Agradecemos a membros do laboratório Huse para aconselhamento e assistência. Suportados pelos institutos nacionais de saúde (R01-AI087644 para MH) e P30-CA008748 ao Memorial Sloan-Kettering Cancer Center dos Estados Unidos.

1. preparação de superfícies de vidro estimulatórios
<ol><li>Casaco oito poços septada lamela com biotinilado poli-L-lisina (Bio-PLL) diluído em água destilada, deionizada (ddH2O) 1: 500. Incube durante 30 min à temperatura ambiente (RT).</li>
	<li>Lavar com H2O.</li>
	<li>Seco por 2h em RT.</li>
	<li>Bloco Bio-PLL revestido de superfícies com tampão de bloqueio (HEPES salino [10 mM HEPES pH 7,4, 150 mM NaCl], com 2% de BSA) durante 30 min à RT<ol>
<li>Dissolva 5 mg de poli-L-lysine hydrobromide em 1 mL de 10mm NaPO4, pH 8,5.</li>
		<li>Adicionar 125 μmol (0,55 mg) de NHS-biotina (a partir de um estoque de 100 mg/mL em DMSO) e verificar o pH da reação. Se o pH estiver abaixo de 8,5, adicione 2 μL de 4 N NaOH para elevá-lo a entre pH 8,5 e 9,5.</li>
		<li>Vórtice por 30 min.</li>
		<li>Saciar a reação com 50 μL de 100mm glicina dissolvida em 20 mM Tris de pH 8.0.</li>
		<li>Rotação em potência máxima por 10 minutos e transferir o sobrenadante para um tubo novo. Nota: Qualidade de Bio-PLL é tipicamente em comparação com anteriores preparações por revestimento dois diluíções do material em uma placa de ELISA de 96 poços e quantificar o conteúdo de biotina, após incubação com fosfatase alcalina acoplado streptavidin.</li>
	</ol>
</li>
	<li>Retire o tampão de bloqueio. Não permita que poços secar. Adicione streptavidin (100 µ g/mL de tampão de bloqueio). Incubar durante 1 h a 4 ° C.</li>
	<li>Lavar em HBS. Preencher e inverter poços de slide de câmara 4 - 5 vezes, removendo HBS dos poços. Não permita que poços secar.</li>
	<li>Adicione o biotinilado pMHC ligantes e moléculas de adesão à superfície. Nota: MCC e óvulos podem ser photocaged pela adição de orto-nitrobenzil com base proteger grupos (por exemplo, nitrophenylethyl (NPE) ou nitroveratryloxycarbonyl (NVOC)) para o grupo ε-amino da chaves lisinas (K12 em MCC, K7 em óvulos). Photocaged MCC e óvulos podem ser obtidos de fornecedores comerciais. Após a reconstituição em tampão aquosa, eles são novamente em modo expressa-eu... Ek e H2-Kb utilizando abordagens estabelecida11,12.</li>
	<li>Validação de peptídeo MCC ou óvulos de photoactivatable<ol>
<li>Decage peptídeos NVOC protegido por irradiação de UV com uma lâmpada UV portátil (ver Tabela de materiais) por 20 min no RT</li>
		<li>Pulso de 1,0 x 105 APCs com 1 µM de peptídeo nonstimulatory (controlo negativo, por exemplo, Hb), não irradiados photoactivatable peptídeo, irradiados photoactivatable peptídeo e peptídeo agonista (controle positivo, por exemplo, MCC) por 1h para durante a noite a 37 ° C.</li>
		<li>Para estimular células T expressam a C. 5 C7/2B4/e TCR, usar células CH12 ou CH27 B. Para estimular as células T OT1, use RMA-s ou células de timoma EL4.</li>
		<li>Misture as APCs pulsados com igual número de células T em 96 poços redondo fundo placas em um volume final de 200 µ l. Incubar a 37 ° C, durante 12-24 h.</li>
		<li>Recuperar os sobrenadantes de cada poço e analisar IL-2 por ELISA com Detecção colorimétrica de peroxidase de rábano-estreptavidina. Nota: A confirmação do status de todos os peptídeos por espectrometria de massa electrospray antes de dobrar em complexos MHC, enjaulamento é altamente recomendável.</li>
	</ol>
</li>
	<li>Execute o dobramento e purificação de MHC, a fim de minimizar a exposição à luz. Por exemplo, envolver reações de dobradura em papel de alumínio e realizar a cromatografia de gel filtração com a lâmpada UV. Nota: Tem sido encontrado que photoactivatable MCC-l-Ok e óvulos - H2-Kb induzem a ativação de célula independente de T UV em alta densidade, em decorrência de enjaulamento funcional incompleta. Portanto, o pMHC photoactivatable é diluída em um excesso de x de 10-30 de nonstimulatory pMHC (por exemplo, eu-Ek contendo o peptídeo de64-76 de hemoglobina (Hb)) durante a etapa de imobilização. Isso melhora a relação sinal / ruído do experimento de imagens subsequente.</li>
	<li>Para Fotoativar CD4+ T células, coletivamente, usar uma mistura de biotinilado Hb-l-Ok (3 µ g/mL), biotinilado photoactivatable MCC-l-Ok (0,1 µ g/mL) e anticorpo biotinilado contra a classe eu MHC H2-Kk (0,5 µ g / mL). O anticorpo anti-H2-Kk incentiva 5 C. Células C7/2B4/e T, que expressam o H2-Kk, para espalhar sobre a superfície de vidro sem sofrer ativação.</li>
	<li>Para CD8+ T células, usar uma mistura de biotinilado H2-Db o peptídeo KAVYDFATL do rolamento (1 µ g/mL), photoactivatable biotinilado óvulos-H2-Kb (0,1 µ g/mL) e o domínio extracelular da molécula de adesão ICAM-1 (2 µ g/mL produzido por cultura de células de inseto13). O ICAM-1 incentiva formação próxima contato acoplando a integrina LFA1 na superfície das células T.</li>
	<li>Aplicar todas as misturas de proteínas em tampão de bloqueio, seguido de incubação por 1h no RT ou pelo menos 2 h a 4 ° C. Nota: A densidade molecular em superfícies desse tipo para ser ~ 8000 por µm2 tem sido previamente determinado6. Dado que photoactivatable pMHC representa ~1/30th da proteína biotinilado na superfície, sua densidade será ~ 267 moléculas por µm2, antes da decaging.</li>
	<li>Lave como na etapa 1.6 e deixar na HBS até estar pronto para usar.</li>
	<li>Adicionar 200.000 CD4+ ou CD8+ T células expressando o TCR apropriado em cada poço e permitir que as células a aderir a 37 ° C por 15 min. Uma vez que as células têm anexados e espalhar sobre a superfície, eles estão prontos para fotoativação e de imagem. Nota: Retroviral transdução de células efetoras T com fluorescentes sondas de imagem é descrita em detalhe elswhere6,7. Sinalização de cálcio pode também ser estudada usando pilhas de T untransduced carregadas com o corante de cálcio sensíveis Fluo-4,6. O corante ratiometric Fura-2 não é recomendado porque requer excitação na faixa de UV, que também induz decaging de pMHC a photoactivatable.</li>
</ol>2. aquisição de imagem
<ol><li>Use um microscópio invertido de TIRF equipado com uma lente objetiva de UV compatível 150 X para aquisição de imagens. UV irradiar regiões definidas pelo usuário, usando um sistema de diafragma digital ligado a uma lâmpada de mercúrio 100 W (HBO). Luz desta lâmpada para a amostra, usando um espelho de passe longo 400 nm direta UV.</li>
	<li>Use o software de análise de imagem para photoactivation localizada e aquisição de lapso de tempo. Na maioria dos experimentos, monitorar sondas em ambos os canais vermelhos e verdes usando 488 nm e 561 lasers de excitação de nm, respectivamente. Luz laser é dirigido para a amostra utilizando um espelho dicroico dual-bandpass que também transmite na faixa de UV (para habilitar decaging). Consulte a Tabela de materiais e a Figura 6 para obter informações adicionais sobre configuração de microscópio.</li>
	<li>Depois de montar o slide de câmara que contém as células T, ajuste as configurações para obter iluminação TIRF ou epifluorescência, conforme necessário. No modo de viver, selecione um campo de células que expressam a fluorescente ição de interesse. Estabelece regiões mícron-escala para photoactivation abaixo usando o software de controle de células individuais.</li>
	<li>Começa a aquisição de lapso de tempo. Normalmente, 80 momentos são adquiridos, com um intervalo de 5 s entre cada ponto de tempo. Isso deixa o tempo mais que suficiente para sequencial 488 nm e 563 exposições nm, no caso de experimentos de cor dupla.</li>
	<li>Após 10 momentos, Fotoativar as regiões selecionadas pela abertura do diafragma digital do obturador para 1-1.5 s.</li>
	<li>Após o lapso de tempo completo, selecione um novo campo de células e repita o processo.</li>
</ol>3. análise de dados
Nota: Photoactivation localizada de ligantes imobilizados cria um bem definido, estacionário estimulatórios que pode ser usado para análise quantitativa de sinalização respostas e eventos de remodelação do citoesqueleto. Análise de protocolos normalmente envolvem ou quantificar a intensidade de fluorescência na região irradiada ou usando a região irradiada como um ponto de extremidade posicional para medições de distância (por exemplo. para avaliar a polarização da centrossoma para o região irradiada). Ambos os protocolos de análise são descritos abaixo. Vários programas de análise de imagem interativo podem ser usados para fazer a intensidade e medições de distância, que podem então ser saída para processamento adicional e análise.
<ol><li>
Intensidade de fluorescência
	<ol>
<li>Determine a intensidade de fluorescência (FI) de uma região de fundo fora da célula (FIb). Isto será usado para a correção de fundo.<ol>
<li>Desenhe uma região quadrada micro-empresas fora da célula e fazer uma máscara. Para determinar a intensidade da fluorescência, clique no Analyze | Mascarar as estatísticas. Selecione Significa intensidade de fluorescência e os valores de exportação. Nota: Detalhes processuais (por exemplo, 3.1.1.1) referem-se ao software Slidebook (ver Tabela de materiais). Implementação do presente protocolo usando outros pacotes de software (ex., FIJI) será ligeiramente diferente.</li>
		</ol>
</li>
		<li>Determine o FI dentro da região de fotoativados por cada ponto de tempo.<ol>
<li>Selecione máscara para destacar a região que foi fotoativados. Para determinar a intensidade da fluorescência, clique no Analyze | Mascarar as estatísticas. Selecione Significa intensidade de fluorescência e os valores de exportação.</li>
		</ol>
</li>
		<li>Subtrai o fundo FI a partir dos valores medidos de FI dentro da região. Então, normalizar as medições FI corrigidas dividindo pela média, fundo corrigida FI dos primeiros 9 quadros antes de fotoativação. ΔF/F = ((FI-FIb)/mean(FI1-9)-FIb)). Nota: Gráfico ΔF/F como uma função do tempo.</li>
	</ol>
</li>
	<li>
Distância
	<ol>
<li>Obter o x e y coordenadas do centro da região fotoativados.<ol>
<li>Para determinar o x e y coordenadas do centro da região fotoativados, selecione máscara para destacar a região que foi fotoativados. Uma vez que a região de fotoativação é realçada, selecione Analyze | Mascarar as estatísticas | Centro de área e exportar os valores.</li>
		</ol>
</li>
		<li>Determinar o x e y coordenadas para a sonda fluorescente de interesse para cada ponto de tempo. Isto geralmente é conseguido através de partícula manual ou automatizada de rastreamento.<ol>
<li>Para determinar o x e y coordenadas da sonda fluorescente de interesse, selecione Manual de controle de partículas e clique sobre a sonda fluorescente de interesse ao longo do tempo. Uma vez que todos os pontos de tempo foram controlados, selecione Analyze | Mascarar as estatísticas | Centro de área e exportar os valores.</li>
		</ol>
</li>
		<li>Calcular a distância entre a proteína fluorescente de interesse e o centro da região fotoativados por cada ponto de tempo usando a equação: distância = √ ((x2x -1)2+ (y2y -1)2), em que X2e y 2 são as coordenadas da proteína fluorescente de interesse e x1 e y1 são as coordenadas do centro da região fotoativados.</li>
		<li>Gráfico da distância em função do tempo.</li>
	</ol>
</li>
</ol>

<ol>
	<li>Dustin, M. L., Chakraborty, A. K., Shaw, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+structure+and+function+of+the+immunological+synapse.">Understanding the structure and function of the immunological synapse.</a> Cold Spring Harb.Perspect.Biol. 2, a002311 (2010).</li><li>Liu, X., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+Synapse+Formation:+Cell+Polarity+During+T+Cell-APC+Interaction.">Immunological Synapse Formation: Cell Polarity During T Cell-APC Interaction.</a> Cell Polarity 1. , 247-275 (2015).</li><li>Bunnell, S. C., Kapoor, V., Trible, R. P., Zhang, W., Samelson, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+actin+polymerization+drives+T+cell+receptor-induced+spreading:+A+role+for+the+signal+transduction+adaptor+LAT.">Dynamic actin polymerization drives T cell receptor-induced spreading: A role for the signal transduction adaptor LAT.</a> Immunity. 14, 315-329 (2001).</li><li>Dustin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+Function+of+the+Immunological+Synapse+from+Studies+with+Supported+Planar+Bilayers.">Insights into Function of the Immunological Synapse from Studies with Supported Planar Bilayers.</a> Current topics in microbiology and immunology. 340, (2010).</li><li>Chauveau, A., Le Floc'h, A., Bantilan, N. S., Koretzky, G. a., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diacylglycerol+kinase+&#945;+establishes+T+cell+polarity+by+shaping+diacylglycerol+accumulation+at+the+immunological+synapse.">Diacylglycerol kinase &#945; establishes T cell polarity by shaping diacylglycerol accumulation at the immunological synapse.</a> Sci. Signal. 7, ra82 (2014).</li><li>Huse, M., 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=Spatial+and+Temporal+Dynamics+of+T+Cell+Receptor+Signaling+with+a+Photoactivatable+Agonist.">Spatial and Temporal Dynamics of T Cell Receptor Signaling with a Photoactivatable Agonist.</a> Immunity. 27, 76-88 (2007).</li><li>Quann, E. J., Merino, E., Furuta, T., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+diacylglycerol+drives+the+polarization+of+the+microtubule-organizing+center+in+T+cells.">Localized diacylglycerol drives the polarization of the microtubule-organizing center in T cells.</a> Nat Immunol. 10, 627-635 (2009).</li><li>Basu, R., Chen, Y., Quann, E. J., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+variable+hinge+region+of+novel+PKCs+determines+localization+to+distinct+regions+of+the+immunological+synapse.">The variable hinge region of novel PKCs determines localization to distinct regions of the immunological synapse.</a> PLoS One. 9, 1-8 (2014).</li><li>Liu, X., Kapoor, T. M., Chen, J. K., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diacylglycerol+promotes+centrosome+polarization+in+T+cells+via+reciprocal+localization+of+dynein+and+myosin+II.">Diacylglycerol promotes centrosome polarization in T cells via reciprocal localization of dynein and myosin II.</a> Proc. Natl. Acad. Sci. U. S. A. 110, 11976-11981 (2013).</li><li>Quann, E. J., Liu, X., Altan-Bonnet, G., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cascade+of+protein+kinase+C+isozymes+promotes+cytoskeletal+polarization+in+T+cells.">A cascade of protein kinase C isozymes promotes cytoskeletal polarization in T cells.</a> Nat. Immunol. 12, 647-654 (2011).</li><li>Boniface, J. J., Reich, Z., Lyons, D. S., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamics+of+T+cell+receptor+binding+to+peptide-MHC:+evidence+for+a+general+mechanism+of+molecular+scanning.">Thermodynamics of T cell receptor binding to peptide-MHC: evidence for a general mechanism of molecular scanning.</a> Proc. Natl. Acad. Sci. U. S. A. 96, 11446-11451 (1999).</li><li>Garboczi, D. N., Hung, D. T., Wiley, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLA-A2-peptide+complexes:+refolding+and+crystallization+of+molecules+expressed+in+Escherichia+coli+and+complexed+with+single+antigenic+peptides.">HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides.</a> Proc. Natl. Acad. Sci. U. S. A. 89, 3429-3433 (1992).</li><li>Huse, M., Lillemeier, B. F., Kuhns, M. S., Chen, D. S., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cells+use+two+directionally+distinct+pathways+for+cytokine+secretion.">T cells use two directionally distinct pathways for cytokine secretion.</a> Nat. Immunol. 7, 247-255 (2006).</li><li>Abeyweera, T. P., Merino, E., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitory+signaling+blocks+activating+receptor+clustering+and+induces+cytoskeletal+retraction+in+natural+killer+cells.">Inhibitory signaling blocks activating receptor clustering and induces cytoskeletal retraction in natural killer cells.</a> J. Cell Biol. 192, 675-690 (2011).</li><li>Nguyen Duc, T., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Generalizable+Platform+for+the+Photoactivation+of+Cell+Surface+Receptors.">A Generalizable Platform for the Photoactivation of Cell Surface Receptors.</a> ACS Chem. Biol. 10, 2435-2440 (2015).</li><li>Airan, R. D., Thompson, K. R., Fenno, L. E., Bernstein, H., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+precise+in+vivo+control+of+intracellular+signalling.">Temporally precise in vivo control of intracellular signalling.</a> Nature. 458, 1025-1029 (2009).</li><li>Xu, 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=Optogenetic+control+of+chemokine+receptor+signal+and+T-cell+migration.">Optogenetic control of chemokine receptor signal and T-cell migration.</a> Proc. Natl. Acad. Sci. 111, 6371-6376 (2014).</li><li>Levskaya, A., Weiner, O. D., Lim, W. A., Voigt, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+control+of+cell+signalling+using+a+light-switchable+protein+interaction.">Spatiotemporal control of cell signalling using a light-switchable protein interaction.</a> Nature. 461, 997-1001 (2009).</li><li>Guntas, 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=Engineering+an+improved+light-induced+dimer+(iLID)+for+controlling+the+localization+and+activity+of+signaling+proteins.">Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins.</a> Proc. Natl. Acad. Sci. 112, 112-117 (2015).</li><li>Wu, Y. I., 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+genetically+encoded+photoactivatable+Rac+controls+the+motility+of+living+cells.">A genetically encoded photoactivatable Rac controls the motility of living cells.</a> Nature. 461, 104-108 (2009).</li><li>Pathak, G. P., Vrana, J. D., Tucker, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+control+of+cell+function+using+engineered+photoreceptors.">Optogenetic control of cell function using engineered photoreceptors.</a> Biol. cell/under auspices Eur. Cell Biol. Organ. 105, 59-72 (2014).</li><li>Grusch, M., 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=Spatio-temporally+precise+activation+of+engineered+receptor+tyrosine+kinases+by+light.">Spatio-temporally precise activation of engineered receptor tyrosine kinases by light.</a> EMBO J. 33, 1713-1726 (2014).</li><li>Yi, 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=Centrosome+repositioning+in+T+cells+is+biphasic+and+driven+by+microtubule+end-on+capture-shrinkage.">Centrosome repositioning in T cells is biphasic and driven by microtubule end-on capture-shrinkage.</a> J. Cell Biol. 202, 779-792 (2013).</li></ol>

Os autores não têm nada para divulgar.

Nos últimos anos, a luz tem emergido como uma excelente ferramenta para activação spatiotemporally controlada dos processos celulares. Várias metodologias têm sido desenvolvidos, cada um com as desvantagens e vantagens associadas. O sistema descrito aqui, que se baseia o decaging de ligantes extracelulares, imobilizados, é ideal para a análise das respostas de sinalização rápidas, subcellular, polarizadas. Esta abordagem tem sido aplicada para examinar a formação é em células T conforme descrito acima. Adic...

Linfócitos T (células T) desempenham um papel central na imunidade celular, reconhecendo peptídeos antigênicos exibidos no contexto da superfície de células MHC. Reconhecimento do antígeno, que é mediado pelo TCR, dirige a diferenciação de células T de ingênuo e promove a entrega de respostas citolíticas e comunicativas por populações efetoras. Noivado de TCR também induz mudanças dramáticas na arquitetura celular. Em poucos minutos, as células T cisma no lado da célula apresentadora de antígeno (APC), formando uma interface polarizada conhecida como sinapse imunológica (IS)1,2. A IS potencializa respostas de células T efetoras, permitindo o lançamento direcional de citocinas ou, no caso dos linfócitos T citotóxicos (CTLs), proteínas líticas que destruir o APC.
Noivado de TCR por pMHC induz a rápida fosforilação de várias moléculas de adaptador a jusante, incluindo o vinculador para as células de ativação de T (LAT), que, finalmente, promove a remodelação robusto do citoesqueleto sináptica2. Actina filamentosa cortical (F-Actina) conduz a célula T, espalhando sobre a superfície da APC e então resolve em uma estrutura anular caracterizada pelo acúmulo de F-Actina na periferia IS e depleção do centro. Formação de anel F-Actina é rigidamente acoplada a reorientação do centro organizador de microtúbulos (MTOC, também chamado a centrossoma em células T) para uma posição abaixo do centro da interface. Ambos os eventos ocorrem em poucos minutos de reconhecimento inicial do antígeno e estabelecer o contexto arquitetônico em que eventos de ativação subsequente e efetoras respostas ocorrem.
Para estudar a formação de IS, vários laboratórios desenvolveram abordagens em que o APC é substituído por uma superfície de vidro que contém ligantes TCR imobilizados ou suporta uma bicapa lipídica que em si contém o ligantes3,4. Células T formam IS-como contatos nestas superfícies que pode ser fotografada por microscópio de fluorescência de reflexão interna total (TIRF) ou microscopia confocal, permitindo estudos de alta resolução do início da ativação de células T e formação de IS.
Embora essas abordagens têm permitido para excelente visualização do IS totalmente montado, grande parte da sinalização da seguinte ligadura TCR:pMHC ocorre em segundos, complicando os esforços para determinar a sequência de eventos após a ativação de TCR com precisão . Para contornar esse problema, uma abordagem de fotoativação foi desenvolvida, em que photoactivatable pMHC é usado para obter um controlo spatiotemporal de TCR ativação5,6,7. Neste sistema, as células T estão conectadas em superfícies de vidro contendo photoactivatable pMHC que é não-estimulatório de TCR até irradiadas com luz ultravioleta (UV). Irradiação UV de uma região de mícron de tamanho da superfície abaixo da célula T remove o photocage criando uma zona estimulatório que pode ser reconhecida pela célula T. Sinalização de eventos subsequentes e remodelação do citoesqueleto são então monitorados usando repórteres fluorescentes geneticamente codificados. Duas versões de photoactivatable de peptídeos antigênicos, traça citocromo c88-103 (MCC) e no257-264 ovalbumina (OVA), que são apresentados no contexto da classe II MHC-eu... Ek e classe de MHC H2-Kb, respectivamente, foram desenvolvido (Figura 1). Isto permite a análise de ambos CD4+ T células específicas para MCC-l-Ok (expressando o 5 C. C7, 2B4, ou e TCRs) e CD8+ T células específicas para óvulos-H2-Kb (expressando o TCR OT1).
Na última década, a abordagem de fotoativação e imagem latente de TCR tem sido utilizada para estabelecer a cinética precisa de TCR primeiros passos de sinalização e também para identificar as vias moleculares que regem a remodelação do citoesqueleto polarizada5, 6 , 7 , 8 , 9 , 10. por exemplo, o ensaio foi fundamental na determinação que centrossoma reorientação em direção a APC é mediada por um gradiente localizado do lipídios segundo mensageiro diacilglicerol centrado no IS. Prevê-se que esta metodologia continuará a ser valioso para aplicações que exigem análise de imagens de alta resolução da função da célula T.

<table><tbody><tr><td>Nunc Lab-Tek Chambered Coverglass</td><td>Thermofischer Scientific</td><td>155361</td><td></td></tr><tr><td>Poly-L-lysine hydrobromide</td><td>Sigma-Aldrich</td><td>P2636</td><td>Will need to make Biotinylated Poly-L-Lysine</td></tr><tr><td>EZ-Link NHS-Biotin</td><td>Thermofischer Scientific</td><td>20217</td><td>Will need to make Biotinylated Poly-L-Lysine</td></tr><tr><td>Streptavidin</td><td>Thermofischer Scientific</td><td>434301</td><td></td></tr><tr><td>BirA-500: BirA biotin-protein ligase standard reaction kit</td><td>Avidity</td><td>BirA500</td><td>Will be used to biotinylate proteins</td></tr><tr><td>Biotinylated Hb I-E&lt;sup&gt;K&amp;nbsp;&lt;/sup&gt;</td><td></td><td></td><td>For protein folding, see reference 6. For biotinylation, use BirA kit</td></tr><tr><td>Biotinylated NPE-MCC I-E&lt;sup&gt;K&amp;nbsp;&lt;/sup&gt;</td><td>Anaspec</td><td></td><td>Custom NPE-MCC (H-ANERADLIAYL-K(Nvoc)-QATK-OH) can be purchased from Anaspec</td></tr><tr><td>Biotinylated &amp;alpha;H2-K&lt;sup&gt;k&lt;/sup&gt; antibody</td><td>BD Biosciences</td><td>553591</td><td></td></tr><tr><td>Biotinylated NPE-OVA H2-K&lt;sup&gt;b&lt;/sup&gt;</td><td>Anaspec</td><td></td><td>Custom NPE-OVA (H-SIINFE-K(Nvoc)-L-OH) can be purchased from Anaspec</td></tr><tr><td>Biotinylated KAVY H2-D&lt;sup&gt;b&lt;/sup&gt;&amp;nbsp;</td><td>Anaspec</td><td></td><td>Custom synthesized protein (KAVYDFATL) can be purchased from Anaspec</td></tr><tr><td>Biotinylated ICAM1&amp;nbsp;</td><td></td><td></td><td>For protein folding, see reference in protocol. For biotinylation, use BirA kit</td></tr><tr><td>Hand held UV lamp</td><td>UVP</td><td>UVGL-25</td><td>Lamp is held &amp;lt; 1 cm from the sample.&amp;nbsp; 30 s of 365 light is sufficient for detectable decaging, 20 min for quantitative decaging.</td></tr><tr><td>Olympus IX-81 OMAC TIRF system.</td><td>Olympus</td><td></td><td>Additional information about the imaging system can be found in &lt;strong class=&quot;xfig&quot;&gt;Figure 6&lt;/strong&gt;</td></tr><tr><td>Mosaic digital diaphragm</td><td>Andor</td><td></td><td></td></tr><tr><td>Slidebook software</td><td>Intelligent Imaging Innovations</td><td></td><td></td></tr></tbody></table>

spatial and temporal control of t cell activation using a photoactivatable agonist

Linfócitos T envolver-se na rápida, polarizado sinalização, ocorrendo dentro de minutos após a ativação do TCR. Isto induz a formação de sinapse imunológica, uma junção de célula-célula estereotipadas que regula a ativação de células T e metas direcionalmente respostas efetoras. Para estudar estes processos efetivamente, é necessária uma abordagem de imagem que é adaptada para a captura de respostas rápidas, polarizadas. Este protocolo descreve um sistema, que é baseado em um photoactivatable do peptide-major formando um complexo (pMHC) que é não-estimulatórios até que ele é exposto à luz ultravioleta. Decaging alvo estes reagentes durante experimentos de tele-microscopia permite controle spatiotemporal preciso da ativação do TCR e monitoramento de alta resolução de respostas celulares subsequentes por reflexão interna total (TIRF) de imagem. Essa abordagem também é compatível com estratégias de perturbação genética e farmacológicas. Isso permite que o conjunto das vias moleculares bem definidos que link TCR sinalização para a formação das estruturas do citoesqueleto polarizadas que fundamentam a sinapse imunológica.

A abordagem de fotoativação e de imagem permite a observação e facile quantificação das respostas de sinalização rápidas, polarizadas. Para ilustrar suas capacidades, reproduzidas aqui, é um experimento examinando a spatiotemporal correlação entre acumulação de DAG TCR-induzida e de reorientação centrossoma. 5 C. Explosões de célula C7 T retrovirally foram transfectadas com dois repórteres fluorescentes: um biossensor DAG que contém os domínios de C1 em tandem da prot...

Watch this Scientific Journal Video about Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist at JoVE.com

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

Controle espacial e Temporal da ativação de células T usando um agonista Photoactivatable

Este protocolo descreve um método baseado em imagem para ativar os linfócitos T usando photoactivatable peptídeo-MHC, permitindo um controle preciso da ativação de células T spatiotemporal.

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

spatial-temporal-control-t-cell-activation-using-photoactivatable

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

6.2K visualizações.  Memorial Sloan-Kettering Cancer Center. Este protocolo descreve um método baseado em imagem para ativar os linfócitos T usando photoactivatable peptídeo-MHC, permitindo um controle preciso da ativação de células T spatiotemporal.

Vídeo: Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. 
To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac4, Cdc424, RhoA4 and Ras5, in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells6. Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level6.

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

Espaço-Temporal Manipulação de Atividade GTPase Pequeno em nível subcelular e em escala de tempo de segundos em células vivas

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

Bioengenharia

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Um método de Optogenetic para controlar e analisar os padrões de expressão de Gene em interações de célula para célula

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

Genética

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

A técnica de microscopia TIRF para imagens em tempo real, simultâneo da TCR e seus associados proteínas sinalizadoras

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Imunologia e Infecção

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Em tempo real de imagens ao vivo de células T Sinalização de Formação de Complexos

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

Two-Photon-Based fotoativação no Live Embriões Zebrafish

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Biologia

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

Migração intersticial imagens de células CD4 T na derme inflamadas

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Controlo óptico de uma proteína neuronal utilizando um ácido Unnatural Amino geneticamente codificados em neurónios

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Neurociência

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

Uso da tecnologia de MHC única cadeia para investigar Co-Genealogia na ativação de células T humanas CD8 +

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist ...

Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity

Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain unanswered. One requirement for advancements in our understanding of the underlying disease mechanisms and cellular dynamics is the precise coupling of the microanatomical location of cell subsets with downstream molecular or functional analyses; a goal that has traditionally been difficult to achieve. The development of stable photoactivatable biological fluorophores and their integration into reporter strains has recently enabled precise microanatomical labeling and tracking of cellular subsets in murine models. Here, we describe how the ability to analyze autoreactive lymphocytes from single germinal centers may help to provide novel insights into autoimmunity, using the combination of a novel chimeric model of autoimmunity with a photoactivatable reporter as an example. We demonstrate a procedure for generating mixed chimeras with spontaneous autoreactive germinal centers populated by lymphocytes carrying a photoactivatable green fluorescent protein reporter. Using in vivo labeling strategies, single germinal centers can be visualized in explanted lymphoid tissues and their cellular constituents photoactivated by two-photon microscopy. Photoactivated lymphocytes from single germinal centers can then be analyzed or sorted flow cytometrically, as single cells or in bulk, and may be subjected to additional downstream molecular and functional analyses. This approach may directly be applied to provide renewed insights in the field of autoimmunity, but the procedure for generating bone marrow chimeras and the photoactivation procedure may additionally find broad application in studies of infectious diseases and tumor metastases.

This protocol describes the generation of mixed murine bone marrow chimeras with spontaneous autoimmune germinal centers, in which autoreactive lymphocytes carry a photoactivatable green fluorescent protein (PA-GFP) reporter. This provides the ability to link cellular location in tissues with downstream molecular and functional analyses.

Interrogando os centros germinativos auto-reativas individuais por Photoactivation em um modelo misto de quimérico de auto-imunidade

Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain ...

<meta charset="utf8"></head><body>Controle optogenético da atividade enzimática usando interruptor alostérico regulado por luz

Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation

Optogenetics offers the potential for mimicking complex spatiotemporal control of enzyme activity down to a subcellular resolution. However, most optogenetic approaches often face significant challenges in integrating multiple capabilities in a single tool applicable to a wide range of target proteins. Achieving precise control over ON/OFF kinetics, ensuring minimum leakiness in the dark, and demonstrating efficient performance in mammalian cells with subcellular precision are some of the most common challenges faced in this field. A promising solution lies in the application of rationally designed light-sensitive domains to allosterically control protein activity. Using that strategy, we generated an optogenetic method combining all the desired features. The approach involves the incorporation of the Light-regulated allosteric switch module (LightR) in the target protein to regulate enzyme activity using blue (465 nm) light. The LightR domain is generated by linking two Vivid (VVD) photoreceptor domains, creating a light-sensitive clamp that can be incorporated into a small flexible loop within the catalytic domain of an enzyme. In its dark state, LightR clamp is open, thus distorting the enzyme&#39;s catalytic domain and inactivating it. Upon exposure to blue light, the LightR domain closes and restores the catalytic domain&#39;s structure and enzyme activity. In this manuscript, we discuss design strategies&#160;to generate a light-regulated protein kinase and demonstrate its control by blue light, reversibility, kinetics, and precise regulation at the subcellular level, enabling tight spatiotemporal precision. Utilizing Src tyrosine kinase as a model, we showcase a protocol for effectively regulating LightR-Src kinase activity. We also demonstrate LightR applicability across different enzyme classes, expanding the utility of the tool system in addressing mechanistic questions of signaling pathways in different diseases.

<meta charset="utf8"></head><body>Controle espaço-temporal da atividade proteica por meio da regulação alostérica optogenética

This protocol describes the application of an engineered blue-light-activated allosteric switch (LightR) domain for reversible spatiotemporal control of protein activity. Utilizing Src tyrosine kinase as a model, this study offers an elaborate protocol for developing and characterizing light-regulated Src (LightR-Src). It demonstrates the versatility of this approach across enzyme classes.