O plasmídeo GFP-β-actina foi generosamente fornecidos pelo Dr. Wayne Orr, LSUHSC-S Departamento de Patologia, e foi amplificado no laboratório do Dr. Becky Worthylake, LSUHSC-NO Departamento de Farmacologia. Este trabalho foi financiado por concessões do National Institutes of Health (P20 RR-018766) e da 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>

Não há conflitos de interesse declarados.

A imagem de GFP-actina em células endoteliais ao vivo permite uma análise detalhada da dinâmica do citoesqueleto de actina em resposta a estímulos inflamatórios. Este método também pode ser útil para construir em cima descobertas anteriores que mostram remodelação do citoesqueleto em resposta às forças físicas, como tensão de cisalhamento 11. Além disso, este método permite uma avaliação detalhada da contribuição da dinâmica do citoesqueleto de actina para diversas atividades das células endoteliais, incluindo a migração, mitose, formação de junções intercelulares e maturação juncional, e manutenção da função de barreira. Nos dados apresentados, o comportamento do citoesqueleto de actina endotelial pode ser observado antes e após o tratamento com trombina. Lamellipodia locais, todos ao longo das bordas das células endoteliais foram observados formando e regredindo ao longo do tempo em monocamadas de células tanto nonconfluent e confluente. Tratamento com trombina brevemente interrompido forma lamellipodiação e volume de negócios. Trombina também causou as células a contrair-se ligeiramente, de acordo com relatos anteriores de que a trombina causas actina formação das fibras de estresse e desenvolvimento crescente tensão centrípeta nas células endoteliais 12-14. No entanto, a partir de estudos de células vivas de imagens como esta, a origem das fibras de estresse pode agora ser determinada. Em HUVEC, a maioria das fibras de estresse originam na periferia celular e assemelham-se fibras de arco transversal na migração de células 8, 9. Outro ponto forte deste método sobre o uso de células fixas é que o número de fibras de stress podem ser quantificados em células individuais antes e após o tratamento de trombina, eliminando o viés de seleção entre os grupos experimentais. Com este protocolo nós avaliamos movimento dinâmico da borda de células e fibras de stress de actina. Para entender a dinâmica de monômero de actina em células endoteliais, as técnicas mais avançadas, como a recuperação de fluorescência após a fotodegradação (FRAP) ou fluorescência speckle microscópia (FSM) pode ser aplicado 15, 16. Além disso, como células endoteliais microvasculares pode representar um modelo melhor da função de barreira microvascular, otimização de protocolos de transfecção de efetivamente expressar GFP-actina em células endoteliais microvasculares representa uma direção futura lógica. Em resumo, a imagem latente de viver células endoteliais expressando GFP-actina representa uma ferramenta poderosa para determinar como o citoesqueleto de actina da célula endotelial responde a vários tipos de estímulos. Estudos utilizando fortemente confluentes monocamadas endotelial irá ajudar a determinar as funções de estruturas dinâmicas, tais como actina-ricos lamellipodia e fibras arco transversal estresse em função de barreira endotelial. Além disso, imagens de células vivas de células endoteliais expressando GFP-actina ou proteínas de fusão outras que permitem a visualização de outras estruturas subcelulares irá fornecer informações detalhadas espaço-temporal necessária para entender a sinalização e os mecanismos estruturais que determine integridade da barreira.

<table border="1"><tbody><tr><td colspan="4"> 1. Ringer stock 5x </td></tr><tr><td> Químico </td><td> Companhia </td><td> Número de catálogo </td><td> Quantidade </td></tr><tr><td> Cloreto de sódio </td><td> EMD </td><td> SX0420-3 </td><td> 35 g </td></tr><tr><td> Cloreto de Potássio </td><td> JT Baker </td><td> 3040 </td><td> 1,75 g </td></tr><tr><td> Cloreto de cálcio </td><td> Sigma </td><td> C-3881 </td><td> 1,47 g </td></tr><tr><td> Sulfato de magnésio </td><td> Sigma </td><td> M-9397 </td><td> 1,44 g </td></tr><tr><td> Água filtrada estéril </td><td> N / A </td><td> N / A </td><td> Trazer para 1 L </td></tr><tr><td colspan="4"> Filtro estéril em frascos autoclavados e lojas a 4 ° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 2. Tampão MOPS </td></tr><tr><td> Químico </td><td>Companhia </td><td> Número de catálogo </td><td> Quantidade </td></tr><tr><td> MOPS </td><td> Sigma </td><td> M3183 </td><td> 125,6 g </td></tr><tr><td> Água filtrada estéril </td><td> N / A </td><td> N / A </td><td> Trazer para 1 L </td></tr><tr><td colspan="4"> Filtro estéril em frascos autoclavados e lojas a 4 ° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 3. Solução Sal albumina Physiological (APSS) </td></tr><tr><td> Químico </td><td> Companhia </td><td> Número de catálogo </td><td> Quantidade </td></tr><tr><td> Ringer estoque (5x) </td><td> N / A </td><td> N / A </td><td> 200 mL </td></tr><tr><td> Tampão Mops </td><td> N / A </td><td> N / A </td><td> 5 mL </td></tr><tr><td> Fosfato de sódio </td><td> Sigma </td><td> S-9638 </td><td> 0,168 g </td></tr><tr><td> Piruvato de sódio &lt;/ Td&gt; <td> Sigma </td><td> P5280 </td><td> 0,22 g </td></tr><tr><td> EDTA sal de sódio </td><td> Sigma </td><td> ED2SS </td><td> 0,0074 g </td></tr><tr><td> Glicose </td><td> Sigma </td><td> G7528 </td><td> 0,901 g </td></tr><tr><td> Albumina Bovina </td><td> USB </td><td> 10856 </td><td> 10 g </td></tr><tr><td> Água filtrada estéril </td><td> N / A </td><td> N / A </td><td> Trazer para 1 L </td></tr><tr><td colspan="4"> Ajustar o pH para 7,4 a 37 ° C, seguida de filtração estéril em frascos autoclavados e armazenar a 4 ° C. </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 4. Saline 0,9% </td></tr><tr><td> Químico </td><td> Companhia </td><td> Número de catálogo </td><td> Quantidade </td></tr><tr><td> Cloreto de sódio </td><td> EMD </td><td> SX0420-3 </td><td> 9 g </td></tr><tr><td> Água filtrada estéril </td><td> N / A </td><td> N / A </td><td> Trazer para 1 L </td></tr><tr><td colspan="4"> Filtro estéril em frascos autoclavados e lojas a 4 ° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 5. 1,5% Solução Gelatina </td></tr><tr><td> Gelatina de pele suína </td><td> Sigma </td><td> G2500 </td><td> 15 g </td></tr><tr><td> Saline 0,9% </td><td> N / A </td><td> N / A </td><td> Trazer para 1 L </td></tr><tr><td colspan="4"> Aquecer a solução a 37 ° C para dissolver a gelatina suficientemente. Enquanto ainda quente filtro, estéril em frascos autoclavados e armazenar a 4 ° C </td></tr></tbody></table>

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

O endotélio microvascular desempenha um papel importante como uma barreira seletiva permeável a fluidos e solutos. As junções adesivas entre as células endoteliais regulam a permeabilidade do endotélio, e muitos estudos têm indicado a importante contribuição do citoesqueleto de actina para determinar a integridade juncional 1-5. Um cinto de actina cortical é pensado para ser importante para a manutenção das junções estável 1, 2, 4, 5. Em contraste, as fibras de estresse actina são pensados ​​para gerar tensão centrípeta no interior das células endoteliais que enfraquece junções 2-5. Grande parte desta teoria foi baseada em estudos em que as células endoteliais são tratados com mediadores inflamatórios conhecidos por aumentar a permeabilidade endotelial e fixação das células e rotulagem F-actina para observação microscópica. No entanto, estes estudos fornecem uma compreensão muito limitada do papel do citoesqueleto de actina porque as imagens de células fixos proporcionam snapshots apenas emtempo com nenhuma informação sobre a dinâmica das estruturas de actina 5. Live-célula de imagem permite a incorporação da natureza dinâmica do citoesqueleto de actina nos estudos dos mecanismos de determinação integridade da barreira endotelial. Uma grande vantagem deste método é que o impacto de vários estímulos inflamatórios em estruturas de actina em células endoteliais pode ser avaliada no mesmo conjunto de células vivas, antes e após o tratamento, a remoção viés potencial que podem ocorrer quando se observa os espécimes fixos. Células endoteliais da veia umbilical humana (HUVEC) são transfectadas com GFP-β-actina plasmídeo e cresceu para confluência em lamínulas. Lapso de tempo imagens de GFP-actina em HUVEC confluentes são capturados antes e após a adição de mediadores inflamatórios que provocam mudanças dependentes do tempo na integridade da barreira endotelial. Estes estudos permitem a observação visual da seqüência fluida de mudanças no citoesqueleto de actina, que contribuem para endotéliol rompimento da barreira e restauração. Nossos resultados mostram consistentemente local, rico em actina lamellipodia formação e volume de negócios nas células endoteliais. A formação e movimento das fibras de actina estresse também pode ser observado. Uma análise da frequência de formação e volume de negócios do lamellipodia local, antes e após o tratamento com estímulos inflamatórios podem ser documentadas por meio de análises quimógrafo. Estes estudos fornecem informações importantes sobre a natureza dinâmica do citoesqueleto de actina em células endoteliais, que pode ser usado para descobrir previamente não identificados mecanismos moleculares importantes para a manutenção da integridade da barreira 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

Imagens microscópicas de vida das células endoteliais expressando GFP-actina permite a caracterização de mudanças dinâmicas em estruturas do citoesqueleto. Ao contrário das técnicas que utilizam amostras fixadas, este método fornece uma avaliação detalhada das mudanças temporais no citoesqueleto de actina nas mesmas celas antes, durante e depois de vários estímulos físicos, farmacológicos, ou inflamatórias.

Estudo do citoesqueleto de actina em Live Células Endoteliais Expressando 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 visualizações.  Louisiana State University Health Sciences Center. Imagens microscópicas de vida das células endoteliais expressando GFP-actina permite a caracterização de mudanças dinâmicas em estruturas do citoesqueleto. Ao contrário das técnicas que utilizam amostras fixadas, este método fornece uma avaliação detalhada das mudanças temporais no citoesqueleto de actina nas mesmas celas antes, durante e depois de vários estímulos físicos, farmacológicos, ou inflamatórias.

Vídeo: 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.

Teste de sensibilidade visual para a velocidade e direção do movimento em Lagartos

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

Biologia

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.

Gerados por computador Modelo Estímulos 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 Ensaio Co Sedimentação; para a Análise da proteína de ligação a 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).

Imagens de células vivas de F-actina Dynamics via Microscopia 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.

Imagens ao vivo da motilidade celular e citoesqueleto de actina de neurônios individuais e células da crista neural em embriões Zebrafish

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.

Transplante de GFP-expressando Blastômeros for Imaging ao vivo de Desenvolvimento da retina e do cérebro em embriões Zebrafish Quimérico

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 Dispositivo de Recuperação de amostras criopreservadas cair acidentalmente em Tanques de armazenamento criogênico

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.

Imagens ao vivo de Apuramento celular por apoptose durante<em&gt; Drosophila</em&gt; Embriogênese

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 Estruturas de imagem Subcelulares em ratos vivos Expressando fluorescentes Proteínas

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.

Endotelial Tubo celular Formação ensaio para a<em&gt; In Vitro</em&gt; Estudo da angiogênese

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