Name: Author Spotlight: Investigating Viral Disruption of Intestinal Epithelial Signaling &#8211; Research Insights and Future Directions
Uploaded: 2024-01-19T14:05:00
Description: Watch this Scientific Journal Video about Investigating Viral Disruption of Intestinal Epithelial Signaling â€“ Research Insights and Future Directions at JoVE.com

Dette arbejde blev støttet af tilskud R01DK115507 og R01AI158683 (PI: J. M. Hyser) fra National Institutes of Health (NIH). Praktikantstøtte blev ydet af NIH-tilskud F30DK131828 (PI: J.T. Gebert), F31DK132942 (PI: F. J. Scribano) og F32DK130288 (PI: K.A. Engevik). Vi vil gerne takke Texas Medical Center Digestive Diseases Enteroid Core for at levere organoid vedligeholdelsesmedier.

Alle de humane tarmorganoider (HIO'er), der anvendes i denne protokol, og de repræsentative eksperimenter blev afledt af humant væv opnået og vedligeholdt af Texas Medical Center Digestive Diseases Enteroid Core. Alle prøver blev indsamlet i overensstemmelse med en protokol godkendt af Institutional Review Board ved Baylor College of Medicine.
1. Fremstilling af materialer og reagenser
<ol><li>Til organoid vedligeholdelse samles cellekulturbehandlede 24-brøndplader, kæ...

Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers

<ol>
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Ændringer i cytosoliske Ca2+ niveauer kan være både en årsag og virkning af patologier i epitelet 10,16,17. Stigninger i cytosolisk calcium kan direkte drive sekretion via aktivering af den calciumafhængige chloridkanal TMEM16A18,19. Aktivering af TMEM16A som reaktion på Ca2+ muliggør apikal udstrømning af chlorid og etablerer en osmotisk gr...

Calcium er en bredt bevaret anden budbringer, der spiller en kritisk rolle i reguleringen af cellulær fysiologi1. På grund af sin stærke ladning, lille størrelse og høje opløselighed under fysiologiske forhold er calcium en ideel manipulator af proteinkonformation. Dette gør calcium til et kraftfuldt middel til at transducere elektrokemiske signaler til enzymatiske, transkriptionelle eller posttranskriptionelle ændringer. De strenge calciumkoncentrationsgradienter over det endoplasmatiske retikulum (ER) og plasmamembraner skaber en høj drivkraft, der muliggør hurtige ændringer i cytosolisk calciumkoncentration. Flere mekanismer, herunder både buffering og aktiv transport, opretholder denne gradient tæt. Selvom det er nødvendigt for normale cellulære funktioner, er denne vedligeholdelse energisk dyr, hvilket gør den særlig modtagelig i stresstilstande 2.
Som sådan er dysregulering af calcium i cytosolen et næsten universelt signal om mange slags cellulær stress. Metaboliske forstyrrelser, toksiner, patogener, mekaniske skader og genetiske forstyrrelser kan alle forstyrre calciumsignalering. Uanset stimulus kan vedvarende, ukontrollerede stigninger i cytosolisk calcium på helcelleniveau fremme apoptose og til sidst nekrose 3,4. Ændringer i cytosoliske calciumniveauer med lavere amplitude eller højere frekvens har imidlertid varierende virkninger2. Ligeledes kan resultaterne af calciumfluktuationer variere baseret på det rumlige mikrodomæne, hvor de forekommer5. Overvågning af calciumniveauer kan derfor give indsigt i dynamiske signalprocesser, men dette kræver prøveudtagning med relativt høj tidsmæssig og rumlig opløsning.
Genetisk kodede calciumindikatorer (GECI'er) er effektive værktøjer til kontinuerlig prøveudtagning i levende cellesystemer6. Nogle af de mest anvendte GECI'er er GFP-baserede calcium-responsive fluorescerende proteiner kendt som GCaMPs7. Den kanoniske GCaMP er en fusion af tre forskellige proteindomæner: en cirkulært permuteret GFP (cpGFP), calmodulin og M136. Calmodulindomænet gennemgår en konformationsændring ved binding af calcium, hvilket tillader dets interaktion med M13. Calmodulin-M13-interaktionen inducerer en konformationsændring i cpGFP, der øger dens fluorescerende emission ved excitation. Som sådan korrelerer en stigning i calciumkoncentration med en stigning i GCaMP-fluorescensintensitet. Disse sensorer kan være cytosoliske eller målrettet mod specifikke organeller8.
I lighed med de fleste væv regulerer calcium en række funktioner i mave-tarmepitelet. Tarmepitelet er integreret for næringsstof- og væskeabsorption, men skal også danne en tæt barriere og immungrænseflade for at undgå patogeninvasion eller giftige fornærmelser. Calciumafhængige veje påvirker næsten alle disse vitale funktioner 9,10,11. Imidlertid forbliver calciumsignalering i tarmepitelet en underudforsket grænse med lovende potentiale som et terapeutisk mål. Mens overvågning af calciumdynamikken i tarmepitelet in vivo fortsat giver udfordringer, tilbyder humane tarmorganoider (HIO'er) et tilpasningsdygtigt ex vivo-system til eksperimenter12. HIO'er er 3-dimensionelle (3D) sfæroider afledt af humane tarmstamceller og rekapitulerer efter differentiering meget af den cellulære mangfoldighed af det indfødte tarmepitel12.
Denne protokol beskriver omfattende metoder til at konstruere HIO'er, der udtrykker GECI'er og derefter forberede konstruerede HIO'er som monolag til levende celle calciumbilleddannelse. Det tilbyder virusinfektion som et eksempel på en patologisk manipulation, der forstyrrer calciumsignalering og giver en analytisk tilgang til kvantificering af disse ændringer.

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			<td>Advanced DMEM F12</td>
			<td>Gibco</td>
			<td>12634028</td>
			<td></td>
		</tr>
		<tr>
			<td>[Leu15]-Gastrin I</td>
			<td>Sigma-Aldrich</td>
			<td>G9145</td>
			<td></td>
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			<td>0.05% Trypsin EDTA&#160;</td>
			<td>Gibco&#160;</td>
			<td>25300054</td>
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			<td>0.05% Trypsin EDTA&#160;</td>
			<td>Gibco&#160;</td>
			<td>25300054</td>
			<td></td>
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			<td>1.5mL microcentrifuge tubes</td>
			<td>Fisherbrand</td>
			<td>5408137</td>
			<td></td>
		</tr>
		<tr>
			<td>15mL conical tubes</td>
			<td>Thermofisher Scientific</td>
			<td>0553859A</td>
			<td></td>
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			<td>16% formaldehyde</td>
			<td>Thermofisher Scientific</td>
			<td>28906</td>
			<td></td>
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			<td>1M HEPES</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
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			<td>1M HEPES</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
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			<td>1X PBS</td>
			<td>Corning&#160;</td>
			<td>21-040-CV</td>
			<td></td>
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			<td>25 gauge needle</td>
			<td>Thermofisher Scientific</td>
			<td>1482113D</td>
			<td></td>
		</tr>
		<tr>
			<td>A-83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td></td>
		</tr>
		<tr>
			<td>ADP</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A2754</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM F12</td>
			<td>Gibco</td>
			<td>12634028</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycocytic&#160;</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic&#160;</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>FisherScientific&#160;</td>
			<td>BP1600100</td>
			<td></td>
		</tr>
		<tr>
			<td>CellView Cell Culture Slide, PS, 75/25 MM, Glass Bottom, 10 compartments</td>
			<td>Greiner</td>
			<td>543979</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Sigma Aldrich</td>
			<td>C5533</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermofisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Corning</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>Corning&#160;</td>
			<td>35010CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>Corning&#160;</td>
			<td>35010CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorobrite</td>
			<td>Gibco</td>
			<td>A1896701</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX&#160;</td>
			<td>Gibco&#160;</td>
			<td>35050079</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX&#160;</td>
			<td>Gibco&#160;</td>
			<td>35050079</td>
			<td></td>
		</tr>
		<tr>
			<td>Human epidermal growth factor</td>
			<td>ProteinTech</td>
			<td>HZ-1326</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentivirus</td>
			<td>VectorBuilder</td>
			<td>(variable)</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosceicen</td>
			<td>356231/CB40230C</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Cell Culture Treated 24-well Plates</td>
			<td>Thermofisher Scientific</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>MilliporeSigma</td>
			<td>TR1003G</td>
			<td></td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Sigma-Aldrich</td>
			<td>S70767</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher BioReagents</td>
			<td>BP151100</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme, no phenol red</td>
			<td>Thermofisher Scientific</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Worthington Biochemical</td>
			<td>NC9811754</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
		</tr>
	</tbody>
</table>

live calcium imaging of virus-infected human intestinal organoid monolayers using genetically encoded calcium indicators

Calciumsignalering er en integreret regulator af næsten alle væv. Inden for tarmepitelet er calcium involveret i reguleringen af sekretorisk aktivitet, actindynamik, inflammatoriske reaktioner, stamcelleproliferation og mange andre ukarakteriserede cellulære funktioner. Som sådan kan kortlægning af calciumsignaldynamik i tarmepitelet give indsigt i homeostatiske cellulære processer og afsløre unikke reaktioner på forskellige stimuli. Humane tarmorganoider (HIO'er) er en højkapacitetsmodel, der er afledt af mennesker, til at studere tarmepitelet og repræsenterer således et nyttigt system til at undersøge calciumdynamikken. Dette papir beskriver en protokol til stabilt at transducere HIO'er med genetisk kodede calciumindikatorer (GECI'er), udføre levende fluorescensmikroskopi og analysere billeddannelsesdata for meningsfuldt at karakterisere calciumsignaler. Som et repræsentativt eksempel blev 3-dimensionelle HIO'er transduceret med lentivirus for stabilt at udtrykke GCaMP6'er, en grøn fluorescerende proteinbaseret cytosolisk GECI. De konstruerede HIO'er blev derefter spredt i en enkeltcellesuspension og podet som monolag. Efter differentiering blev HIO-monolagene inficeret med rotavirus og/eller behandlet med lægemidler, der vides at stimulere et calciumrespons. Et epifluorescensmikroskop udstyret med et temperaturstyret, befugtet levende billeddannelseskammer tillod langtidsbilleddannelse af inficerede eller lægemiddelbehandlede monolag. Efter billeddannelse blev erhvervede billeder analyseret ved hjælp af den frit tilgængelige analysesoftware, ImageJ. Samlet set etablerer dette arbejde en tilpasningsdygtig pipeline til karakterisering af cellulær signalering i HIO'er.

Calcium is a widely conserved second messenger that plays a critical role in regulating cellular physiology1. Given its strong charge, small size, and high solubility in physiological conditions, calcium is an ideal manipulator of protein conformation. This makes calcium a powerful means to transduce electrochemical signals into enzymatic, transcriptional, or post-transcriptional alterations. The strict calcium concentration gradients across the endoplasmic reticulum (ER) and plasma membranes create a high driving force that allows for rapid changes in cytosolic calcium concentration. Multiple mechanisms, including both buffering and active transport, tightly maintain this gradient. While necessary for normal cellular functions, this maintenance is energetically expensive, making it particularly susceptible in states of stress 2.
As such, dysregulation of calcium within the cytosol is a near-universal signal of many kinds of cellular stress. Metabolic disturbances, toxins, pathogens, mechanical damage, and genetic perturbations can all disrupt calcium signaling. Regardless of the stimulus, on a whole-cell level, sustained, uncontrolled rises in cytosolic calcium can promote apoptosis and&#160;eventually necrosis3,4. Alterations in cytosolic calcium levels of lower amplitude or higher frequency, however, have varying effects2. Likewise, the outcomes of calcium fluctuations may differ based on the spatial microdomain in which they occur5. Monitoring calcium levels can&#160;therefore offer insight into dynamic signaling processes, but this requires sampling with relatively high temporal and spatial resolution.
Genetically encoded calcium indicators (GECIs) are powerful tools for continuous sampling in live-cell systems6. Some of the most widely used GECIs are GFP-based calcium-responsive fluorescent proteins known as GCaMPs7. The canonical GCaMP is a fusion of three distinct protein domains: a circularly permuted GFP (cpGFP), calmodulin, and M136. The calmodulin domain undergoes a conformation change upon binding calcium, allowing its interaction with M13. The calmodulin-M13 interaction induces a conformational change in the cpGFP that increases its fluorescent emission upon excitation. As such, an increase in calcium concentration correlates with an increase in GCaMP fluorescence intensity. These sensors can be cytosolic or targeted to specific organelles8.
Similar to most tissues, calcium regulates a variety of functions within the gastrointestinal epithelium. The intestinal epithelium is integral for nutrient and fluid absorption but also must form a tight barrier and immune interface to avoid pathogen invasion or toxic insults. Calcium-dependent pathways influence nearly all of these vital functions9,10,11. However, calcium signaling within the intestinal epithelium remains an underexplored frontier with promising potential as a therapeutic target. While monitoring calcium dynamics within the intestinal epithelium in vivo continues to present challenges, human intestinal organoids (HIOs) offer an adaptable ex vivo system for experimentation12. HIOs are 3-dimensional (3D) spheroids derived from human intestinal stem cells and, upon differentiation, recapitulate much of the cellular diversity of the native intestinal epithelium12.
This protocol describes comprehensive methods to engineer HIOs that express GECIs and then prepare engineered HIOs as monolayers for live-cell calcium imaging. It offers viral infection as an example of a pathologic manipulation that disrupts calcium signaling and provides an analytic approach to quantify these changes.

Author Spotlight: Investigating Viral Disruption of Intestinal Epithelial Signaling &#8211; Research Insights and Future Directions 

Levende calciumbilleddannelse af virusinficerede humane tarmorganoidmonolag ved hjælp af genetisk kodede calciumindikatorer

Figur 1A viser en BMM-kuppel indeholdende 3-dimensionelle humane tarmorganoider, der er blevet transduceret for stabilt at udtrykke GCaMP6'er. Figur 1B viser den samme linje af organoid forgyldt som et monolag ved 24, 48 og 72 timer efter såning. For at validere funktionen af GCaMP6s blev monolaget afbildet ved fluorescensmikroskopi hver 2. s i 4 minutter, og 100 nM ADP blev tilføjet til medierne efter ~ 20 s. ADP fremkalder calciumfrigivelse fra det endoplasm...

Watch this Scientific Journal Video about Investigating Viral Disruption of Intestinal Epithelial Signaling â€“ Research Insights and Future Directions at JoVE.com

Denne protokol beskriver en tilgang til udførelse af calciumbilleddannelse i virusinficerede humane tarmorganoider og tilbyder en tilgang til analyse.

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory ...

Gennem vores arbejde sigter vi mod at forstå, hvordan vira forstyrrer signalering i tarmepitelet. Vi er især interesserede i at forstå de måder, hvorpå virusinficerede celler dysregulerer nærliggende uinficerede celler for at bidrage til patogenese. Vi har for nylig opdaget, at rotavirusinficerede celler frigiver pulser af ADP, som dysregulerer nærliggende uinficerede celler og letter viral spredning og forværrer sygdommen.Mens calciumbilleddannelse er meget populær inden for områder som neurovidenskab, har den ikke været meget brugt til at studere epitelbiologi eller virologi. Denne protokol tilbyder en tilgang til tilpasning af eksisterende teknologier og modelsystemer til undersøgelse af calciumsignalering og levende virusinficeret epitelvæv. Ved at konstruere organoider til at udtrykke genetisk kodede calciumindikatorer giver vores tilgang mulighed for en konsistent, reproducerbar langsigtet billeddannelse i et modelsystem, der rekapitulerer den cellulære mangfoldighed af det menneskelige tarmepitel.Vi sigter mod at forstå virkningerne af virusinduceret parakrin purinerg signalering for både værten og virussen. Vores fremtidige arbejde vil også afgøre, om denne type signalering er bredt bevaret på tværs af andre vira, og om virkningerne er ens.