Name: Auteur in de schijnwerpers: beoordeling van mitofagieflux in pancreas-β-cellen met behulp van effectieve en robuuste complementaire benaderingen
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. erkent de steun van de NIH (T32-AI007413 en T32-AG000114). SAS erkent steun van de JDRF (COE-2019-861), de NIH (R01 DK135268, R01 DK108921, R01 DK135032, R01 DK136547, U01 DK127747), het Department of Veterans Affairs (I01 BX004444), de familie Brehm en de familie Anthony.

De dierstudies die in dit protocol worden gepresenteerd, zijn beoordeeld en goedgekeurd door de University of Michigan Institutional Animal Care and Use Committee. Voor deze studie werden mannelijke C57BL/6J-muizen van twintig weken oud, die ofwel een 15 weken durend normaal vetdieet (RFD) of een vetrijk dieet (HFD) volgden, gebruikt.
1. Beoordeling van mitofagie via de op kleurstof gebaseerde MtPhagy-benadering (methode 1)
<ol><li>Bereiding en behandeling van muiseilandjes<...

Kwantitatieve analyse van mitofagie in β-cellen van de pancreas van muizen

<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=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. 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Dit protocol beschreef twee complementaire methoden om mitofagieflux in gedissocieerde primaire muiseilandjes te kwantificeren. Met behulp van de mt-Keima-methode werd een toename van mitofagie gekwantificeerd als een verhoogde verhouding van zure (561 nm)/neutrale (405 nm) cellen, terwijl in de MtPhagy-methode een verhoogde mitofagieflux werd gekwantificeerd als een toename van de FluozinhogeMtPhagyhogeTMRElage celpopulatie. Deze methoden maken snelle, kwantitatieve en β-celspecifieke b...

Pancreas-β-cellen produceren en scheiden insuline af om aan de metabole eisen te voldoen, en β-celdisfunctie is verantwoordelijk voor hyperglykemie en het ontstaan van diabetes bij zowel type 1- als type 2-diabetes. β-cellen koppelen het glucosemetabolisme aan insulinesecretie via mitochondriale energetica en metabole output, die afhankelijk zijn van een reserve van functionele mitochondriale massa 1,2,3. Om een optimale functie van β cellen te behouden, vertrouwen β-cellen op mitochondriale kwaliteitscontrolemechanismen om verouderde of beschadigde mitochondriën te verwijderen en functionele mitochondrialemassa te behouden. Selectieve mitochondriale autofagie, ook bekend als mitofagie, is een belangrijk onderdeel van de mitochondriale kwaliteitscontroleroute.
Beoordelingen van mitofagie in levende cellen zijn vaak afhankelijk van veranderingen in de mitochondriale pH die optreden tijdens mitofagie. Mitochondriën hebben een licht alkalische pH en gezonde mitochondriën bevinden zich normaal gesproken in het pH-neutrale cytosol. Tijdens mitofagie worden beschadigde of disfunctionele mitochondriën selectief opgenomen in autofagosomen en uiteindelijk opgeruimd in zure lysosomen5. Verschillende in vivo transgene mitofagie-reportermuismodellen, zoals mt-Keima6, mitoQC7 en CMMR8, evenals transfecteerbare mitofagie-sondes, zoals de Cox8-EGFP-mCherry-plasmide9, maken gebruik van deze pH-verandering om kwantitatieve beoordelingen van mitofagie te geven. Het gebruik van transgene muizen die de mt-Keima pH-gevoelige dual-excitatie ratiometrische sonde tot expressie brengen, is geoptimaliseerd voor mitofagiebeoordelingen in eilandjes en β-cellen via flowcytometrie10,11. De verhouding tussen zure en neutrale mt-Keima-golflengte-emissies (de verhouding tussen zure 561 nm en neutrale 480 nm excitatie) kan worden gebruikt om mitofagie 6,12 robuust te kwantificeren.
Dit protocol beschrijft een geoptimaliseerde benadering om mitofagieflux te beoordelen in primaire eilandjes en β-cellen geïsoleerd uit mt-Keima transgene muizen 10,11. Hoewel mt-Keima een zeer gevoelige sonde is, vereist het ofwel gecompliceerde fokprogramma's voor dieren of de transfectie van cellen, wat vaak een uitdaging kan zijn wanneer het werkt in combinatie met andere genetische modellen of met primaire menselijke eilandjes. Bovendien kan het gebruik van meerdere fluorescentielasers en -detectoren om neutrale en zure celpopulaties te identificeren, het combinatorische gebruik van andere fluorescerende reporters beperken.
Om deze uitdagingen het hoofd te bieden, beschrijft dit protocol ook een aanvullende, op kleurstof gebaseerde methode met één fluorescerend kanaal voor robuuste detectie van mitofagie in β-cellen van geïsoleerde muiseilandjes. Deze benadering, ook wel de MtPhagy-methode genoemd, maakt gebruik van een combinatie van drie celdoorlatende kleurstoffen om β-cellen te selecteren, de celpopulaties te kwantificeren die actief mitofagie ondergaan en tegelijkertijd het mitochondriale membraanpotentieel (MMP of Δψm) te beoordelen.
De eerste van deze kleurstoffen is Fluozin-3-AM, een celdoorlatende Zn2+ indicator met een Ex/Em 494/516 nm13. Muizeneilandjes bestaan uit een heterogene populatie van functioneel verschillende cellen, waaronder α-, β-, δ- en PP-cellen. β-cellen omvatten ongeveer 80% van de cellen in het muiseilandje en kunnen worden onderscheiden van andere eilandjesceltypen door hun hoge Zn2+-concentratie in insulinekorrels14,15, waardoor β-cellen kunnen worden geïdentificeerd als de Fluozin-3-AMhoge populatie. De MtPhagy-kleurstof, een pH-gevoelige kleurstof die via een chemische binding op mitochondriën wordt geïmmobiliseerd en zwakke fluorescentie afgeeft, wordt ook in dit protocol gebruikt16. Bij mitofagie-inductie worden beschadigde mitochondriën opgenomen in het zure lysosoom en verhoogt de MtPhagy-kleurstof de fluorescentie-intensiteit in de omgeving met een lage pH (Ex/Em 561/570-700 nm).
Daarnaast wordt tetramethylrhodamine, ethylester (TMRE), gebruikt om MMP te beoordelen. TMRE is een celdoorlatende positief geladen kleurstof (Ex/Em 552/575 nm) die wordt afgezonderd door gezonde mitochondriën vanwege de relatieve negatieve lading die wordt ondersteund door hun membraanpotentiaal17. Beschadigde of ongezonde mitochondriën verdrijven hun membraanpotentieel, wat resulteert in een verminderd vermogen om TMRE te sekwestreren. Door deze kleurstoffen samen te gebruiken, kunnen β-cellen die mitofagie ondergaan, worden geïdentificeerd als de FluozinhogeMtPhagyhogeTMRElage populatie via flowcytometrie. Aangezien mitofagie een dynamisch in plaats van statisch proces is, is dit protocol geoptimaliseerd om mitofagieflux te beoordelen met behulp van valinomycine, een K+-ionofoor die mitofagie induceert na dissipatie van MMP18. Vergelijking van mitofagie in de aan- en afwezigheid van valinomycine maakt het mogelijk om de mitofagieflux in verschillende steekproefgroepen te beoordelen.
Het op kleurstoffen gebaseerde karakter van de huidige aanpak maakt het mogelijk om het te extrapoleren naar menselijke eilandjes en andere moeilijk te transfecten celtypen en omzeilt de noodzaak van gecompliceerde fokprogramma's voor dieren, in tegenstelling tot het mt-Keima-protocol. Het overkoepelende doel van dit protocol is om mitofagie in β-cellen op het niveau van één cel te kwantificeren via twee onafhankelijke op flowcytometrie gebaseerde methoden. Alles bij elkaar beschrijft dit protocol twee krachtige en complementaire methoden die zowel precisie als flexibiliteit mogelijk maken in de kwantitatieve studie van mitochondriale kwaliteitscontrole.

<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

Mitofagie is een kwaliteitscontrolemechanisme dat nodig is om een optimale mitochondriale functie te behouden. Disfunctionele β-cel mitofagie resulteert in onvoldoende insulineafgifte. Geavanceerde kwantitatieve beoordelingen van mitofagie vereisen vaak het gebruik van genetische verslaggevers. Het mt-Keima-muismodel, dat een mitochondriën-gerichte pH-gevoelige dual-excitatie ratiometrische sonde uitdrukt voor het kwantificeren van mitofagie via flowcytometrie, is geoptimaliseerd in β-cellen. De verhouding tussen zure en neutrale mt-Keima-golflengte-emissies kan worden gebruikt om mitofagie robuust te kwantificeren. Het gebruik van genetische mitofagieverslaggevers kan echter een uitdaging zijn bij het werken met complexe genetische muismodellen of moeilijk te transfecteren cellen, zoals primaire menselijke eilandjes. Dit protocol beschrijft een nieuwe complementaire, op kleurstof gebaseerde methode om β-celmitofagie in primaire eilandjes te kwantificeren met behulp van MtPhagy. MtPhagy is een pH-gevoelige, celdoorlatende kleurstof die zich ophoopt in de mitochondriën en de fluorescentie-intensiteit verhoogt wanneer mitochondriën zich in omgevingen met een lage pH bevinden, zoals lysosomen tijdens mitofagie. Door de MtPhagy-kleurstof te combineren met Fluozin-3-AM, een Zn2+ -indicator die selecteert voor β-cellen, en Tetramethylrhodamine, ethylester (TMRE) om het mitochondriale membraanpotentieel te beoordelen, kan mitofagieflux specifiek in β-cellen worden gekwantificeerd via flowcytometrie. Deze twee benaderingen zijn zeer complementair en zorgen voor flexibiliteit en precisie bij het beoordelen van de mitochondriale kwaliteitscontrole in tal van β-celmodellen.

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.

Auteur in de schijnwerpers: beoordeling van mitofagieflux in pancreas-β-cellen met behulp van effectieve en robuuste complementaire benaderingen 

Complementaire benaderingen om mitofagieflux in pancreas-β-cellen te ondervragen

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.

Beoordeling van mitofagie via de op kleurstof gebaseerde MtPhagy-benadering Deze op kleurstof gebaseerde benadering is geoptimaliseerd om mitofagieflux in primaire β-cellen van muizen te analyseren zonder dat er een genetische reporter nodig is, met behulp van Fluozin-3-AM, TMRE en MtPhagy en DAPI om dode cellen uit te sluiten. Door deze kleurstoffen te combineren met valinomycine om mitofagie te induceren, schetst dit protocol een op kleurstoffen gebaseerde methode om selectief de mitofagieflux te m...

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

Dit protocol schetst twee methoden voor de kwantitatieve analyse van mitofagie in pancreas-β-cellen: ten eerste een combinatie van celdoorlatende mitochondriën-specifieke kleurstoffen en ten tweede een genetisch gecodeerde mitofagie-reporter. Deze twee technieken vullen elkaar aan en kunnen worden ingezet op basis van specifieke behoeften, waardoor flexibiliteit en precisie mogelijk zijn bij het kwantitatief aanpakken van mitochondriale kwaliteitscontrole.

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

Ons protocol beschrijft twee complementaire methoden om bètacelmitofagieflux te bestuderen bij de resolutie van één cel via flowcytometrie. Het mt-Keima-protocol maakt gebruik van een genetische mitofagie-reporter, terwijl het MtPhagy-protocol drie fluorescerende kleurstoffen combineert, waardoor het gemakkelijk te extrapoleren is naar moeilijk te transfecteren cellen in menselijke monsters. Beoordelingen van mitofagie met behulp van traditionele biochemische benaderingen en beeldvormingsbenaderingen zijn zowel tijdrovend als uitdagend.Het is dus belangrijk om nieuwe, robuuste en effectieve methoden te ontwikkelen om mitofagieflux in bètacellen te bestuderen. Zowel de mt-Keima- als de MtPhagy-methode zijn kwantitatieve beoordelingen die efficiënt zijn voor het bestuderen van mitofagieflux in bètacellen van de alvleesklier. Het Soleimanpour-lab richt zich dus op de verschillende mechanismen die het falen van bètacellen van de alvleesklier en diabetes veroorzaken.En we richten ons specifiek op de verschillende aspecten van de mitochondriale levenscyclus, waaronder mitofagie, om te begrijpen hoe defecten in de mitochondriale functie de ontwikkeling van diabetes stimuleren.

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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.

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.

Eencellige dissociatie van muizeneilandjes voor flowcytometrische analyse van β-cel mitofagie

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.

Flowcytometrische analyse van mitofagie in β-cellen van de pancreas van muizen met behulp van MtPhagy-kleurstof

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.