3 </td><td> 9 g </td></tr><tr><td> El agua filtrada estéril </td><td> N / A </td><td> N / A </td><td> Llevar a 1 L </td></tr><tr><td colspan="4"> Filtro estéril en botellas autoclave y se almacena a 4 º C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 5. 1,5% solución de gelatina </td></tr><tr><td> La gelatina de piel porcina </td><td> Sigma </td><td> G2500 </td><td> 15 g </td></tr><tr><td> 0,9% </td><td> N / A </td><td> N / A </td><td> Llevar a 1 L </td></tr><tr><td colspan="4"> Caliente la solución a 37 ° C para disolver la gelatina suficiente. Mientras aún está caliente, filtro estéril en botellas autoclave y se almacena a 4 º C </td></tr></tbody></table>

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

El endotelio microvascular juega un papel importante como una barrera selectivamente permeable a los líquidos y solutos. Las uniones adhesivas entre las células endoteliales regulan la permeabilidad del endotelio, y muchos estudios han indicado la importante contribución del citoesqueleto de actina a la determinación de la integridad de la unión 1-5. Una banda de actina cortical se cree que es importante para el mantenimiento de la estabilidad uniones 1, 2, 4, 5. Por el contrario, las fibras de actina estrés se cree que generan tensión centrípeta dentro de las células endoteliales, que debilita las uniones 2-5. Gran parte de esta teoría se ha basado en estudios en los que las células endoteliales son tratados con mediadores inflamatorios que aumentan la permeabilidad endotelial, y la fijación de las células y el etiquetado de F-actina para la observación microscópica. Sin embargo, estos estudios proporcionan una comprensión muy limitada de la función del citoesqueleto de actina de las células ya que las imágenes fijas proporcionan instantáneas sólo entiempo que no tienen información sobre la dinámica de las estructuras de actina 5. Imágenes de células vivas, permite la incorporación de la naturaleza dinámica del citoesqueleto de actina en los estudios de los mecanismos que determinan la integridad de la barrera endotelial. Una gran ventaja de este método es que el impacto de los diversos estímulos inflamatorios en las estructuras de actina en las células endoteliales puede ser evaluada en el mismo conjunto de células vivas, antes y después del tratamiento, la eliminación de los posibles sesgos que pueden ocurrir al observar las muestras fijadas. Humanos vena umbilical células endoteliales (HUVEC) se transfectaron con GFP-β-actina de plásmidos y crecido a la confluencia en cubreobjetos de vidrio. Time-lapse imágenes de GFP-actina en HUVEC confluentes son capturados antes y después de la adición de mediadores inflamatorios que provocan cambios dependientes del tiempo en la integridad de la barrera endotelial. Estos estudios permiten la observación visual de la secuencia fluida de los cambios en el citoesqueleto de actina que contribuyen al endoteliol alteración de la barrera y la restauración. Nuestros resultados muestran de forma consistente local, rica en actina formación lamellipodia y la rotación en las células endoteliales. La formación y el movimiento de las fibras de actina estrés también puede ser observado. Un análisis de la frecuencia de formación y la rotación de los locales lamellipodia, antes y después del tratamiento con estímulos inflamatorios pueden ser documentados por los análisis quimógrafo. Estos estudios proporcionan información importante sobre la naturaleza dinámica del citoesqueleto de actina en las células endoteliales que pueden utilizarse para descubrir los mecanismos moleculares previamente no identificado importante para el mantenimiento de la integridad de la barrera endotelial.

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

Imágenes microscópicas de vida las células endoteliales que expresan GFP-actina permite la caracterización de los cambios dinámicos en las estructuras del citoesqueleto. A diferencia de las técnicas que utilizan las muestras fijadas, este método proporciona una evaluación detallada de los cambios temporales en el citoesqueleto de actina en las células del mismo, antes, durante y después de diversos estímulos físicos, farmacológicos o inflamatorios.

Estudio del citoesqueleto de actina en vivo las células endoteliales que expresan GFP-actina

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 vistas.  Louisiana State University Health Sciences Center. Imágenes microscópicas de vida las células endoteliales que expresan GFP-actina permite la caracterización de los cambios dinámicos en las estructuras del citoesqueleto. A diferencia de las técnicas que utilizan las muestras fijadas, este método proporciona una evaluación detallada de los cambios temporales en el citoesqueleto de actina en las células del mismo, antes, durante y después de diversos estímulos físicos, farmacológicos o inflamatorios.

Video: 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.

Prueba de sensibilidad visual a la velocidad y dirección del movimiento en lagartos

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

Investigación

Biología

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.

Los estímulos generados por ordenador Modelo 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.

Actina Co-sedimentación de ensayo, pues el análisis de proteína de unión a la F-actina

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).

Imágenes de células vivas de la F-actina Dynamics a través de Microscopía Fluorescente Speckle (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.

Imágenes en directo de la motilidad celular y citoesqueleto de actina de las neuronas individuales y las células de la cresta neural en embriones de pez cebra

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.

El trasplante de las buenas prácticas agrarias-que expresa Blastómeros de imágenes en vivo del desarrollo de la retina y el cerebro en embriones de pez cebra quiméricos

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 de dispositivos para la recuperación de las muestras criopreservadas de caída accidental en los tanques de almacenamiento criogénicos

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.

Imágenes de Apoptotic Liquidación de células vivas durante<em&gt; Drosophila</em&gt; Embriogénesis

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.

Microscopia intravital para formar imágenes de estructuras subcelulares en ratones vivos que expresan proteínas 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.

Ensayo de formación de tubos de células endoteliales para la<em&gt; In Vitro</em&gt; Estudio de la angiogénesis

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