Name: imaging ca2+ responses during shigella infection of epithelial cells
Uploaded: 2018-05-24T14:45:00
Description: Watch this Scientific Journal Video about Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells at JoVE.com

Vi takker Jenny-Lee Thomassin for hendes hjælp i redigering håndskriftet. Arbejdet var støttet af ANR grants MITOPATHO og PATHIMMUN, tilskud fra Labex Memolife og PSL IDEX Shigaforce. CHUNHUI Sun er en modtager af en Ph.D. bevilling fra Kina stipendium Rådet. Laurent Combettes og Guy Tran Van Nhieu er modtagere af en WBI-Frankrig exchange Tournesol program N ° 31268YG (Wallonie-Bruxelles International, Fonds de la Recherche Scientifique, Ministère Français des Affaires étrangères et européennes, Ministère de l'Enseignement supérieur et de la Recherche dans le cadre des partnerskabsaftalerne Hubert Curien).

1. præparater
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Bakterier forberedelse
	<ol>
<li>Plade bakterier —Shigella vildtype stamme udtryk for AfaE adhesin (M90T-AfaE) — på en trypticase soja (TCS) agar plade indeholdende 0,01% Congo rød (CR) og dem der inkuberes i 18 timer ved 37 ° C. Bemærk: For at øge deres reproducerbarhed, Shigella plader fremstillet i trin 1.1.1 opbevares ved 4 ° C og bør anvendes inden for en uge, fordi alle kolonier på CR medium vil i sidste ende bliver røde over tid.</li>
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<ol>
	<li>Ashida, H., Ogawa, M., Kim, M., Mimuro, H., Sasakawa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+and+host+interactions+in+the+gut+epithelial+barrier.">Bacteria and host interactions in the gut epithelial barrier.</a> Nature Chemical Biology. 8 (1), 36-45 (2012).</li><li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signaling.">The versatility and universality of calcium signaling.</a> Nature Reviews Molecular Cell Biology. 1 (1), 11-21 (2000).</li><li>Strehler, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+calcium+ATPases:+from+generic+Ca(2+)+sump+pumps+to+versatile+systems+for+fine-tuning+cellular+Ca(2).">Plasma membrane calcium ATPases: from generic Ca(2+) sump pumps to versatile systems for fine-tuning cellular Ca(2).</a> Biochemical and Biophysical Research Communications. 460 (1), 26-33 (2015).</li><li>Muallem, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+Ca2++signals:+a+question+of+timing.">Decoding Ca2+ signals: a question of timing.</a> Journal of Cell Biology. 170 (2), 173-175 (2005).</li><li>Uhlen, P., Fritz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry+of+calcium+oscillations.">Biochemistry of calcium oscillations.</a> Biochemical and Biophysical Research Communications. 396 (1), 28-32 (2010).</li><li>Carneiro, L. 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=Shigella+induces+mitochondrial+dysfunction+and+cell+death+in+nonmyleoid+cells.">Shigella induces mitochondrial dysfunction and cell death in nonmyleoid cells.</a> Cell Host &amp; Microbe. 5 (2), 123-136 (2009).</li><li>Horng, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+and+mitochondrial+destabilization+in+the+triggering+of+the+NLRP3+inflammasome.">Calcium signaling and mitochondrial destabilization in the triggering of the NLRP3 inflammasome.</a> Trends in Immunology. 35 (6), 253-261 (2014).</li><li>Galan, J. E., Lara-Tejero, M., Marlovits, T. C., Wagner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+type+III+secretion+systems:+specialized+nanomachines+for+protein+delivery+into+target+cells.">Bacterial type III secretion systems: specialized nanomachines for protein delivery into target cells.</a> Annual Review of Microbiology. 68, 415-438 (2014).</li><li>Ashida, H., Mimuro, H., Sasakawa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shigella+manipulates+host+immune+responses+by+delivering+effector+proteins+with+specific+roles.">Shigella manipulates host immune responses by delivering effector proteins with specific roles.</a> Frontiers in Immunology. 6, 219 (2015).</li><li>Tran Van Nhieu, 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=Actin-based+confinement+of+calcium+responses+during+Shigella+invasion.">Actin-based confinement of calcium responses during Shigella invasion.</a> Nature Communications. 4, 1567 (2013).</li><li>Niebuhr, 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=Conversion+of+PtdIns(4,5)P(2)+into+PtdIns(5)P+by+the+S.+flexneri+effector+IpgD+reorganizes+host+cell+morphology.">Conversion of PtdIns(4,5)P(2) into PtdIns(5)P by the S. flexneri effector IpgD reorganizes host cell morphology.</a> The EMBO Journal. 21 (19), 5069-5078 (2002).</li><li>Konradt, C., 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+Shigella+flexneri+type+three+secretion+system+effector+IpgD+inhibits+T+cell+migration+by+manipulating+host+phosphoinositide+metabolism.">The Shigella flexneri type three secretion system effector IpgD inhibits T cell migration by manipulating host phosphoinositide metabolism.</a> Cell Host &amp; Microbe. 9 (4), 263-272 (2011).</li><li>Friedrich, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intriguing+Ca2++requirement+of+calpain+activation.">The intriguing Ca2+ requirement of calpain activation.</a> Biochemical and Biophysical Research Communications. 323 (4), 1131-1133 (2004).</li><li>Thomas, D., 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+comparison+of+fluorescent+Ca2++indicator+properties+and+their+use+in+measuring+elementary+and+global+Ca2++signals.">A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals.</a> Cell Calcium. 28 (4), 213-223 (2000).</li><li>Sun, C. 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=The+Shigella+type+III+effector+IpgD+recodes+Ca2++signals+during+invasion+of+epithelial+cells.">The Shigella type III effector IpgD recodes Ca2+ signals during invasion of epithelial cells.</a> The EMBO Journal. 36 (17), 2567-2580 (2017).</li><li>Allaoui, A., Menard, R., Sansonetti, P. J., Parsot, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+Shigella+flexneri+IpgD+and+IpgF+genes,+which+are+located+in+the+proximal+part+of+the+mxi+locus.">Characterization of the Shigella flexneri IpgD and IpgF genes, which are located in the proximal part of the mxi locus.</a> Infection and Immunity. 61 (5), 1707-1714 (1993).</li></ol>

Dette manuskript beskriver den protokol, vi manipuleret til at følge lokale Ca2 + signaler i den relativt korte kinetik af en Shigella invasion, samt global Ca2 + svar under den udvidede kinetik af Shigella. Nedenfor signaler nøglen spørgsmål kan findes der skal løses for at optimere påvisning af Ca2 + samtidig minimere enhver indblanding med de biologiske processer.
Kemiske vs kodet genetisk Ca2 + sonder:
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Ca2 + regulerer alle kendte celle processer, herunder cytoskeletal reorganisering, inflammatoriske respons og celle død veje relateret til vært-patogen interaktioner1,2,3. Fysiologiske betingelser, basal cytosole Ca2 + koncentrationerne er lave, i hundredvis af nM rækkevidde, men kan være udsat for forbigående stigninger på agonist stimulation. Disse variationer viser ofte oscillerende adfærd gennem handling af pumper og kanaler på plasma og endoplasmatiske reticulum membraner. Mønstret for disse svingninger er karakteriseret ved periode, varigheden og amplitude af Ca2 + stiger, og er dekrypteret af celler, som igen udløser specifikke svar på hvad er kendt som den Ca2 + kode4,5 . En vedvarende stigning i det cytosole Ca2 + koncentration patologiske betingelser kan føre til celledød er forbundet med permeabilization af mitokondrie membraner og udgivelsen af pro-apoptotiske eller nekrotisk faktorer6, 7.
Shigella, det agens af dysenteri, invaderer epitelceller ved at indsprøjte effektorer i værtsceller ved hjælp af en type III sekretion system (T3SS)8,9. Shigella invasion af værtsceller er forbundet med lokale og globale Ca2 + signaler fremkaldes ved T3SS. Hvad angår poreformede toksiner, T3SS translocon, der indsættes i vært cellemembraner og er nødvendige for injektion af T3SS effektorer er sandsynligvis ansvarlige for aktivering af PLC og inositol (1, 4, 5) trisphosphate (InsP3)-afhængige Ca2 + Release. Kombinationen af lokalisere PLC stimulering og ophobning af polymeriseret actin på lokaliteter af Shigella invasion resultatet i en atypisk langvarig InsP3-afhængige Ca2 + frigive10. Type III effektor IpgD, en phosphatidylphosphatid 4,5 bisfosfat (PIP2) -4-fosfatase, begrænser den lokale mængde PIP2, derved kontrollere mængden af tilgængelig substrat for PLC til at generere InsP3, som bidrager til indkapsling af lokale Ca2 + svar på bakteriel invasion websteder11,12. Disse lokale Ca2 + svar sandsynligvis bidrage til aktin polymerisering på Shigella invasion sites10. Globale Ca2 + svar, der er også fremkaldes af Shigella, dog er undværes for bakteriel invasion proces men udløse åbningen af connexin hemichannels på plasma membran og udgivelsen af ATP i den ekstracellulære rum. Frigivne ATP handler på en paracrine måde, stimulerer til gengæld Ca2 + oscillerende svar i celler ved siden af den inficerede celle. IpgD er også ansvarlige for udformningen af globale Ca2 + svar i uregelmæssig isolerede svar med langsom dynamics. I sidste ende, efter en langvarig bakteriel infektion fører IpgD til hæmning af InsP3-medieret Ca2 + signaler. Gennem sin interferens med Ca2 + signalering, IpgD forsinker en Ca2 +-afhængige calpain aktivering fører til afmontering af fokale vedhæftning strukturer og tidlig udstationering af inficerede celler13.
Mens Ca2 + signaler er involveret i kritiske aspekter af patogenese, rejser anvendelse af en mikroorganisme en række tekniske udfordringer, der ikke er fundet i klassisk agonist undersøgelser. De protokoller er beskrevet bruger her den almindeligt anvendte fluorescerende Ca2 + kemiske indikator Fluo-4, der vi manipuleret til at karakterisere lokale Ca2 + signaler under en Shigella infektionen. Trin kritisk til påvisning af disse signaler er diskuteret, samt procedurer, der gennemføres for deres kvantitative analyser, der kræves til at karakterisere rollen af bakteriel effektorer i Ca2 + signalering.

<table border="0" cellpadding="0" cellspacing="0" style="width:693px;" width="694">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fluo-4 AM</td>
			<td style="width:120px;">Invitrogen</td>
			<td style="width:136px;">F14201</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Metamorph version 7.7</td>
			<td style="width:120px;">Universal Imaging</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">CoolLED illumination system pE-2</td>
			<td style="width:120px;">Roper Scientific</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">micro-dish 35 mm, high&#160;</td>
			<td style="width:120px;">IBIDI</td>
			<td>81156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Trypticase Soy (TCS) broth</td>
			<td style="width:120px;">Thermofisher</td>
			<td style="width:136px;"></td>
			<td>B11768</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">TCS agar</td>
			<td style="width:120px;">Thermofisher</td>
			<td style="width:136px;"></td>
			<td>B11043</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Congo red</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:136px;"></td>
			<td>75768</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:247px;">M90T-AfaE</td>
			<td style="width:120px;">Sun et al. 2017</td>
			<td style="width:136px;">Shigella flexneri serotype V. expressing the AfaE adhesin</td>
			<td></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:247px;">ipgD-AfaE</td>
			<td style="width:120px;">Sun et al. 2017</td>
			<td style="width:136px;">isogenic ipgD mutant strain expressing the AfaE adhesin</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging ca2+ responses during shigella infection of epithelial cells

Ca2 + er en allestedsnærværende ion involveret i alle kendte cellulære processer. Mens globale Ca2 + svar kan påvirke celle skæbne, regulere lokale variationer i gratis Ca2 + cytosole koncentrationer, knyttet til at frigive fra interne butikker eller en tilstrømning gennem plasmamembran kanaler, kortikale celle processer. Patogener, overholde eller invadere vært celler udløser en reorganisering af actin cytoskelettet underliggende vært plasmamembran, som sandsynligvis påvirker både globale og lokale Ca2 + signalering. Fordi disse begivenheder kan forekomme ved lave frekvenser i et pseudo stokastiske måde over udvidede kinetik, rejser analyse af Ca2 + signaler induceret af patogener store tekniske udfordringer, der skal løses.
Her rapporterer vi protokoller til påvisning af globale og lokale Ca2 + signaler på en Shigella infektionen af epitelceller. I disse protokoller, artefakter knyttet til en langvarig eksponering og solskader tilknyttet excitation af Ca2 + fluorescerende sonder er foretaget fejlfinding ved nøje at kontrollere parametrene erhvervelse over definerede perioder under en Shigella invasion. Procedurer er implementeret for at nøje analysere amplitude og frekvens af globale cytosole Ca2 + signaler under udvidede infektion kinetik ved hjælp af kemiske sonden Fluo-4.

Shigella invasion er forbundet med atypiske langvarig lokale Ca2 + svar:
Efter den protokol, der er nævnt ovenfor, Fluo-4-loaded HeLa celler blev udfordret med WT Shigella og stream opkøb blev udført for at analysere Ca2 + signaler. En repræsentativ eksperiment er vist i figur 1, med time-lapse billeder serie af fluorescens-intensiteten af Fluo-4 sonden i gen...

Watch this Scientific Journal Video about Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells at JoVE.com

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Vi præsenterer her, protokoller for at visualisere calcium (Ca2 +) svar fremkaldes ved HeLa celler inficeret af Shigella. Ved at optimere parametre af bakteriel infektion og imaging med Ca2 + fluorescerende sonder, er atypiske globale og lokale Ca2 + signaler fremkaldt af bakterier over en lang række infektion kinetik karakteriseret.

Imaging Ca2 + svar under Shigella infektionen af epitelceller

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

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Immunology and Infection

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

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