Name: quantitative analysis of cell edge dynamics during cell spreading
Uploaded: 2021-05-22T15:00:00
Description: Watch this Scientific Journal Video about Quantitative Analysis of Cell Edge Dynamics during Cell Spreading at JoVE.com

Este trabalho foi apoiado pelo Connaught Fund New Investigator Award to S.P., Canada Foundation for Innovation, NSERC Discovery Grant Program (bolsas RGPIN-2015-05114 e RGPIN-2020-05881), University of Manchester and University of Toronto Joint Research Fund e University of Toronto XSeed Program.

1. Semeadura celular
NOTA: O protocolo de disseminação de células descrito foi realizado utilizando fibroblastos embrionários de camundongos (MEFs) expressando PH-Akt-GFP (um marcador fluorescente para PIP3/PI(3,4)P2). Esta linha celular foi gerada pela integração genomicamente de uma construção de expressão para PH-Akt-GFP (Addgene #21218) pela edição de genes mediada pelo CRISPR. No entanto, outros marcadores fluorescentes que são expressos transitoriamente ou...

<ol>
	<li>Mullins, R. D., Heuser, J. A., Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+Arp2/3+complex+with+actin:+Nucleation,+high+affinity+pointed+end+capping,+and+formation+of+branching+networks+of+filaments.">The interaction of Arp2/3 complex with actin: Nucleation, high affinity pointed end capping, and formation of branching networks of filaments.</a> Proceedings of the National Academy of Sciences. 95 (11), 6181-6186 (1998).</li><li>Yang, C., Czech, L., Gerboth, S., Kojima, S., Scita, G., Svitkina, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Roles+of+Formin+mDia2+in+Lamellipodia+and+Filopodia+Formation+in+Motile+Cells.">Novel Roles of Formin mDia2 in Lamellipodia and Filopodia Formation in Motile Cells.</a> PLoS Biology. 5 (11), 317 (2007).</li><li>Mogilner, A., Oster, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+motility+driven+by+actin+polymerization.">Cell motility driven by actin polymerization.</a> Biophysical Journal. 71 (6), 3030-3045 (1996).</li><li>Mogilner, A., Oster, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+Generation+by+Actin+Polymerization+II:+The+Elastic+Ratchet+and+Tethered+Filaments.">Force Generation by Actin Polymerization II: The Elastic Ratchet and Tethered Filaments.</a> Biophysical Journal. 84 (3), 1591-1605 (2003).</li><li>Pollard, T. D., Borisy, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Motility+Driven+by+Assembly+and+Disassembly+of+Actin+Filaments.">Cellular Motility Driven by Assembly and Disassembly of Actin Filaments.</a> Cell. 112 (4), 453-465 (2003).</li><li>Wu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arp2/3+is+critical+for+lamellipodia+and+response+to+extracellular+matrix+cues+but+is+dispensable+for+chemotaxis.">Arp2/3 is critical for lamellipodia and response to extracellular matrix cues but is dispensable for chemotaxis.</a> Cell. 148 (5), 973-987 (2012).</li><li>Steffen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac+function+is+crucial+for+cell+migration+but+is+not+required+for+spreading+and+focal+adhesion+formation.">Rac function is crucial for cell migration but is not required for spreading and focal adhesion formation.</a> Journal of cell science. 126, 4572-4588 (2013).</li><li>Gupton, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+without+a+lamellipodium.">Cell migration without a lamellipodium.</a> The Journal of Cell Biology. 168 (4), 619-631 (2005).</li><li>Dimchev, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+Arp2/3+Complex+Depletion+Increases+FMNL2/3+Formin+Expression+and+Filopodia+Formation.">Induced Arp2/3 Complex Depletion Increases FMNL2/3 Formin Expression and Filopodia Formation.</a> Frontiers in Cell and Developmental Biology. 9, 634708 (2021).</li><li>Leithner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversified+actin+protrusions+promote+environmental+exploration+but+are+dispensable+for+locomotion+of+leukocytes.">Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes.</a> Nature cell biology. 18 (11), 1253-1259 (2016).</li><li>Giannone, G., Dubin-Thaler, B. J., D&#246;bereiner, H. -. G., Kieffer, N., Bresnick, A. R., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodic+Lamellipodial+Contractions+Correlate+with+Rearward+Actin+Waves.">Periodic Lamellipodial Contractions Correlate with Rearward Actin Waves.</a> Cell. 116 (3), 431-443 (2004).</li><li>Dubin-Thaler, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Cell+Edge+Velocities+and+Traction+Forces+Reveals+Distinct+Motility+Modules+during+Cell+Spreading.">Quantification of Cell Edge Velocities and Traction Forces Reveals Distinct Motility Modules during Cell Spreading.</a> PLoS ONE. 3 (11), 3735 (2008).</li><li>Suraneni, P., Rubinstein, B., Unruh, J. R., Durnin, M., Hanein, D., Li, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arp2/3+complex+is+required+for+lamellipodia+extension+and+directional+fibroblast+cell+migration.">The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration.</a> The Journal of cell biology. 197 (2), 239-251 (2012).</li><li>Wang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconvolution+of+subcellular+protrusion+heterogeneity+and+the+underlying+actin+regulator+dynamics+from+live+cell+imaging.">Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging.</a> Nature Communications. 9 (1), 1688 (2018).</li><li>Dimchev, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lamellipodin+tunes+cell+migration+by+stabilizing+protrusions+and+promoting+adhesion+formation.">Lamellipodin tunes cell migration by stabilizing protrusions and promoting adhesion formation.</a> Journal of cell science. 133 (7), 239020 (2020).</li><li>Burnette, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+actin+arcs+in+the+leading-edge+advance+of+migrating+cells.">A role for actin arcs in the leading-edge advance of migrating cells.</a> Nature cell biology. 13 (4), 371-381 (2011).</li><li>Yamada, K. M., Kennedy, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dualistic+nature+of+adhesive+protein+function:+fibronectin+and+its+biologically+active+peptide+fragments+can+autoinhibit+fibronectin+function.">Dualistic nature of adhesive protein function: fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function.</a> The Journal of Cell Biology. 99 (1), 29-36 (1984).</li><li>Cai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonmuscle+Myosin+IIA-Dependent+Force+Inhibits+Cell+Spreading+and+Drives+F-Actin+Flow.">Nonmuscle Myosin IIA-Dependent Force Inhibits Cell Spreading and Drives F-Actin Flow.</a> Biophysical Journal. 91 (10), 3907-3920 (2006).</li><li>Humphries, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+assays.">Cell adhesion assays.</a> Molecular Biotechnology. 18 (1), 57-61 (2001).</li><li>Cavalcanti-Adam, E. A., Volberg, T., Micoulet, A., Kessler, H., Geiger, B., Spatz, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Spreading+and+Focal+Adhesion+Dynamics+Are+Regulated+by+Spacing+of+Integrin+Ligands.">Cell Spreading and Focal Adhesion Dynamics Are Regulated by Spacing of Integrin Ligands.</a> Biophysical Journal. 92 (8), 2964-2974 (2007).</li><li>Dubin-Thaler, B. J., Giannone, G., D&#246;bereiner, H. -. G., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanometer+Analysis+of+Cell+Spreading+on+Matrix-Coated+Surfaces+Reveals+Two+Distinct+Cell+States+and+STEPs.">Nanometer Analysis of Cell Spreading on Matrix-Coated Surfaces Reveals Two Distinct Cell States and STEPs.</a> Biophysical Journal. 86 (3), 1794-1806 (2004).</li><li>Gauthier, N. C., Fardin, M. A., Roca-Cusachs, P., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporary+increase+in+plasma+membrane+tension+coordinates+the+activation+of+exocytosis+and+contraction+during+cell+spreading.">Temporary increase in plasma membrane tension coordinates the activation of exocytosis and contraction during cell spreading.</a> Proceedings of the National Academy of Sciences. 108 (35), 14467-14472 (2011).</li><li>Wolfenson, H., Iskratsch, T., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Events+in+Cell+Spreading+as+a+Model+for+Quantitative+Analysis+of+Biomechanical+Events.">Early Events in Cell Spreading as a Model for Quantitative Analysis of Biomechanical Events.</a> Biophysical Journal. 107 (11), 2508-2514 (2014).</li><li>Guan, J. -. L., Berrier, A. L., LaFlamme, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Migration,+Developmental+Methods+and+Protocols.">Cell Migration, Developmental Methods and Protocols.</a> Methods in molecular biology. 294, 55-68 (2004).</li><li>Raucher, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol+4,5-Bisphosphate+Functions+as+a+Second+Messenger+that+Regulates+Cytoskeleton-Plasma+Membrane+Adhesion.">Phosphatidylinositol 4,5-Bisphosphate Functions as a Second Messenger that Regulates Cytoskeleton-Plasma Membrane Adhesion.</a> Cell. 100 (2), 221-228 (2000).</li><li>Machacek, M., Danuser, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphodynamic+Profiling+of+Protrusion+Phenotypes.">Morphodynamic Profiling of Protrusion Phenotypes.</a> Biophysical Journal. 90 (4), 1439-1452 (2006).</li><li>Zack, G. W., Rogers, W. E., Latt, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+measurement+of+sister+chromatid+exchange+frequency.">Automatic measurement of sister chromatid exchange frequency.</a> The journal of histochemistry and cytochemistry official journal of the Histochemistry Society. 25 (7), 741-753 (1977).</li><li>Bardsley, W. G., Aplin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+cell+spreading.+I.+Theory+and+modelling+of+curves.">Kinetic analysis of cell spreading. I. Theory and modelling of curves.</a> Journal of cell science. 61, 365-373 (1983).</li></ol>

O ensaio de disseminação de células descrito permite o rastreamento contínuo de alterações morfológicas (por exemplo, tamanho e forma da célula) e movimentos de borda celular(ou seja, velocidade de saliência e frequência de retração), que são características ausentes na maioria dos protocolos de disseminaçãocelular 19,24. Embora os ensaios de disseminação de células de ponto final comumente usados permitam a determinação da ...

As saliências lamellipodias são estruturas citoesqueléticos proeminentes formadas na frente de uma célula migratória. Em lamellipodia, a polimerização do actin com o auxílio do complexo Arp2/3 e formins cria um trabalho de malha de actina ramificada de rápido crescimento que empurra contra a membrana plasmática1,2. A força de empurrão gerada pelo trabalho de malha de actina impulsiona fisicamente a célula para a frente1,3,4,5. Esgotamento do complexo Arp2/3 ou interrupção de vias de sinalização essenciais para saliências lamellipodias muitas vezes prejudicam a migração celular6, 7. Embora a migração de células deficientes de lamellipodia também tenha sido relatada8,9, a importância da lamellipodia na migração celular é evidente como o esgotamento dessa estrutura protrusiva perturba a capacidade da célula de se mover através de microambientes biológicos complexos6,10.
Um grande empecilho para entender a regulação da lamellipodia em células migratórias é a variabilidade natural na cinética de protrusão lamellipodial, tamanho e forma11,12,13,14. Além disso, estudos recentes demonstraram que a lamellipodia apresenta comportamentos salitivos complexos, incluindo saliências flutuantes, periódicas e aceleradas14,15. Em comparação com a lamellipodia altamente variável das células migratórias6,16, a lamellipodia formada durante a disseminação celular são mais uniformes12. Uma vez que a atividade strusiva de células disseminadas e migratórias é impulsionada por conjuntos macromoleculares idênticos, que incluem uma rede de actina ramificada, feixes de actomyosina contracttil e aderências de matriz celular baseada em integrin17,18, células disseminadas têm sido amplamente utilizadas como modelo para investigar a regulação da dinâmica da lamellipodia.
A disseminação celular é um processo mecanoquímico dinâmico onde uma célula em suspensão primeiro adere a um substrato através de aderências baseadas em integrin17,19,20 e, emseguida,se espalha estendendo as saliências baseadas em actina21,22,23. Durante a fase de disseminação, a lamellipodia emanando do corpo celular protuberante e persistentemente com pouca ou nenhuma retração ou paralisação12. Os protocolos de disseminação de células mais usados são os ensaios de pontos finais, onde as células de disseminação são fixadas em vários momentos após o revestimento19,24. Estes ensaios, embora rápidos e simples, são limitados em seu poder de diagnóstico para detectar alterações nas características dinâmicas da lamellipodia. Para determinar os mecanismos moleculares que controlam a dinâmica da lamellipodia, o grupo Sheetz foi pioneiro no uso da análise quantitativa de células vivas e descobriu muitas propriedades fundamentais das saliências de borda celular11,12,22. Esses estudos demonstraram que o ensaio de disseminação de células vivas é uma técnica robusta e poderosa na caixa de ferramentas de um laboratório de biologia celular. Apesar disso, um protocolo detalhado e uma ferramenta computacional de código aberto para um ensaio de espalhamento de células vivas estão atualmente indisponíveis para a comunidade de biologia celular. Para isso, nosso protocolo descreve os procedimentos de imagens ao vivo espalhando células e fornece uma ferramenta automatizada de análise de imagens. Para validar este método, utilizamos a inibição de Arp2/3 como tratamento experimental e mostramos que inibir a função do complexo Arp2/3 não dedundo a propagação celular, mas causou uma redução significativa na velocidade de saliência celular, bem como a estabilidade das saliências da borda celular, dando origem a bordas celulares irregulares. Esses dados demonstram que a combinação de imagens de células vivas e análise automatizada de imagens é uma ferramenta útil para analisar a dinâmica das bordas celulares e identificar componentes moleculares que regulam a lamellipodia.

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quantitative analysis of cell edge dynamics during cell spreading

A disseminação celular é um processo dinâmico no qual uma célula suspensa em mídia se conecta a um substrato e se achata de uma forma arredondada para uma forma fina e espalhada. Seguindo o acessório de substrato celular, a célula forma uma fina folha de lamellipodia emanando do corpo celular. Na lamellipodia, os monômeros globulares (G-actin) polimerizam-se em uma densa malha de ato desordenada (F-actin) que empurra contra a membrana plasmática, fornecendo assim as forças mecânicas necessárias para que a célula se espalhe. Notavelmente, os atores moleculares que controlam a polimerização de actina na lamellipodia são essenciais para muitos outros processos celulares, como migração celular e endocitose. 
Uma vez que a disseminação de células forma lamellipodia contínua que abrange toda a periferia celular e se expande persistentemente para fora, ensaios de disseminação celular tornaram-se uma ferramenta eficiente para avaliar a cinética das protrusões lamellipodial. Embora várias implementações técnicas do ensaio de disseminação de células tenham sido desenvolvidas, uma descrição detalhada do fluxo de trabalho, que incluiria um protocolo passo a passo e ferramentas computacionais para análise de dados, está atualmente em falta. Aqui, descrevemos os procedimentos experimentais do ensaio de disseminação celular e apresentamos uma ferramenta de código aberto para análise quantitativa e imparcial da dinâmica da borda celular durante a disseminação. Quando combinado com manipulações farmacológicas e/ou técnicas de silenciamento de genes, este protocolo é favorável a uma tela em larga escala de jogadores moleculares que regulam saliências lamellipodiais.

O protocolo acima descreve os procedimentos experimentais para a imagem de células vivas de células disseminadas e uma ferramenta computacional para a análise quantitativa da dinâmica de disseminação celular. A ferramenta computacional pode ser usada em um formato de baixo ou alto rendimento para identificar os atores moleculares que regulam o maquinário de polimerização de actina na borda principal da célula.
A representação esquemática dos procedimentos experimentais é retratad...

Watch this Scientific Journal Video about Quantitative Analysis of Cell Edge Dynamics during Cell Spreading at JoVE.com

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading

Neste protocolo, apresentamos os procedimentos experimentais de um ensaio de difusão celular que é baseado em microscopia de células vivas. Fornecemos uma ferramenta computacional de código aberto para a segmentação imparcial de células fluorescentes rotuladas e análise quantitativa da dinâmica da lamellipodia durante a disseminação celular.

Análise quantitativa da dinâmica da borda celular durante a disseminação celular

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out ...

O protocolo descrito neste vídeo definirá os procedimentos experimentais necessários para a imagem em tamanho real das células disseminadas e o uso de uma ferramenta computacional que quantifica as células espalhando dinâmicas de forma imparcial e automatizada. O ativo de disseminação de células permite o rastreamento contínuo do movimento da borda celular e alterações morfológicas durante a disseminação celular, que é uma característica que está faltando na maioria dos ensaios de propagação de células existentes. Além disso, este protocolo implementa o uso de processamento e análise automatizada de computadores, fornecendo informações sobre circularidade celular, ciclos de retração de área e saliência de células disseminantes.Esse processamento automatizado reduz o viés na análise de dados e fornece um método robusto de analisar a dinâmica de disseminação celular para um grande número de células. Quando combinado com tratamentos medicamentosos ou ciência genética e técnicas, este protocolo é favorável a velocidades em larga escala de jogadores moleculares que regulam as saliências celulares. Para o protocolo dado, as células utilizadas são fibroblastos embrionários de camundongos que codificam geneticamente PH-Akt-GFP, o que permite a marcação fluorescente da membrana plasmática.Antes do início do ensaio de propagação celular, cultura um prato de células para 90% de confluência. Uma vez que as células tenham atingido a confluência adequada, flame um deslizamento de cobertura de 22 por 22 milímetros e colocá-lo em um prato de cultura celular de 35 milímetros. Cubra o deslizamento com fibronectina que foi diluída em PBS a uma concentração de 2,5 microgramas por mililitro.Coloque o prato com a tampa deslize em uma incubadora de 37 graus por uma hora. Após uma hora aspirar a fibronectina certificando-se de não tocar no deslizamento da tampa com a ponta da pipeta. Lave o prato com PBS, em pipetando suavemente ao redor da tampa, deslize duas a três vezes.Para semeadura celular, comece aspirando a mídia de cultura celular a partir do prato das células. Depois, lave o prato com PBS quente. Adicione 650 microliters de trippsina EDTA ao prato e incline o prato para distribuir uniformemente a enzima.Coloque o prato com o trypsin na incubadora por um minuto. Após a incubação, você precisará primeiro adicionar 10 mililitros de mídia de cultura celular em um tubo de centrífuga. Em seguida, adicione rapidamente mais 10 mililitros de mídia no prato, a fim de saciar o trypsin.Para diluir as células que serão semeadas no deslizamento da tampa, pipeta um mililitro do conteúdo do prato no tubo centrífuga. Do tubo, pipeta aproximadamente 500 a 1.000 microliters de células diluídas no prato com o deslizamento de cobertura. Certifique-se de que o deslizamento da tampa está em uma confluência de 10% ou 50.000 células por mililitro e ajuste o volume de células diluídas conforme necessário.O objetivo dessas células é estabelecer um plano de foco durante a aquisição de imagens. Com as células restantes no prato, passar um quinto das células em um prato pequeno por tratamento. Estas serão as células que serão analisadas para difundir dinâmicas.Coloque os pratos de passagem e o prato de deslizamento de cobertura em uma incubadora por oito a 24 horas. Para testar a importância do Arp2/3 para a disseminação celular, primeiro adicione cinco mililitros de mídia de cultura celular em um controle e um tubo de centrífuga de tratamento. Em seguida, adicione 20 mililitros de dmem que está faltando vermelho fenol em cada um dos dois tubos de centrífuga maiores.Pipeta ou a droga CK-666 que é um inibidor do complexo Arp2/3 ou o tratamento controlado como o DMSO nos tubos pequenos e grandes. Para iniciar o tratamento farmacológico das células, remova os pratos de passagem que estavam incubando durante a noite. Aspire a mídia de cultura celular de todos os pratos e lave os pratos com PBS quente.Pegue os pequenos tubos contendo CK-666 ou controle a mídia suplementada e adicione o conteúdo do tubo relevante em cada um dos pratos de passagem. Rotule cada um dos pratos com o tratamento medicamentoso correto e, em seguida, coloque os pratos de volta na incubadora por mais uma hora Após uma hora de incubação, aspire a mídia suplementada da droga de cada um dos pratos. Em seguida, adicione PBS quente a todos os pratos, a fim de remover completamente todos os demais meios vermelhos de fenol.Adicione 230 microliters de trippsina EDTA e coloque os pratos em uma incubadora por um minuto. Remova os pratos experimentpsinizados da incubadora e continue a adicionar cinco mililitros do dmem suplementado que está sem fenol vermelho em um tubo designado como tubo B.Em seguida, adicione mais cinco mililitros da mesma mídia no prato relevante para saciar a trippsina. Pipeta a mídia para cima e para baixo várias vezes, a fim de separar todas as células do prato.Transfira todo o conteúdo do prato para outro tubo que já tenha sido designado como tubo A.In ordem para preparar uma diluição celular apropriada para a imagem, transfira um mililitro de células do tubo A para o tubo B.Repita todas as etapas de diluição para cada tratamento. Coloque todos os tubos na incubadora por 45 minutos para permitir que as células se recuperem da trippsinização. Certifique-se de que todas as partes de uma câmara magnética celular que possam acomodar um deslizamento de cobertura de 22 por 22 milímetros foram completamente limpas antes de usar.Retire o prato com o deslizamento da tampa da incubadora. Aspire a mídia de cultura celular e lave o deslizamento da tampa com PBS quente. Remova o deslizamento da tampa usando um par de fórceps e coloque suavemente o deslizamento da tampa na placa inferior da câmara magnética.Em seguida, pegue a junta de silicone e coloque-a em cima do deslizamento da tampa. Coloque o corpo principal da câmara magnética na placa inferior. Adicione um mililitro do dmem sem fenol vermelho correspondente ao tratamento relevante à câmara magnética.Pegue um tecido sem fiapos e desça cuidadosamente o invólucro entre o corpo principal e a placa inferior, a fim de verificar se há vazamentos. Para completar a câmara magnética, baixe a tampa transparente sobre o corpo principal. Por fim, limpe o fundo da tampa com um tecido pulverizado com água e outro pulverizado com 70% de etanol.Pré-aqueça a incubadora superior do estágio de um microscópio confocal a 37 graus. Coloque a câmara magnética em cima do objetivo. Antes de adquirir dinâmicas de difusão, defina o foco para as células já polarizadas no canal fluorescente verde.Isso garante que as células que se espalham estarão em foco assim que se anexarem ao deslizamento da tampa. Remova a tampa transparente da câmara magnética e a pipeta 500 microliters do tubo B que foi removido da incubadora. Coloque a tampa transparente de volta por cima.Para identificar células ideais para análise de propagação celular procure halos verdes que representem células que ainda não se anexaram ao deslizamento de cobertura ou estão nos estágios iniciais de apego. Adquira imagens e salve os arquivos. Depois de ter concluído a aquisição de imagem da expansão celular, comece executando um IDE Python compatível, como Spyder.Para análise computacional da dinâmica de disseminação de células, localize e abra o arquivo de GUI de expansão celular fornecido nos pacotes de script relevantes. Pressione o botão executar que abrirá o painel de gui de análise em segundo plano. A GUI abriga todas as configurações necessárias para análise.Para analisar a área da célula e a circularidade, insira o arquivo de aquisição e as configurações necessárias na guia da área de propagação da célula. Os ajustes terão que ser feitos dependendo do tipo de célula e dos parâmetros de aquisição de imagens. Após a pressão de execução, o script criará uma circularidade celular e uma parcela de área versus tempo para todas as células que espalham.Para dados de kymograph, pressione o gerador de kymograph e a guia de análise. Esta análise requer que os arquivos de entrada sejam cortados em pilhas de imagens de célula única. Depois de inserir todas as configurações e pressionar a execução, o script criará vários kymogramas para a célula dada.À esquerda estão células representativas dos tratamentos DMSO e CK-666, segmentados automaticamente usando o script fornecido. Ao contrário da morfologia isotropica e altamente circular demonstrada pelas células de controle, as células inibidas Arp2/3 apresentam diminuição da circularidade. Além disso, os kymogramas gerados automaticamente fornecem resolução temporal que é fundamental para entender a dinâmica das saliências.As células de controle se projetam persistentemente com pouca ou nenhuma retração. Em contraste, a inibição de Arp2/3 interrompe a estabilidade das saliências, como indicado pelo aumento dos eventos de retração ao longo da propagação. Estes vídeos devem fornecer-lhe a compreensão de como seed adequadamente células, realizar tratamentos farmacológicos e preparar imagens e ferramentas computacionais necessárias para a quantificação da dinâmica de disseminação celular.Este protocolo pode ser ainda mais combinado com imagens fluorescentes de citoesqueleto e ensaios de migração para identificar os atores moleculares que regem as saliências celulares. Obrigado por assistir e boa sorte com suas experiências.

quantitative-analysis-of-cell-edge-dynamics-during-cell-spreading

Research

JoVE Journal

Biology

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

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Em células vivas, Imagem e Análise Quantitativa de células embrionárias epiteliais em Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

Determinação do Número celular durante Cultura Celular usando o contador de células Scepter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Quantitativas de células vivas de Análise de microscopia de fluorescência de levedura

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

Um fluxo de trabalho Análise Quantitativa de Fitness

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

Espalhando superfície e Immunostaining de cromossomas da levedura

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Imagens de alta resolução e Análise de individuais Astral microtúbulos Dynamics em brotamento Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Viver a microscopia de fluorescência da célula para observar os processos essenciais durante o crescimento de células microbianas

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

Lente livre de microscopia para a dinâmica e a análise quantitativa de cultura de células aderentes

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

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.