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

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Unit 1

The Cytoskeleton I: Actin and Microfilaments

SCIENTISTS IN ACTION

27.1K 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

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Em células vivas, imagens de células que expressam proteínas fluorescentes Migrando-marcado em uma matriz tridimensional

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Bioengenharia

In Vitro Polymerization of F-actin on Early Endosomes

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto the endosomal membrane. We have established a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to genetic and biochemical manipulations. Endosomal fractions are prepared by floatation in sucrose gradients from cells expressing the early endosomal protein GFP-RAB5. Cytosolic fractions are prepared from separate batches of cells. Both endosomal and cytosolic fractions can be stored frozen in liquid nitrogen, if needed. In the assay, the endosomal and cytosolic fractions are mixed, and the mixture is incubated at 37 &#176;C under appropriate conditions (e.g., ionic strength, reducing environment). At the desired time, the reaction mixture is fixed, and the F-actin is revealed with phalloidin. Actin nucleation and polymerization are then analyzed by fluorescence microscopy. Here, we report that this assay can be used to investigate the role of factors that are involved either in actin nucleation on the membrane, or in the subsequent elongation, branching, or crosslinking of F-actin filaments.

In Vitro Polymerization of F-actin on Early Endosomes

Early endosome functions depend on F-actin polymerization. Here, we describe a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to biochemical and genetic manipulations.

In Vitro Polimerização de F-Actina no início endossomos

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto ...

Bioquímica

Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton3-6. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling7. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant.
Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant8-10. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells11, 12. When rhodamine-actin is added at a concentration above the critical concentration for actin monomer addition to barbed ends, rhodamine-actin assembles onto free barbed ends. If the attractive cue is presented in a gradient, such as being released from a micropipette positioned to one side of a growth cone, the incorporation of rhodamine-actin onto F-actin barbed ends will be greater in the growth cone side toward the micropipette10.
Growth cones are small and delicate cell structures. The procedures of permeabilization, rhodamine-actin incorporation, fixation and fluorescence visualization are all carefully done and can be conducted on the stage of an inverted microscope. These methods can be applied to studying local actin polymerization in migrating neurons, other primary tissue cells or cell lines.

A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed ends.

Rotulagem F-actina Termina farpado com Rodamina-actina em permeabilizadas Cones de Crescimento Neuronal

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally ...

Neurociência

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

analisando<em&gt; In Vivo</em&gt; Migração celular usando transplantes de células e de imagem Time-lapse em embriões Zebrafish

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Biologia do Desenvolvimento

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 ...

Biologia

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

Visualizando a dinâmica de acoplamento de actina e microtúbula in vitro por microscopia de fluorescência interna total (TIRF)

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

Resposta celular ao vivo para Estimulação mecânicas estudadas por Microscopia de Força Atômica e Óptica Integrada

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

No estudo in vivo da Human Interaction Endotelial-pericyte usando o plug Ensaio Matrix Gel no mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

Técnicas de micromanipulação, permitindo a análise da dinâmica morfogenética e rotatividade dos reguladores do citoesqueleto

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly

Aberrant changes in endothelial phenotype and morphology are considered to be initial events in the pathogenesis of atherosclerosis. Direct observation of the intact endothelium will provide valuable information for understanding the cellular and molecular events in the dysfunctional endothelial cells. Here, we describe a modified en face immunofluorescence staining technique which enables scientists to obtain clear images of the intact endothelial surface and analyze the molecule expression patterns in situ. The method is simple and reliable for observing the entire endothelial monolayer at different sites of the aorta. This technique may be a promising tool for understanding the pathophysiology of atherosclerosis, especially at an early stage.

Here, we present a protocol for immunofluorescence staining to observe the endothelial cells of the mouse aorta directly. This technique is useful when studying the cellular and molecular phenotype of endothelial cells in different flow patterns and in the development of atherosclerosis.

Usando a mancha da imunofluorescência da cara do en para observar diretamente pilhas endothelial vasculares

Aberrant changes in endothelial phenotype and morphology are considered to be initial events in the pathogenesis of atherosclerosis. Direct observation of ...