Name: monitoring stub1-mediated pexophagy
Uploaded: 2023-05-12T12:35:00
Description: Watch this Scientific Journal Video about Monitoring Stub1-Mediated Pexophagy at JoVE.com

Este trabalho foi apoiado em parte por uma bolsa de pesquisa MOST 111-2311-B-001-019-MY3 do Conselho Nacional de Ciência e Tecnologia de Taiwan.

1. Preparação de células expressando diKillerRed ou proteínas auto-marcadas (SLPs) no lúmen do peroxissomo
<ol><li>Seme as células desejadas em placas de cultura celular com fundo de vidro. Para o experimento, foram utilizadas 2 x10 5 células SHSY5Y humanas em 840 μL de meio de cultura (DMEM/F-12 suplementado com 10% de soro fetal bovino e 1% de penicilina/estreptomicina) ou 6 x 104 células NIH3T3 de camundongo em 840 μL de meio de cultura (DMEM suplementado com 10% de s...

<ol>
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T., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin+signals+autophagic+degradation+of+cytosolic+proteins+and+peroxisomes.">Ubiquitin signals autophagic degradation of cytosolic proteins and peroxisomes.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (52), 20567-20574 (2008).</li><li>Yamashita, S., Abe, K., Tatemichi, Y., Fujiki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+membrane+peroxin+PEX3+induces+peroxisome-ubiquitination-linked+pexophagy.">The membrane peroxin PEX3 induces peroxisome-ubiquitination-linked pexophagy.</a> Autophagy. 10 (9), 1549-1564 (2014).</li><li>Nordgren, 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=Export-deficient+monoubiquitinated+PEX5+triggers+peroxisome+removal+in+SV40+large+T+antigen-transformed+mouse+embryonic+fibroblasts.">Export-deficient monoubiquitinated PEX5 triggers peroxisome removal in SV40 large T antigen-transformed mouse embryonic fibroblasts.</a> Autophagy. 11 (8), 1326-1340 (2015).</li><li>Zhang, 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=ATM+functions+at+the+peroxisome+to+induce+pexophagy+in+response+to+ROS.">ATM functions at the peroxisome to induce pexophagy in response to ROS.</a> Nature Cell Biology. 17 (10), 1259-1269 (2015).</li><li>Chen, B. -. H., Chang, Y. -. J., Lin, S., Yang, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsc70/Stub1+promotes+the+removal+of+individual+oxidatively+stressed+peroxisomes.">Hsc70/Stub1 promotes the removal of individual oxidatively stressed peroxisomes.</a> Nature Communications. 11 (1), 5267 (2020).</li><li>Hung, Y. H., Chen, L. M., Yang, J. Y., Yang, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporally+controlled+induction+of+autophagy-mediated+lysosome+turnover.">Spatiotemporally controlled induction of autophagy-mediated lysosome turnover.</a> Nature Communications. 4, 2111 (2013).</li><li>Mageswaran, S. K., Yang, W. Y., Chakrabarty, Y., Oikonomou, C. M., Jensen, G. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bit-by-bit+autophagic+removal+of+parkin-labelled+mitochondria.">Bit-by-bit autophagic removal of parkin-labelled mitochondria.</a> Nature Communications. 4, 2428 (2013).</li><li>Los, G. 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=HaloTag:+A+novel+protein+labeling+technology+for+cell+imaging+and+protein+analysis.">HaloTag: A novel protein labeling technology for cell imaging and protein analysis.</a> ACS Chemical Biology. 3 (6), 373-382 (2008).</li><li>Lismont, C., Walton, P. 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Este protocolo detalha como desencadear a pexofagia mediada por Stub1 em culturas de células elevando os níveis de ROS peroxissomais com luz. Como o protocolo depende da geração de ROS assistida por corante, é necessário garantir a expressão suficiente de diKillerRed-VKSKL ou coloração de ligante SLP marcada com corante dentro das células de interesse. Dado que diferentes tipos celulares ou células de diferentes origens genéticas podem abrigar peroxissomas com propriedades ligeiramente diferentes, pode-se pre...

Os peroxissomas são organelas ligadas a uma única membrana presentes na maioria das células eucarióticas. Os peroxissomas são um compartimento metabólico essencial para a realização da beta-oxidação de ácidos graxos de cadeia muito longa, catabolismo de purinas e síntese de fosfolipídios éteres e ácidos biliares1. O acetil-CoA derivado do peroxissomo controla a homeostase lipídica regulando a sinalização central no metabolismo2. Portanto, não é surpresa que o comprometimento das funções peroxissomais esteja implicado em várias doenças, incluindo doenças neurodegenerativas, envelhecimento, cânceres, obesidade e diabetes 3,4,5. Um processo essencial na manutenção da operação peroxissomal é a pexofagia. A puxofagia é um processo catabólico para o turnover seletivo de peroxissomas por autofagia. As células usam a pexofagia para ajudar a controlar a quantidade e a qualidade dos peroxissomos, garantindo assim a função peroxissomal adequada. Um estudo recente demonstrou que a perda de peroxissomo causada por mutações nos fatores 1 e 6 da biogênese peroxissomal PEX1 resulta de pexofagia não controlada6. Notavelmente, 65% de todos os pacientes com transtorno da biogênese do peroxissomo (DBP) apresentam deficiências no complexo ATPase AAA peroxissomal, composto por PEX1, PEX6 e PEX26em células de mamíferos7.
Vários métodos podem ser usados para iniciar e estudar a pexofagia. Em leveduras, a pexofagia é desencadeada quando os nutrientes fornecidos são trocados de fontes de carbono dependentes de peroxissomo para fontes de carbono independentes de peroxissomo (para diminuir o número de peroxissomas celulares)8. Por exemplo, a transferência de células de Pichia pastoris cultivadas com metanol do meio metanol para o meio glicosado e etanol induz micropexofagia e macropexofagia, respectivamente 8,9,10. A micropexofagia sequestra peroxissomas agrupados para degradação, remodelando o vacúolo para formar membranas de sequestro vacuolar em forma de taça e uma estrutura semelhante a uma tampa denominada aparato de membrana específico para micropexofagia (MIPA). Na macropexofagia, os peroxissomas individuais são engolidos por estruturas de membrana dupla conhecidas como pexofagossomos, seguidos de fusão com o vacúolo para degradação 8,9,10. A fosforilação de receptores de pexofagia, como Atg36p em Saccharomyces cerevisiae e Atg30p em Pichia pastoris, é crítica para recrutar maquinaria de autofagia central e facilitar o direcionamento peroxissomal para autofagossomos 8,11.
Em células de mamíferos, a pexofagia pode ser induzida por ubiquitinação. A marcação das proteínas da membrana peroxissomal PMP34 ou PEX3 com ubiquitina no lado citosólico induz a pexofagia12. A superexpressão de PEX3 induz a ubiquitinação do peroxissomo e a eliminação do peroxissomo pelos lisossomos13. Além disso, a fusão de PEX5 com um EGFP C-terminal prejudica a exportação de PEX5 monoubiquitinado e resulta em pexofagia14. Por outro lado, a pexofagia também pode ser desencadeada pelo tratamento com H 2 O2. Os peroxissomas produzem espécies reativas de oxigênio (EROs); especificamente, a enzima peroxissomal Acox1, que catalisa a etapa inicial da beta-oxidação de ácidos graxos de cadeia muito longa (carbono > 22), produz não apenas acetil-CoA, mas também ROS peroxissomal. Em resposta aos níveis elevados de ROS sob o tratamento comH 2 O2, as células de mamíferos ativam a pexofagia para diminuir a produção de ROS e aliviar o estresse. Tem sido relatado que o tratamento com H 2 O2direciona o recrutamento de ataxia-telangiectasia mutada (ATM) para peroxissomas. A ATM fosforila então PEX5 para promover o turnover peroxissomal pela pexofagia15.
Como os peroxissomas são centros geradores de ROS, eles também são propensos a danos de ROS. Lesões peroxissomais provocadas por ERO forçam as células a ativar a pexofagia para iniciar vias de controle de qualidade do peroxissomo (remoção do peroxissomo danificado por autofagia). Aqui, delineamos uma abordagem para o desencadeamento sob demanda de lesão peroxissomal provocada por ROS. O protocolo aproveita a produção de ERO ativadas por luz dentro de organelas 16,17,18,19,20 (Figura 1). Os peroxissomas marcados com corante são iluminados, levando à produção de ERO dentro da luz peroxissomal, o que desencadeia especificamente a lesão peroxissomal. Usando este protocolo, mostra-se que os peroxissomas estressados por ROS são removidos através de uma via de degradação dependente de ubiquitina. Os peroxissomas estressados com ROS recrutam a ubiquitina E3 ligase Stub1 para permitir seu engolfamento em autofagossomos para remoção individual por pexofagia16. Pode-se usar este protocolo para comparar o destino de peroxissomas lesionados e saudáveis dentro de uma mesma célula por microscopia de lapso de tempo. O método também pode ser usado para danificar globalmente todos os peroxissomas (em todas as células) em uma placa de cultura, permitindo a análise bioquímica da via da pexofagia.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm culture dish with a 20 mm diameter glass microwell&#160;</td>
			<td>MatTek</td>
			<td>P35G-1.5-20-C</td>
			<td>20 mm glass bottomed</td>
		</tr>
		<tr>
			<td>3-amino-1,2,4-triazole (3-AT)</td>
			<td>Sigma Aldrich</td>
			<td>A8056</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum</td>
			<td>ThermoFisher Scientific</td>
			<td>16170060</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Nuaire</td>
			<td>NU-4750</td>
			<td></td>
		</tr>
		<tr>
			<td>diKillerRed-PTS1</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>made by appending the KillerRed tandem dimer with PTS1(VKSKL)</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>ThermoFisher Scientific</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12)</td>
			<td>ThermoFisher Scientific</td>
			<td>11330032</td>
			<td></td>
		</tr>
		<tr>
			<td>EGFP-C1</td>
			<td>Clontech</td>
			<td>pEGFP-C1</td>
			<td>The backbone of EGFP-C1 was used for cloning EGFP-Stub1, EGFP-Hsp70, EGFP-p62</td>
		</tr>
		<tr>
			<td>EGFP-Hsp70</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>Hsp70 gene (HSPA1A) PCR amplified from HeLa cDNA and cloned into EGFP-C1</td>
		</tr>
		<tr>
			<td>EGFP-LC3B</td>
			<td>Addgene</td>
			<td>11546</td>
			<td></td>
		</tr>
		<tr>
			<td>EGFP-p62</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>generated by inserting the human SQSTM1 gene (through PCR amplification of the HeLa cell cDNA) into EGFP-C1</td>
		</tr>
		<tr>
			<td>EGFP-Stub1</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>generated by inserting the mouse Stub1 gene (through PCR amplification of the total mouse kidney cDNA) into EGFP-C1</td>
		</tr>
		<tr>
			<td>EGFP-Ub</td>
			<td>Addgene</td>
			<td>11928</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>ThermoFisher Scientific</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>HaloTag TMR ligand&#160;</td>
			<td>Promega</td>
			<td>G8252</td>
			<td></td>
		</tr>
		<tr>
			<td>HaloTag-PTS1</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>PTS1 appended and cloned into EGFP-C1 backbone</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>ThermoFisher Scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Confocal Microscope&#160;</td>
			<td>Olympus</td>
			<td>FV3000RS</td>
			<td>405 nm Ex, 488 nm Ex, 561 nm Ex,&#160; microscope with a TOKAI HIT chamber incubator and the UNIV2-D35 dish attachment</td>
		</tr>
		<tr>
			<td>Janelia Fluor 646 HaloTag Ligand</td>
			<td>Promega</td>
			<td>GA1120</td>
			<td></td>
		</tr>
		<tr>
			<td>LED</td>
			<td>VitaStar</td>
			<td>PAR64</td>
			<td>80 W, 555-570 nm</td>
		</tr>
		<tr>
			<td>lipofectamine 2000&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>11668</td>
			<td>transfection reagent</td>
		</tr>
		<tr>
			<td>NIH3T3 cell</td>
			<td>ATCC</td>
			<td>CRL-1658</td>
			<td>adherent</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>ThermoFisher Scientific</td>
			<td>319850</td>
			<td>reduced serum media</td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>PMP34-TagBFP</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>PMP34 PCR amplified from HeLa cDNA and cloned intoTagBFP-C (Evrogen FP171)</td>
		</tr>
		<tr>
			<td>roGFP2-PTS1</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>generated by appending eroGFP (taken from Addgene plasmid 20131) with the amino acid sequence VKSKL, and cloned into the EGFP-C1</td>
		</tr>
		<tr>
			<td>SHSY5Y cell</td>
			<td>ATCC</td>
			<td>CRL-2266</td>
			<td>adherent</td>
		</tr>
	</tbody>
</table>

monitoring stub1-mediated pexophagy

Células de mamíferos podem transformar peroxissomas através da pexofagia mediada por Stub1. A via potencialmente permite o controle celular da quantidade e qualidade dos peroxissomos. Durante esse processo, a proteína de choque térmico 70 e a ubiquitina E3 ligase, Stub1, translocam-se em peroxissomas para serem viradas para iniciar a pexofagia. A atividade da ligase Stub1 permite o acúmulo de ubiquitina e outros módulos relacionados à autofagia em peroxissomas alvo. A elevação dos níveis de espécies reativas de oxigênio (EROs) dentro da luz peroxissomal pode ativar a pexofagia mediada por Stub1. Pode-se, portanto, usar a geração de ROS assistida por corante para desencadear e monitorar essa via. Este artigo descreve os procedimentos para o uso de duas classes de corantes, proteínas fluorescentes e fluoróforos sintéticos, para iniciar a pexofagia em culturas de células de mamíferos. Esses protocolos baseados na geração de ROS assistida por corante podem não apenas ser usados para atingir todos os peroxissomas dentro de uma população celular globalmente, mas também podem permitir a manipulação de peroxissomas individuais dentro de células únicas. Também descrevemos como a pexofagia mediada por Stub1 pode ser acompanhada usando microscopia de células vivas.

O esquema de indução de pelofagia mediado por Stub1 mostrado aqui aproveita a geração de ROS assistida por corante dentro do lúmen do peroxissomo. Esta operação requer intensidades luminosas mínimas. Peroxissomas contendo proteínas fluorescentes ou corantes podem, portanto, ser iluminados usando microscópios confocais de varredura a laser padrão. A iluminação focal leva à produção instantânea e localizada de ERO dentro de peroxissomas individuais, como indicado pelo repórter fluorescente roGFP2-VKSKL (<...

Watch this Scientific Journal Video about Monitoring Stub1-Mediated Pexophagy at JoVE.com

Monitoring Stub1-Mediated Pexophagy

Este protocolo fornece instruções para o desencadeamento e monitoramento da pexefagia mediada por Stub1 em células vivas.

Monitoramento da Coxofagia Mediada por Stub1

Mammalian cells can turn over peroxisomes through Stub1-mediated pexophagy. The pathway potentially permits cellular control of the quantity and quality ...

Os peroxissomas realizam a beta oxidação e são propensos a danos de ROS. Este método permite investigar como as células lidam com peroxissomas estressados por ROS. Este protocolo não é usado apenas para atingir globalmente todos os peroxissomas dentro de uma população celular, mas também pode permitir a manipulação dos peroxissomas individuais dentro de células únicas.A geração de ROS assistida por corante também pode ser usada para atingir organelas fora dos peroxissomas para investigar como as células lidam com lesões de organelas. Para começar, semeie duas vezes 10 para a quinta célula SHSY5Y humana ou seis vezes 10 para a quarta célula NIH H3T3 de camundongo em 840 microlitros de meio de cultura em placas de cultura de 35 milímetros com fundo de vidro com um micropoço de vidro de 20 milímetros de diâmetro. Cultivar as células sob 5% de dióxido de carbono a 37 graus Celsius por 24 horas.Em seguida, transfectar as células com plasmídeos desejados usando um reagente de transfecção ou transfecção de células SHSY5Y. Diluir plasmídeos em 55 microlitros de DMEM/F-12 sem soro e diluir um microlitro de reagente de transfecção em 55 microlitros de DMEM/F-12 sem soro. Combinar o ADN diluído com o reagente diluído.Misture delicadamente e incube por 30 minutos à temperatura ambiente. Adicione o complexo ao micropoço de 20 milímetros previamente banhado com 100 microlitros de meio de cultura contendo células SHSY5Y sem antibióticos. Agite suavemente o prato para frente e para trás.Realizar a transfecção de células NIH 3T3 como demonstrado antes, mas substituir o meio de soro reduzido em vez de DMEM/F-12 livre de soro para diluição de plasmídios e reagente de transfecção. Após duas horas da transfecção, remova o meio contendo reagente de transfecção e adicione um mililitro de meio de cultura fresco. Incubar as células NIH 3T3 ou SHSY5Y a 37 graus Celsius e 5% de dióxido de carbono por 24 horas.Para a produção de ROS ativadas por luz, em 24 horas após a transfecção remova o meio de cultura e adicione 10 microlitros de 200 nanomolares HaloTag TMR ligante ou 200 nanomolares Janelia Fluor 646 HaloTag ligante no micropoço de vidro de 20 milímetros. Transfira o prato para uma incubadora de 37 graus Celsius e 5% de dióxido de carbono e deixe por uma hora para manchar as células. Após uma hora, remova bem o meio de coloração e adicione um mililitro de meio de cultura fresco.Coloque um prato com fundo de vidro contendo as células desejadas em um microscópio confocal de varredura a laser equipado com uma incubadora de palco. No software, clique em Janela de ferramentas, escolha Ocular e clique no botão DIA para acender a luz transmitida. Visualize o prato através da ocular do microscópio e coloque as células em foco girando o botão de foco.Escolha LSM e clique no botão Dye Detector Select. Clique duas vezes nos corantes desejados na janela pop-up para selecionar as configurações apropriadas de laser e filtro para gerar imagens das células. Clique em Live4x no painel Live para iniciar a varredura rápida a laser para iniciar a triagem dos sinais fluorescentes das células.Mova o palco usando o controlador de joystick e localize as células médias que expressam diKillerRed VKSKL ou SLP-VKSKL para aplicar estresse ROS nos peroxissomos. Adquira uma imagem da célula desejada. Clique em Janela de ferramentas e escolha Estimulação LSM.Em seguida, clique no botão de elipse e, na janela Live Image, use o mouse para desenhar uma ROI de 15 micrômetros de diâmetro contendo os peroxissomas desejados aos quais a tensão ROS será aplicada. Para aplicar o estresse ROS, use 561 nanômetros para células com o ligante SLP marcado com diKillerRed VKSKL ou TMR e use 640 nanômetros para células coradas com o ligante SLP marcado com Janelia Fluor 646. Selecione o comprimento de onda do laser para aplicar a tensão ROS.Insira a porcentagem de laser desejada e a duração. Para a produção de ROS nos peroxissomos, clique em Adquirir e, em seguida, no botão Estimulação para iniciar a varredura com 561 ou 640 nanômetros de luz através das ROIs por 30 segundos cada. Essa estimulação corresponde a aproximadamente 5% de potência do laser para 561 e 640 nanômetros.Após 30 segundos de iluminação a laser, os peroxissomas dentro das ROIs perdem seus sinais diKillerRed VKSKL, TMR ou Janelia Fluor 646. Esta perda de sinal permite distinguir os peroxissomas iluminados e não iluminados dentro da célula. Para monitorar as células vivas de pexófagos, siga a translocação de Stub1, Hsp70, ubiquitina, p62 ou LC3B marcados com EGFP para os peroxissomas iluminados ou estressados por ROS, por meio de imagens de lapso de tempo usando intervalos de 10 minutos entre os quadros.A iluminação focal leva à produção instantânea e localizada de ROS dentro de peroxissomas individuais, como indicado pelo repórter fluorescente roGFP2-VKSKL. A imagem diKillerRed VKSKL foi obtida após todas as medidas de roGFP2-VKSKL terem sido feitas para indicar a localização dos peroxissomas danificados. Os dados também mostram que os peroxissomas não iluminados não são afetados, indicando a precisão da metodologia.Esta metodologia foi utilizada para monitorar a pexofagia mediada por Stub1. Durante esse processo, Hsp70, Stub1, proteínas ubiquitinadas, adaptadores de autofagia p62 e LC3B aparecem subsequentemente em peroxissomas estressados por ROS para conduzir a pexofagia. Usando a mesma estratégia, foi possível danificar simultaneamente todos os peroxissomas em uma placa de cultura para a caracterização bioquímica da pexofagia mediada por Stub1.Antes da iluminação por LED, a fluorescência EGFP-ubiquitina era homogênea no citoplasma e não colocalizava com os peroxissomos. Após nove horas de iluminação LED, os sinais de EGFP-ubiquitina se acumularam em todos os peroxissomas nas células cultivadas em uma placa confocal. Usando este protocolo, descobrimos que os peroxissomas estressados por ROS são removidos através de uma via de degradação dependente de ubiquitina.Os peroxissomas estressados por ROS recrutam a ubiquitina E3 ligase Stub1 para permitir sua remoção por pexofagia.

Research

JoVE Journal

Biology

Monitoring Actin Disassembly with Time-lapse Microscopy

Monitoramento Actina Desmontagem com a Time-lapse Microscopia

Biologia

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, simple perceptual tasks are combined with appetitive and aversive reinforcement.  However, the use of reinforcers such as juice and airpuffs can create challenges for fMRI.  Reinforcer delivery can cause head movement, which creates artifacts in the fMRI signal.  Reinforcement can also lead to changes in heart rate and respiration that are mediated by autonomic pathways.  Changes in heart rate and respiration can directly affect the fMRI (BOLD) signal in the brain and can be confounded with signal changes that are due to neural activity.  In this presentation, we demonstrate methods for administering reinforcers in a controlled manner, for stabilizing the head, and for measuring pulse and respiration.

This presentation demonstrates the use of fMRI to study neural circuits that underlie decision-making. Simple perceptual tasks are combined with appetitive and aversive reinforcements to investigate how outcomes affect decision processes.

Imagem Funcional com reforço, eyetracking e Monitoramento Fisiológico

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, ...

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Métodos única molécula para acompanhar as alterações nas propriedades elásticas bicamada

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Monitoring Plant Hormones During Stress Responses

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.

A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.

Monitoramento hormônios vegetais Durante Respostas ao Estresse

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

Monitoring Acupuncture Effects on Human Brain by fMRI

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate that acupuncture mobilizes a limbic-paralimbic-neocortical network and its anti-correlated sensorimotor/paralimbic network at multiple levels of the brain and that the hemodynamic response is influenced by the psychophysical response.  Physiological monitoring may be performed to explore the peripheral response of the autonomic nerve function. This video describes the studies performed at LI4 (hegu), ST36 (zusanli) and LV3 (taichong), classical acupoints that are commonly used for modulatory and pain-reducing actions. Some issues that require attention in the applications of fMRI to acupuncture investigation are noted.

FMRI and physiological monitoring is used to study the effects of Acupuncture on the central and peripheral nervous systems. Acupuncture mobilizes a limbic-paralimbic-neocortical network, with great overlap with the default mode network, to modulate neurological activity, possibly related to its autonomic effect in the peripheral nervous system.

Monitoramento Effects Acupuntura em Cérebro Humano por fMRI

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

Função de monitorização cardíaca em larval Drosophila melanogaster Para estudos fisiológicos

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

Bioensaios de Resistência a Inseticidas Monitoramento

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A longo prazo de Teste de toxicidade letal com o crustáceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Ensaios para Monitoramento juntamente redobramento de proteínas em<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Reação selecionada Monitoramento Espectrometria de Massa para Absolute Quantificação de Proteína

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...