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 trabajo fue apoyado en parte por una beca de investigación MOST 111-2311-B-001-019-MY3 del Consejo Nacional de Ciencia y Tecnología de Taiwán.

1. Preparación de células que expresan diKillerRed o proteínas automarcantes (SLP) en la luz del peroxisoma
<ol><li>Siembra las células deseadas en placas de cultivo celular con fondo de vidrio. Para el experimento aquí, sembrar 2 x 105 células SHSY5Y humanas en 840 μL de medio de cultivo (DMEM / F-12 suplementado con 10% de suero bovino fetal y 1% de penicilina / estreptomicina) o 6 x 10 4 células NIH3T3de ratón en 840 μL de medio de cultivo (DMEM suplementado con 10% d...

<ol>
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M., Lodhi, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisomal+dysfunction+in+age-related+diseases.">Peroxisomal dysfunction in age-related diseases.</a> Trends in Endocrinology &amp; Metabolism. 28 (4), 297-308 (2017).</li><li>Fransen, M., Nordgren, M., Wang, B., Apanasets, O., Veldhoven, P. P. 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B., 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=The+peroxisomal+AAA+ATPase+complex+prevents+pexophagy+and+development+of+peroxisome+biogenesis+disorders.">The peroxisomal AAA ATPase complex prevents pexophagy and development of peroxisome biogenesis disorders.</a> Autophagy. 13 (5), 868-884 (2017).</li><li>Nazarko, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pexophagy+is+responsible+for+65%+of+cases+of+peroxisome+biogenesis+disorders.">Pexophagy is responsible for 65% of cases of peroxisome biogenesis disorders.</a> Autophagy. 13 (5), 991-994 (2017).</li><li>Eberhart, T., Kovacs, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pexophagy+in+yeast+and+mammals:+An+update+on+mysteries.">Pexophagy in yeast and mammals: An update on mysteries.</a> Histochemistry and Cell Biology. 150 (5), 473-488 (2018).</li><li>Manjithaya, R., Nazarko, T. Y., Farr&#233;, J. -. C., Subramani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+and+physiological+role+of+pexophagy.">Molecular mechanism and physiological role of pexophagy.</a> FEBS Letters. 584 (7), 1367-1373 (2010).</li><li>Till, A., Lakhani, R., Burnett, S. F., Subramani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pexophagy:+The+selective+degradation+of+peroxisomes.">Pexophagy: The selective degradation of peroxisomes.</a> International Journal of Cell Biology. 2012, 512721 (2012).</li><li>Germain, K., Kim, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pexophagy:+A+model+for+selective+autophagy.">Pexophagy: A model for selective autophagy.</a> International Journal of Molecular Sciences. 21 (2), 578 (2020).</li><li>Kim, P. K., Hailey, D. W., Mullen, R. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cryo-electron+tomography+workflow+reveals+protrusion-mediated+shedding+on+injured+plasma+membrane.">A cryo-electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane.</a> Science Advances. 7 (13), eabc6345 (2021).</li><li>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+initiation+of+parkin-mediated+mitophagy+within+single+cells.">Spatiotemporally controlled initiation of parkin-mediated mitophagy within single cells.</a> Autophagy. 7 (10), 1230-1238 (2011).</li><li>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=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. A., Fransen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+monitoring+of+subcellular+redox+dynamics+in+living+mammalian+cells+using+RoGFP2-based+probes.">Quantitative monitoring of subcellular redox dynamics in living mammalian cells using RoGFP2-based probes.</a> Methods in Molecular Biology. 1595, 151-164 (2017).</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=Potential+limitations+in+the+use+of+KillerRed+for+fluorescence+microscopy.">Potential limitations in the use of KillerRed for fluorescence microscopy.</a> Journal of Microscopy. 245 (3), 229-235 (2012).</li></ol>

Este protocolo detalla cómo desencadenar la pexofagia mediada por Stub1 dentro de cultivos celulares elevando los niveles de ROS peroxisomales con luz. Como el protocolo se basa en la generación de ROS asistida por colorante, es necesario garantizar una expresión suficiente de diKillerRed-VKSKL o tinción de ligando SLP marcada con colorante dentro de las células de interés. Dado que diferentes tipos de células o células de diferentes antecedentes genéticos pueden albergar peroxisomas con propiedades ligeramente ...

Los peroxisomas son orgánulos unidos a una sola membrana presentes en la mayoría de las células eucariotas. Los peroxisomas son un compartimento metabólico esencial para llevar a cabo la beta-oxidación de ácidos grasos de cadena muy larga, catabolismo de purina y síntesis de fosfolípidos y ácidos biliareséter 1. La acetil-CoA derivada de peroxisomas controla la homeostasis lipídica regulando la señalización central en el metabolismo2. Por lo tanto, no es de extrañar que las funciones peroxisomales comprometidas estén implicadas en diversas enfermedades, incluyendo trastornos neurodegenerativos, envejecimiento, cáncer, obesidad y diabetes 3,4,5. Un proceso esencial en el mantenimiento de la operación peroxisomal es la pexofagia. La pexofagia es un proceso catabólico para el recambio selectivo de peroxisomas por autofagia. Las células utilizan la pexofagia para ayudar a controlar la cantidad y calidad de los peroxisomas, asegurando así una función peroxisomal adecuada. Un estudio reciente demostró que la pérdida de peroxisomas causada por mutaciones en los factores de biogénesis peroxisomal PEX1 1 y 6 resulta de la pexofagia no controlada6. En particular, el 65% de todos los pacientes con trastorno de biogénesis de peroxisomas (PBD) albergan deficiencias en el complejo ATPasa AAA peroxisomal, compuesto por PEX1, PEX6 y PEX26 en células de mamíferos7.
Se pueden utilizar varios métodos para iniciar y estudiar la pexofagia. En la levadura, la pexofagia se desencadena cuando los nutrientes suministrados se cambian de fuentes de carbono dependientes del peroxisoma a fuentes de carbono independientes del peroxisoma (para reducir el número de peroxisomas celulares)8. Por ejemplo, la transferencia de células de Pichia pastoris cultivadas con metanol del medio metanol al medio de glucosa y al medio de etanol induce micropexofagia y macropexofagia, respectivamente 8,9,10. Los secuestros de micropexofagia agrupan los peroxisomas para su degradación mediante la remodelación de la vacuola para formar membranas de secuestro vacuolar en forma de copa y una estructura similar a una tapa denominada aparato de membrana específico de micropexofagia (MIPA). En la macropexofagia, los peroxisomas individuales son engullidos por estructuras de doble membrana conocidas como pexofagosomas, seguidas de fusión con la vacuola para la degradación 8,9,10. La fosforilación de receptores de pexofagia, como Atg36p en Saccharomyces cerevisiae y Atg30p en Pichia pastoris, es crítica para que los receptores recluten maquinaria central de autofagia y faciliten la orientación peroxisomal a los autofagosomas 8,11.
En las células de mamíferos, la pexofagia puede ser inducida por ubiquitinación. El marcado de las proteínas de membrana peroxisomal PMP34 o PEX3 con ubiquitina en el lado citosólico induce pexofagia12. La sobreexpresión de PEX3 induce la ubiquitinación de los peroxisomas y la eliminación de los peroxisomas por los lisosomas13. Además, la fusión de PEX5 con un EGFP C-terminal perjudica la exportación de PEX5 monoubiquitinado y da lugar a pexofagia14. Por otro lado, la pexofagia también puede ser desencadenada por el tratamiento conH2O2. Los peroxisomas producen especies reactivas de oxígeno (ROS); específicamente, la enzima peroxisomal Acox1, que cataliza el paso inicial de la beta-oxidación de ácidos grasos de cadena muy larga (> 22 carbono), produce no solo acetil-CoA sino también ROS peroxisomal. En respuesta a los niveles elevados de ROS bajo el tratamiento conH2O2, las células de mamíferos activan la pexofagia para reducir la producción de ROS y aliviar el estrés. Se ha informado que el tratamiento conH2O2impulsa el reclutamiento de ataxia-telangiectasia mutada (ATM) a peroxisomas. ATM luego fosforila PEX5 para promover el recambio peroxisomal por pexofagia15.
Dado que los peroxisomas son centros generadores de ROS, también son propensos al daño de ROS. Las lesiones peroxisomales provocadas por ROS obligan a las células a activar la pexofagia para iniciar las vías de control de calidad del peroxisoma (eliminación del peroxisoma dañado por autofagia). Aquí, describimos un enfoque para el desencadenamiento a demanda de la lesión peroxisomal provocada por ROS. El protocolo aprovecha la producción de ROS activadas por luz dentro de los orgánulos 16,17,18,19,20 (Figura 1). Los peroxisomas marcados con colorante se iluminan, lo que lleva a la producción de ROS dentro de la luz peroxisomal, lo que desencadena específicamente la lesión peroxisomal. Usando este protocolo, se muestra que los peroxisomas estresados por ROS se eliminan a través de una vía de degradación dependiente de ubiquitina. Los peroxisomas estresados por ROS reclutan la ubiquitina E3 ligasa Stub1 para permitir su inmersión en autofagosomas para su eliminación individual por pexofagia16. Se puede usar este protocolo para comparar el destino de los peroxisomas lesionados y sanos dentro de la misma célula mediante microscopía de lapso de tiempo. El método también se puede utilizar para dañar globalmente todos los peroxisomas (en todas las células) en una placa de cultivo, lo que permite el análisis bioquímico de la vía de 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

Las células de mamíferos pueden voltear los peroxisomas a través de la pexofagia mediada por Stub1. La vía potencialmente permite el control celular de la cantidad y calidad de los peroxisomas. Durante este proceso, la proteína de choque térmico 70 y la ubiquitina E3 ligasa, Stub1, se translocan en los peroxisomas para ser volteados para iniciar la pexofagia. La actividad de la ligasa Stub1 permite la acumulación de ubiquitina y otros módulos relacionados con la autofagia en peroxisomas específicos. La elevación de los niveles de especies reactivas de oxígeno (ROS) dentro de la luz peroxisomal puede activar la pexofagia mediada por Stub1. Por lo tanto, se puede utilizar la generación de ROS asistida por colorante para desencadenar y monitorear esta vía. Este artículo describe los procedimientos para usar dos clases de colorantes, proteínas fluorescentes y fluoróforos sintéticos, para iniciar la pexofagia dentro de cultivos celulares de mamíferos. Estos protocolos basados en la generación de ROS asistidos por colorante no solo se pueden usar para atacar todos los peroxisomas dentro de una población celular a nivel mundial, sino que también pueden permitir la manipulación de peroxisomas individuales dentro de células individuales. También describimos cómo se puede seguir la pexofagia mediada por Stub1 utilizando microscopía de células vivas.

El esquema de inducción de pexofagia mediado por Stub1 que se muestra aquí aprovecha la generación de ROS asistida por colorante dentro de la luz del peroxisoma. Esta operación requiere intensidades de luz mínimas. Por lo tanto, los peroxisomas que contienen proteínas o colorantes fluorescentes pueden iluminarse utilizando microscopios confocales estándar de barrido láser. La iluminación focal conduce a la producción instantánea y localizada de ROS dentro de los peroxisomas individuales, como lo indica el repo...

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

Monitoring Stub1-Mediated Pexophagy

Este protocolo proporciona instrucciones para desencadenar y monitorear la pexofagia mediada por Stub1 en células vivas.

Monitorización de la pexofagia mediada por Stub1

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

Los peroxisomas llevan a cabo la oxidación beta y son propensos al daño de ROS. Este método permite investigar cómo las células lidian con los peroxisomas estresados por ROS. Este protocolo no solo se utiliza para atacar globalmente todos los peroxisomas dentro de una población celular, sino que también puede permitir la manipulación de los peroxisomas individuales dentro de células individuales.La generación de ROS asistida por colorante también se puede usar para atacar orgánulos fuera de los peroxisomas para investigar cómo las células lidian con las lesiones de orgánulos. Para comenzar, siembre dos veces 10 a la quinta células SHSY5Y humanas o seis veces 10 a la cuarta células NIH H3T3 de ratón en 840 microlitros de medio de cultivo en placas de cultivo de 35 milímetros con fondo de vidrio con un micropocillo de vidrio de 20 milímetros de diámetro. Cultive las células por debajo del 5% de dióxido de carbono a 37 grados centígrados durante 24 horas.A continuación, transfecte las células con los plásmidos deseados utilizando un reactivo de transfección o transfección de células SHSY5Y. Diluir plásmidos en 55 microlitros de DMEM/F-12 sin suero y diluir un microlitro de reactivo de transfección en 55 microlitros de DMEM/F-12 sin suero. Combine el ADN diluido con el reactivo diluido.Mezclar suavemente e incubar durante 30 minutos a temperatura ambiente. Agregue el complejo al micropocillo de 20 milímetros previamente chapado con 100 microlitros de medio de cultivo que contiene células SHSY5Y sin antibióticos. Agite suavemente el plato hacia adelante y hacia atrás.Realice la transfección de células NIH 3T3 como se demostró antes, pero sustituya el medio sérico reducido en lugar de DMEM / F-12 sin suero por dilución de plásmidos y reactivo de transfección. Después de dos horas de transfección, retire el medio que contiene reactivo de transfección y agregue un mililitro de medio de cultivo fresco. Incubar las células NIH 3T3 o SHSY5Y a 37 grados centígrados y 5% de dióxido de carbono durante 24 horas.Para la producción de ROS activadas por luz, a las 24 horas posteriores a la transfección, retire el medio de cultivo y agregue 10 microlitros de 200 nanomolares del ligando HaloTag TMR o 200 nanomolares Janelia Fluor 646 HaloTag en el micropocillo de vidrio de 20 milímetros. Transfiera el plato a una incubadora de 37 grados centígrados y 5% de dióxido de carbono y déjelo durante una hora para teñir las células. Después de una hora, retire bien el medio de tinción y agregue un mililitro de medio de cultivo fresco.Coloque un plato con fondo de vidrio que contenga las células deseadas en un microscopio confocal de escaneo láser equipado con una incubadora de etapa. En el software, haga clic en Ventana de herramientas, elija Ocular y haga clic en el botón DIA para encender la luz transmitida. Vea el plato a través del ocular del microscopio y enfoque las células girando la perilla de enfoque.Elija LSM y haga clic en el botón Seleccionar detector de tinte. Haga doble clic en los tintes deseados en la ventana emergente para seleccionar la configuración adecuada de láser y filtro para obtener imágenes de las células. Haga clic en Live4x en el panel Live para iniciar un escaneo láser rápido para comenzar a detectar las señales fluorescentes de las células.Mueva el escenario con el controlador del joystick y localice las células diKillerRed VKSKL o SLP-VKSKL medianas para aplicar tensión ROS en los peroxisomas. Adquirir una imagen de la célula deseada. Haga clic en Ventana de herramientas y elija LSM Stimulation.Luego haga clic en el botón de elipse y en la ventana Live Image use el mouse para dibujar un ROI de 15 micrómetros de diámetro que contenga los peroxisomas deseados a los que se aplicará la tensión ROS. Para aplicar estrés ROS, use 561 nanómetros para células con el ligando SLP marcado con diKillerRed VKSKL o TMR y use 640 nanómetros para células teñidas con el ligando SLP marcado con Janelia Fluor 646. Seleccione la longitud de onda del láser para aplicar la tensión ROS.Introduzca el porcentaje de láser deseado y la duración. Para la producción de ROS en los peroxisomas, haga clic en Adquirir y luego en el botón Estimulación para iniciar el escaneo con 561 o 640 nanómetros de luz a través de los ROI durante 30 segundos cada uno. Esta estimulación corresponde a aproximadamente 5% de ajustes de potencia láser para 561 y 640 nanómetros.Después de 30 segundos de iluminación láser, los peroxisomas dentro de los ROI pierden sus señales diKillerRed VKSKL, TMR o Janelia Fluor 646. Esta pérdida de señal permite distinguir los peroxisomas iluminados y no iluminados dentro de la célula. Para monitorear las células vivas pexófagas, siga la translocación de Stub1, Hsp70, ubiquitina, p62 o LC3B marcados con EGFP en los peroxisomas iluminados o estresados por ROS mediante imágenes de lapso de tiempo utilizando intervalos de 10 minutos entre cuadros.La iluminación focal conduce a la producción instantánea y localizada de ROS dentro de los peroxisomas individuales, como lo indica el reportero fluorescente roGFP2-VKSKL. La imagen de diKillerRed VKSKL se tomó después de que se realizaron todas las mediciones de roGFP2-VKSKL para indicar la ubicación de los peroxisomas dañados. Los datos también muestran que los peroxisomas no iluminados no se ven afectados, lo que indica la precisión de la metodología.Esta metodología se utilizó para monitorizar la pexofagia mediada por Stub1. Durante este proceso, Hsp70, Stub1, proteínas ubiquitinadas, adaptadores de autofagia p62 y LC3B aparecen posteriormente en peroxisomas estresados por ROS para impulsar la pexofagia. Usando la misma estrategia, fue posible dañar simultáneamente todos los peroxisomas en una placa de cultivo para la caracterización bioquímica de la pexofagia mediada por Stub1.Antes de la iluminación LED, la fluorescencia de EGFP-ubiquitina era homogénea en el citoplasma y no colocalizaba con los peroxisomas. Después de nueve horas de iluminación LED, las señales de EGFP-ubiquitina se acumularon en todos los peroxisomas en las células cultivadas en una placa confocal. Usando este protocolo, encontramos que los peroxisomas estresados por ROS se eliminan a través de una vía de degradación dependiente de ubiquitina.Los peroxisomas estresados por ROS reclutan la ubiquitina E3 ligasa Stub1 para permitir su eliminación por pexofagia.

Research

JoVE Journal

Biology

Monitoring Actin Disassembly with Time-lapse Microscopy

Monitoreo de actina desmontaje con la Microscopía de Time-lapse

Investigación

Biología

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.

Imágenes funcionales con Refuerzo, Eyetracking y Monitoreo 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 de una sola molécula para vigilar los cambios en las propiedades elásticas bicapa

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.

Monitoreo hormonas vegetales en las respuestas de estrés

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.

Efectos de la acupuntura en vigilancia por fMRI del cerebro humano

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.

Monitorización de la función del corazón en las larvas Drosophila melanogaster Para los estudios 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.

Bioensayos para vigilar la resistencia insecticida

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 largo plazo Prueba de toxicidad letal con el 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.

Los ensayos acoplados para Monitoreo replegamiento de proteínas en<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.

Reacción seleccionada Monitoreo espectrometría de masas para Absolute Proteína cuantificación

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