Name: Autor destacado: Evaluación del flujo de mitofagia en las células β pancreáticas utilizando enfoques complementarios efectivos y robustos
Uploaded: 2023-09-15T14:40:00
Description: Watch this Scientific Journal Video about Assessment of Mitophagy Flux in Pancreatic β-Cells Using Effective and Robust Complementary Approaches at JoVE.com

E.L-D. agradece el apoyo de los NIH (T32-AI007413 y T32-AG000114). SAS agradece el apoyo de la JDRF (COE-2019-861), los NIH (R01 DK135268, R01 DK108921, R01 DK135032, R01 DK136547, U01 DK127747), el Departamento de Asuntos de Veteranos (I01 BX004444), la familia Brehm y la familia Anthony.

Los estudios en animales presentados en este protocolo fueron revisados y aprobados por el Comité Institucional de Cuidado y Uso de Animales de la Universidad de Michigan. Para este estudio se utilizaron ratones machos C57BL/6J de veinte semanas de edad, ya sea con una dieta regular de grasas (RFD) de 15 semanas o una dieta alta en grasas (HFD).
1. Evaluación de la mitofagia a través del enfoque MtPhagy basado en colorantes (Método 1)
<ol><li>Preparación y tratamiento d...

Análisis cuantitativo de la mitofagia en células β pancreáticas murinas

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diabetes+susceptibility+gene+Clec16a+regulates+mitophagy.">The diabetes susceptibility gene Clec16a regulates mitophagy.</a> Cell. 157 (7), 1577-1590 (2014).</li><li>Pearson, 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=Clec16a,+Nrdp1,+and+USP8+form+a+ubiquitin-dependent+tripartite+complex+that+regulates+&#946;-cell+mitophagy.">Clec16a, Nrdp1, and USP8 form a ubiquitin-dependent tripartite complex that regulates &#946;-cell mitophagy.</a> Diabetes. 67 (2), 265-277 (2018).</li><li>Hattori, N., Saiki, S., Imai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+by+mitophagy.">Regulation by mitophagy.</a> International Journal of Biochemistry and Cell Biology. 53, 147-150 (2014).</li><li>Berezhnov, A. 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=Intracellular+pH+modulates+autophagy+and+mitophagy.">Intracellular pH modulates autophagy and mitophagy.</a> Journal of Biological Chemistry. 291 (16), 8701-8708 (2016).</li><li>Sun, N., 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=Measuring+in+vivo+mitophagy.">Measuring in vivo mitophagy.</a> Molecular Cell. 60 (4), 685-696 (2015).</li><li>McWilliams, T. 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=mito-QC+illuminates+mitophagy+and+mitochondrial+architecture+in+vivo.">mito-QC illuminates mitophagy and mitochondrial architecture in vivo.</a> Journal of Cell Biology. 214 (3), 333-345 (2016).</li><li>Aoyagi, K., 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+new+beta+cell-specific+mitophagy+reporter+mouse+shows+that+metabolic+stress+leads+to+accumulation+of+dysfunctional+mitochondria+despite+increased+mitophagy.">A new beta cell-specific mitophagy reporter mouse shows that metabolic stress leads to accumulation of dysfunctional mitochondria despite increased mitophagy.</a> Diabetologia. 66 (1), 147-162 (2023).</li><li>Ma, X., Ding, W. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescence+imaging+based-assay+to+monitor+mitophagy+in+cultured+hepatocytes+and+mouse+liver.">A fluorescence imaging based-assay to monitor mitophagy in cultured hepatocytes and mouse liver.</a> Liver Research. 5 (1), 16-20 (2021).</li><li>Sidarala, 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=Mitophagy+protects+&#946;+cells+from+inflammatory+damage+in+diabetes.">Mitophagy protects &#946; cells from inflammatory damage in diabetes.</a> JCI Insight. 5 (24), 141138 (2020).</li><li>Gingerich, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intrinsically+disordered+protein+region+encoded+by+the+human+disease+gene+CLEC16A+regulates+mitophagy.">An intrinsically disordered protein region encoded by the human disease gene CLEC16A regulates mitophagy.</a> Autophagy. 19 (2), 525-543 (2023).</li><li>Sun, N., Malide, D., Liu, J., Rovira, I. I., Combs, C. A., Finkel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescence-based+imaging+method+to+measure+in+vitro+and+in+vivo+mitophagy+using+mt-Keima.">A fluorescence-based imaging method to measure in vitro and in vivo mitophagy using mt-Keima.</a> Nature Protocols. 12 (8), 1576-1587 (2017).</li><li>Zhao, J., Bertoglio, B. A., Gee, K. R., Kay, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zinc+indicator+FluoZin-3+is+not+perturbed+significantly+by+physiological+levels+of+calcium+or+magnesium.">The zinc indicator FluoZin-3 is not perturbed significantly by physiological levels of calcium or magnesium.</a> Cell Calcium. 44 (4), 422-426 (2008).</li><li>Da Silva Xavier, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cells+of+the+Islets+of+Langerhans.">The Cells of the Islets of Langerhans.</a> Journal of Clinical Medicine. 7 (3), 54 (2018).</li><li>Thapa, 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=Imaging+&#946;-cell+function+using+a+zinc-responsive+MRI+contrast+agent+may+identify+first+responder+islets.">Imaging &#946;-cell function using a zinc-responsive MRI contrast agent may identify first responder islets.</a> Frontiers in Endocrinology. 12, 809867 (2022).</li><li>Iwashita, H., 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=Live+cell+imaging+of+mitochondrial+autophagy+with+a+novel+fluorescent+small+molecule.">Live cell imaging of mitochondrial autophagy with a novel fluorescent small molecule.</a> ACS Chemical Biology. 12 (10), 2546-2551 (2017).</li><li>Crowley, L. C., Christensen, M. E., Waterhouse, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mitochondrial+transmembrane+potential+by+TMRE+staining.">Measuring mitochondrial transmembrane potential by TMRE staining.</a> Cold Spring Harbor Protocols. 2016 (12), 087361 (2016).</li><li>Klein, B., W&#246;rndl, K., L&#252;tz-Meindl, U., Kerschbaum, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perturbation+of+intracellular+K(+)+homeostasis+with+valinomycin+promotes+cell+death+by+mitochondrial+swelling+and+autophagic+processes.">Perturbation of intracellular K(+) homeostasis with valinomycin promotes cell death by mitochondrial swelling and autophagic processes.</a> Apoptosis. 16 (11), 1101-1117 (2011).</li><li>Ji, 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=Toll-like+receptors+TLR2+and+TLR4+block+the+replication+of+pancreatic+&#946;+cells+in+diet-induced+obesity.">Toll-like receptors TLR2 and TLR4 block the replication of pancreatic &#946; cells in diet-induced obesity.</a> Nature Immunology. 20 (6), 677-686 (2019).</li><li>Sauvat, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+cellular+viability+by+automated+microscopy+and+flow+cytometry.">Quantification of cellular viability by automated microscopy and flow cytometry.</a> Oncotarget. 6 (11), 9467-9475 (2015).</li><li>Liu, Y. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mt-Keima+detects+PINK1-PRKN+mitophagy+in+vivo+with+greater+sensitivity+than+mito-QC.">Mt-Keima detects PINK1-PRKN mitophagy in vivo with greater sensitivity than mito-QC.</a> Autophagy. 17 (11), 3753-3762 (2021).</li><li>Mauro-Lizcano, 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=New+method+to+assess+mitophagy+flux+by+flow+cytometry.">New method to assess mitophagy flux by flow cytometry.</a> Autophagy. 11 (5), 833-843 (2015).</li></ol>

Este protocolo describe dos métodos complementarios para cuantificar el flujo mitofagia en islotes primarios disociados de ratón. Utilizando el método mt-Keima, un aumento en la mitofagia se cuantificó como un aumento de la proporción de células ácidas (561 nm)/neutras (405 nm), mientras que en el método MtPhagy, el aumento del flujo de mitofagia se cuantificó como un aumento en la población de célulasbajas de FluozinaltaMtPhagyaltaTMRE. Estos métodos permiten realizar evaluac...

Las células β pancreáticas producen y secretan insulina para satisfacer las demandas metabólicas, y la disfunción de las células β es responsable de la hiperglucemia y la aparición de diabetes tanto en la diabetes tipo 1 como en la diabetes tipo 2. Las células β acoplan el metabolismo de la glucosa con la secreción de insulina a través de la energía mitocondrial y la producción metabólica, que dependen de una reserva de masa mitocondrial funcional 1,2,3. Para mantener una función óptima de las células β, las células β dependen de los mecanismos de control de calidad mitocondrial para eliminar las mitocondrias envejecidas o dañadas y preservarla masa mitocondrial funcional. La autofagia mitocondrial selectiva, también conocida como mitofagia, es un componente clave de la vía de control de calidad mitocondrial.
Las evaluaciones de la mitofagia en células vivas a menudo se basan en los cambios en el pH mitocondrial que ocurren durante la mitofagia. Las mitocondrias tienen un pH ligeramente alcalino, y las mitocondrias sanas normalmente residen en el citosol de pH neutro. Durante la mitofagia, las mitocondrias dañadas o disfuncionales se incorporan selectivamente a los autofagosomas y, finalmente, se eliminan dentro de los lisosomas ácidos5. Varios modelos de ratones reporteros de mitofagia transgénica in vivo, como mt-Keima6, mitoQC7 y CMMR8, así como sondas de mitofagia transfectables, como el plásmido Cox8-EGFP-mCherry9, utilizan este cambio de pH para proporcionar evaluaciones cuantitativas de la mitofagia. El uso de ratones transgénicos que expresan la sonda radiométrica de doble excitación sensible al pH mt-Keima se ha optimizado para las evaluaciones de mitofagia en islotes y células β mediante citometría de flujo10,11. La relación entre las emisiones de longitud de onda mt-Keima ácidas y neutras (la relación entre la excitación ácida de 561 nm y la neutra de 480 nm) puede utilizarse para cuantificar de forma robusta la mitofagia 6,12.
Este protocolo describe un enfoque optimizado para evaluar el flujo mitofagia en islotes primarios y células β aisladas de ratones transgénicos mt-Keima10,11. Si bien mt-Keima es una sonda altamente sensible, requiere complicados esquemas de cría de animales o la transfección de células, lo que a menudo puede ser un desafío cuando se trabaja en combinación con otros modelos genéticos o con islotes humanos primarios. Además, el uso de múltiples láseres y detectores de fluorescencia para identificar poblaciones celulares neutras y ácidas puede limitar el uso combinatorio de otros reporteros fluorescentes.
Para superar estos desafíos, este protocolo también describe un método complementario, basado en colorantes de un solo canal fluorescente, para la detección robusta de mitofagia en células β de islotes de ratón aislados. Este enfoque, conocido como el método MtPhagy, utiliza una combinación de tres colorantes permeables a las células para seleccionar las células β, cuantificar las poblaciones celulares que se someten activamente a la mitofagia y evaluar el potencial de membrana mitocondrial (MMP o Δψm) simultáneamente.
El primero de estos colorantes es Fluozin-3-AM, un indicador de Zn2+ permeable a las células con un Ex/Em 494/516 nm13. Los islotes de ratón comprenden una población heterogénea de células funcionalmente distintas, incluidas las células α, β, δ y PP. Las células β comprenden aproximadamente el 80% de las células dentro del islote de ratón y se pueden distinguir de otros tipos de células de islotes debido a su alta concentración de Zn2+ dentro de los gránulos de insulina14,15, lo que permite la identificación de las células β como laalta población de Fluozin-3-AM. El colorante MtPhagy, un colorante sensible al pH que se inmoviliza en las mitocondrias a través de un enlace químico y emite fluorescencia débil, también se utiliza en este protocolo16. Tras la inducción de la mitofagia, las mitocondrias dañadas se incorporan al lisosoma ácido, y el colorante MtPhagy aumenta su intensidad de fluorescencia en el entorno de pH bajo (Ex/Em 561/570-700 nm).
Además, la tetrametilrodamina, éster etílico (TMRE), se utiliza para evaluar la MMP. TMRE es un colorante permeable a las células con carga positiva (Ex/Em 552/575 nm) que es secuestrado por las mitocondrias sanas debido a la carga negativa relativa sostenida por su potencial de membrana17. Las mitocondrias dañadas o poco saludables disipan su potencial de membrana, lo que resulta en una disminución de la capacidad para secuestrar TMRE. Utilizando estos colorantes juntos, las células de β que se someten a mitofagia se pueden identificar como la poblaciónaltade Fluozin MtPhagyaltaTMREbaja a través de la citometría de flujo. Dado que la mitofagia es un proceso dinámico y no estático, este protocolo se optimizó para evaluar el flujo de mitofagia utilizando valinomicina, un ionóforo de K+ que induce la mitofagia después de la disipación de MMP18. La comparación de la mitofagia en presencia y ausencia de valinomicina permite evaluar el flujo de mitofagia en diferentes grupos de muestras.
La naturaleza basada en colorantes del enfoque actual permite extrapolarlo a islotes humanos y otros tipos de células difíciles de transfectar y evita la necesidad de complicados esquemas de cría de animales, a diferencia del protocolo mt-Keima. El objetivo general de este protocolo es cuantificar la mitofagia en células β a nivel de una sola célula a través de dos métodos independientes basados en citometría de flujo. En conjunto, este protocolo describe dos métodos potentes y complementarios que permiten tanto precisión como flexibilidad en el estudio cuantitativo del control de calidad mitocondrial.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Attune NxT Flow Cytometer</td>
			<td>Thermofisher Scientific</td>
			<td>A24858</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Thermofisher Scientific</td>
			<td>D1306</td>
			<td>DAPI reconstituted in ddH2O to reach 0.2 &#181;g/mL stock</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>317275</td>
			<td></td>
		</tr>
		<tr>
			<td>Fatty Acid Free heat shock BSA powder</td>
			<td>Equitech</td>
			<td>BAH66</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gemini Bio</td>
			<td>900-108</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluozin-3AM&#160;</td>
			<td>Thermofisher Scientific&#160;</td>
			<td>F24195</td>
			<td>100 &#956;g Fluozin-3AM powder reconstituted in 51 &#956;L DMSO and 51 &#956;L Pluronic F-127 to reach 1 mM stock.&#160;</td>
		</tr>
		<tr>
			<td>Gibco&#160;RPMI 1640 Medium</td>
			<td>Fisher Scientific</td>
			<td>11-875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Life Technologies</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>MtPhagy dye</td>
			<td>Dojindo</td>
			<td>MT02-10</td>
			<td>5 &#956;g MtPhagy powder reconstituted with 50 &#956;L DMSO to reach 100 &#956;M stock.&#160;</td>
		</tr>
		<tr>
			<td>MtPhagy dye</td>
			<td>Dojindo</td>
			<td>MT02-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100x)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>1x Solution used in procotol by diluting 1:10 in ddH2O</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, 10x</td>
			<td>Fisher Scientific</td>
			<td>BP399-20</td>
			<td>1x Solution used in procotol by diluting 1:10 in ddH2O</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100x)</td>
			<td>Life Technologies</td>
			<td>11360-070</td>
			<td>5 &#956;g MtPhagy powder reconstituted with 50 &#956;L DMSO to reach 100 &#956;M stock.&#160;</td>
		</tr>
		<tr>
			<td>TMRE [Tetramethylrhodamine, ethyl ester, perchlorate]</td>
			<td>Anaspec</td>
			<td>AS-88061</td>
			<td>TMRE powder reconstituted in DMSO to reach 100 &#956;M stock.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Thermofisher Scientific</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Valinomycin</td>
			<td>Sigma</td>
			<td>V0627</td>
			<td>Valinomycin powder reconsituted in DMSO to reach 250 nM stock.</td>
		</tr>
	</tbody>
</table>

complementary approaches to interrogate mitophagy flux in pancreatic &#946;-cells

La mitofagia es un mecanismo de control de calidad necesario para mantener una función mitocondrial óptima. La mitofagia disfuncional de las células β da como resultado una liberación insuficiente de insulina. Las evaluaciones cuantitativas avanzadas de la mitofagia a menudo requieren el uso de indicadores genéticos. El modelo de ratón mt-Keima, que expresa una sonda radiométrica de doble excitación sensible al pH dirigida a las mitocondrias para cuantificar la mitofagia mediante citometría de flujo, se ha optimizado en células β. La relación entre las emisiones de longitud de onda de mt-Keima ácidas y neutras se puede utilizar para cuantificar de forma robusta la mitofagia. Sin embargo, el uso de reporteros genéticos de mitofagia puede ser un desafío cuando se trabaja con modelos genéticos complejos de ratones o células difíciles de transfectar, como los islotes humanos primarios. Este protocolo describe un nuevo método complementario basado en colorantes para cuantificar la mitofagia de células β en islotes primarios mediante MtPhagy. MtPhagy es un colorante sensible al pH y permeable a las células que se acumula en las mitocondrias y aumenta su intensidad de fluorescencia cuando las mitocondrias se encuentran en entornos de pH bajo, como los lisosomas durante la mitofagia. Al combinar el colorante MtPhagy con Fluozin-3-AM, un indicador de Zn2+ que selecciona para las células β, y tetrametilrodamina, éster etílico (TMRE) para evaluar el potencial de la membrana mitocondrial, el flujo de mitofagia se puede cuantificar específicamente en las células β mediante citometría de flujo. Estos dos enfoques son altamente complementarios, lo que permite flexibilidad y precisión en la evaluación del control de calidad mitocondrial en numerosos modelos de células β.

Pancreatic &#946;-cells produce and secrete insulin to meet metabolic demands, and &#946;-cell dysfunction is responsible for hyperglycemia and diabetes onset in both type 1 and type 2 diabetes. &#946;-Cells couple glucose metabolism with insulin secretion via mitochondrial energetics and metabolic output, which depend on a reserve of functional mitochondrial mass1,2,3. To maintain optimal &#946;-cell function, &#946;-cells rely on mitochondrial quality control mechanisms to remove aged or damaged mitochondria and preserve functional mitochondrial mass4. Selective mitochondrial autophagy, also known as mitophagy, is a key component of the mitochondrial quality control pathway.
Assessments of mitophagy in live cells often rely on changes in mitochondrial pH that occur during mitophagy. Mitochondria have a slightly alkaline pH, and healthy mitochondria normally reside in the pH-neutral cytosol. During mitophagy, damaged or dysfunctional mitochondria are selectively incorporated into autophagosomes and eventually cleared within acidic lysosomes5. Several in vivo transgenic mitophagy reporter mouse models, such as mt-Keima6, mitoQC7, and CMMR8, as well as transfectable mitophagy probes, such as the Cox8-EGFP-mCherry plasmid9, utilize this pH change to provide quantitative assessments of mitophagy. Use of transgenic mice expressing the mt-Keima pH-sensitive dual-excitation ratiometric probe has been optimized for mitophagy assessments in islets and &#946;-cells via flow cytometry10,11. The ratio of acidic-to-neutral mt-Keima wavelength emissions (the ratio of acidic 561 nm to neutral 480 nm excitation) can be used to robustly quantify mitophagy6,12.
This protocol describes an optimized approach to assess mitophagy flux in primary islets and &#946;-cells isolated from mt-Keima transgenic mice10,11. While mt-Keima is a highly sensitive probe, it requires either complicated animal breeding schemes or the transfection of cells, which can often be challenging when working in combination with other genetic models or with primary human islets. Additionally, the use of multiple fluorescence lasers and detectors to identify neutral and acidic cell populations can limit the combinatorial use of other fluorescent reporters.
To overcome these challenges, this protocol also describes a complementary, single fluorescent channel, dye-based method for robust detection of mitophagy in &#946;-cells from isolated mouse islets. This approach, referred to as the MtPhagy method, utilizes a combination of three cell-permeable dyes to select for &#946;-cells, quantify the cell populations actively undergoing mitophagy, and assess mitochondrial membrane potential (MMP or &#916;&#968;m) simultaneously.
The first of these dyes is Fluozin-3-AM, a cell-permeable Zn2+ indicator with an Ex/Em 494/516 nm13. Mouse islets comprise a heterogeneous population of functionally distinct cells, including &#945;-, &#946;-, &#948;-, and PP cells. &#946;-Cells comprise approximately 80% of cells within the mouse islet and can be distinguished from other islet cell types due to their high Zn2+ concentration within insulin granules14,15, allowing for identification of &#946;-cells as the Fluozin-3-AMhigh population. The MtPhagy dye, a pH-sensitive dye that is immobilized on mitochondria via a chemical bond and emits weak fluorescence, is also utilized in this protocol16. Upon mitophagy induction, damaged mitochondria are incorporated into the acidic lysosome, and the MtPhagy dye increases its fluorescence intensity within the low pH environment (Ex/Em 561/570-700 nm).
Additionally, Tetramethylrhodamine, ethyl ester (TMRE), is used to assess MMP. TMRE is a cell-permeable positively charged dye (Ex/Em 552/575 nm) that is sequestered by healthy mitochondria due to the relative negative charge upheld by their membrane potential17. Damaged or unhealthy mitochondria dissipate their membrane potential, resulting in decreased ability to sequester TMRE. Utilizing these dyes together, &#946;-cells undergoing mitophagy can be identified as the FluozinhighMtPhagyhighTMRElow population via flow cytometry. Since mitophagy is a dynamic rather than static process, this protocol was optimized to assess mitophagy flux using valinomycin, a K+-ionophore that induces mitophagy following dissipation of MMP18. Comparison of mitophagy in the presence and absence of valinomycin allows for assessment of mitophagy flux in different sample groups.
The dye-based nature of the current approach allows it to be extrapolated to human islets and other difficult-to-transfect cell types and circumvents the need for complicated animal breeding schemes, unlike the mt-Keima protocol. The overarching goal of this protocol is to quantify mitophagy in &#946;-cells at the single-cell level via two independent flow cytometry-based methods. Taken together, this protocol describes two powerful and complementary methods that allow for both precision and flexibility in the quantitative study of mitochondrial quality control.

Autor destacado: Evaluación del flujo de mitofagia en las células β pancreáticas utilizando enfoques complementarios efectivos y robustos 

Enfoques complementarios para interrogar el flujo de mitofagia en las células β pancreáticas

Our protocol describes two complementary methods to study beta-cell mitophagy flux at the single-cell resolution via flow cytometry. The mt-Keima protocol utilizes a genetic mitophagy reporter, whereas the MtPhagy protocol combines three fluorescent dyes, which makes it easy to extrapolate to difficult-to-transfect cells in human samples. Assessments of mitophagy using traditional biochemical approaches and imaging approaches are both time consuming and challenging.Thus, it's important to develop new, robust, and effective methods to study mitophagy flux in beta-cells. Both the mt-Keima and MtPhagy method are quantitative assessments that are efficient for studying mitophagy flux in pancreatic beta cells. So, the Soleimanpour lab focuses on the different mechanisms that drive pancreatic beta-cell failure and diabetes.And we specifically focus on the different aspects of mitochondrial lifecycle, including mitophagy, to understand how defects in mitochondrial function drive the development of diabetes.

Evaluación de la mitofagia a través del enfoque MtPhagy basado en colorantes Este enfoque basado en colorantes se optimizó para analizar el flujo de mitofagia dentro de las células primarias de ratón β sin la necesidad de un reportero genético, utilizando Fluozin-3-AM, TMRE y MtPhagy, así como DAPI para excluir las células muertas. Al combinar estos colorantes con valinomicina para inducir la mitofagia, este protocolo describe un método basado en colorantes para medir selectivamente el flujo...

Watch this Scientific Journal Video about Assessment of Mitophagy Flux in Pancreatic β-Cells Using Effective and Robust Complementary Approaches at JoVE.com

Este protocolo describe dos métodos para el análisis cuantitativo de la mitofagia en las células β pancreáticas: en primer lugar, una combinación de colorantes específicos de mitocondrias permeables a las células y, en segundo lugar, un indicador de mitofagia codificado genéticamente. Estas dos técnicas son complementarias y pueden implementarse en función de necesidades específicas, lo que permite flexibilidad y precisión para abordar cuantitativamente el control de calidad mitocondrial.

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient ...

Nuestro protocolo describe dos métodos complementarios para estudiar el flujo de mitofagia de células beta a la resolución de una sola célula mediante citometría de flujo. El protocolo mt-Keima utiliza un indicador genético de mitofagia, mientras que el protocolo MtPhagy combina tres colorantes fluorescentes, lo que facilita la extrapolación a células difíciles de transfectar en muestras humanas. Las evaluaciones de la mitofagia mediante enfoques bioquímicos tradicionales y enfoques de imagen requieren mucho tiempo y son un desafío.Por lo tanto, es importante desarrollar métodos nuevos, robustos y efectivos para estudiar el flujo de mitofagia en las células beta. Tanto el método mt-Keima como el método MtPhagy son evaluaciones cuantitativas que son eficientes para estudiar el flujo de mitofagia en las células beta pancreáticas. Por lo tanto, el laboratorio de Soleimanpour se centra en los diferentes mecanismos que impulsan la insuficiencia de las células beta pancreáticas y la diabetes.Y nos enfocamos específicamente en los diferentes aspectos del ciclo de vida mitocondrial, incluida la mitofagia, para comprender cómo los defectos en la función mitocondrial impulsan el desarrollo de la diabetes.

complementary-approaches-to-interrogate-mitophagy-flux-pancreatic

Research

JoVE Journal

Biology

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in &#946;-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify &#946;-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for &#946;-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in &#946;-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous &#946;-cell models.

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic &#946;-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

Investigación

Biología

Single-Cell Dissociation of Murine Islets for Flow Cytometric Analysis of &#946;-Cell Mitophagy

Begin by culturing the eyelet samples isolated from regular fat diet or high fat diet mice overnight at 37 degrees Celsius. Design the desired experimental conditions for the study. For each experimental condition, using a pipette, pick 100 eyelets into a six centimeter Petri dish containing two milliliters of eyelet medium.To induce Mitophagy, treat the eyelets in the appropriate Petri dishes with two microliters of 250 nano molar Valinomycin stock for three hours. Isolate the eyelets and using a pipette, transfer the eyelets from each Petri dish to a separate micro centrifuge tube. Then, spin the eyelets at 350 G for one minute at 10 degrees Celsius and using a pipette, discard the supernatant.Wash the obtained sample twice with one milliliter of PBS. Between the washes, spin the sample and discard the supernatant as demonstrated previously. To dissociate eyelets into single cells, add 500 microliters of 0.05%Trypsin prewarmed to 37 degrees celsius, to one sample tube.Pipette the contents of the tube up and down repeatedly until the eyelets are visibly dispersed. Immediately add one milliliter of prewarmed eyelet medium to neutralize trypsin. Spin the samples at 500 G for five minutes at 10 degrees Celsius.Remove the supernatant carefully without disturbing the pellet. Then, wash the samples two times with eyelet flow medium. The cell samples are now ready to be stained for flow cytometric analysis of beta cell mitophagy.

Disociación unicelular de islotes murinos para el análisis citométrico de flujo de la mitofagia de β células

Flow Cytometric Analysis of Mitophagy in Murine Pancreatic &#946;-Cells Using MtPhagy Dye

Proceed to perform flow cytometric analysis of beta cell mitophagy, with a single cell suspension of about 100 isolated mirroring islets prepared, corresponding to each desired experimental condition to be studied. First, resuspend the islet samples for each condition in 500 microliters of islet flow medium. Add 0.25 microliters of MtPhagy stock to tubes designated to receive the MtPhagy dye.Similarly, add 0.25 microliters of TMRE stock and fluozin-3-AM stock to the respective designated tubes. Wrap the tubes in aluminum foil and vortex each tube at low speed for five seconds to mix the contents. Then incubate the tubes at 37 degrees Celsius for 30 minutes.Halfway through incubation, vortex each sample at low speed for five to 10 seconds. After incubation, centrifuge the tubes at 350 G for one minute at 10 degrees Celsius. Discard the supernatant.And resuspend the samples in 500 microliters of islet flow medium. Add DAPI to the microcentrifuge tubes containing samples that require DAPI treatment. Centrifuge again and discard the supernatant as demonstrated previously.Resuspend the residue in 500 microliters of islet flow medium. Finally, place the samples on ice and then proceed with flow cytometry. Adjust the voltages for forward scatter or FSC and side scatter or SSC to ensure cell populations are at the center of the scatter plot.To exclude multiplets, at a rectangular gate on FSC height versus FSC width, followed by SSC height versus SSC width. Adjust voltages and compensation for DAPI to filter for live beta cells. Set fluorescence gates for each fluorophore used based on the unstained RFD sample.Once gates are established, collect 10, 000 events per sample. Beta cells with high utilization of mitophagy were defined as the fluozin high MtPhagy high TMRE low population in quadrant three or Q3.Basal and valinomycin-induced mitophagy levels were characterized in both RFD and HFD islets.

Análisis citométrico de flujo de la mitofagia en células β pancreáticas murinas utilizando el colorante MtPhagy

Flow Cytometric Analysis of Mitophagy in Murine Pancreatic &#946;-Cells Using the Genetically Encoded mt-Keima Reporter

To assess mitophagy in murine pancreatic beta cells using the genetically encoded mitophagy reporter, culture pancreatic islets isolated from wild type and Mt-Keima mice overnight at 37 degrees Celsius. The following day, add 100 islets into a six centimeter Petri dish containing two milliliters of islet medium for each experimental condition used in this protocol. Next, to dissociate the eyelets into single cells and stain with FluoZin-3-AM and DAPI as demonstrated previously for the Mt-Phagy dye based approach.Then proceed with the flow cytometric analysis of the samples. Adjust the forward scatter or FSC and side scatter or SSC voltages to attain an even distribution of cells on an SSCA versus FSCA plot. To select single cells and exclude multiplets, add a rectangular gate on FSC height versus FSC width, followed by SSC height versus SSE width.The multiplets are excluded due to their higher width signal values. Then set up a DAPI negative gate to exclude dead cells. After that, set up FluoZin-3-AM positive gates to filter for live beta cells.Set up a triangle gating scheme using the Mt-Keima positive sample to identify acidic and neutral cell populations. Once the primary and fluorescence gates were established, assess mitophagy flux using basal and valinomycin-induced changes in Mt-Keima fluorescence.