Name: live imaging assay for assessing the roles of ca2+ and sphingomyelinase in the repair of pore-forming toxin wounds
Uploaded: 2013-08-25T14:00:00
Description: Watch this Scientific Journal Video about Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds at JoVE.com

Dette arbejde blev støttet af tilskud fra NIH R37 AI34867 og R01 GM064625 til NWA Vi takker Dr. R. Tweten fra University of Oklahoma for SLO ekspressionsplasmid.

1.. Transcriptional undertrykkelse af ASM <ol><li> Seed 1,5 x 10 5 HeLa-celler i 2 ml DMEM vækstmedier (DMEM høj glucose med 10% føtalt bovint serum (FBS), 2 mM L-glutamin, 1% Penn-Strep) på 35 mm glasbund retter (Mattek) og inkuberes natten over ved 37 ° C i en 5% CO 2-inkubator. </li><li> Den følgende dag, aspirat fra medierne vækst og erstatte det med 2 ml DMEM reduceret serum medium (DMEM med 4% FBS), mindst 1 time, før du tilføjer siRNA transfektion blanding. </li><li> Forbered 4 r?...

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+proximal+lysosomes+are+the+major+vesicles+responsible+for+calcium-dependent+exocytosis+in+nonsecretory+cells.">Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells.</a> J. Cell Biol. 159, 625-635 (2002).</li><li>Chakrabarti, S., 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=Impaired+membrane+resealing+and+autoimmune+myositis+in+synaptotagmin+VII-deficient+mice.">Impaired membrane resealing and autoimmune myositis in synaptotagmin VII-deficient mice.</a> J. Cell Biol. 162, 543-549 (2003).</li><li>Walev, I., 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=Delivery+of+proteins+into+living+cells+by+reversible+membrane+permeabilization+with+streptolysin-O.">Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 3185-3190 (2001).</li><li>Iacovache, I., van der Goot, F. G., Pernot, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pore+formation:+an+ancient+yet+complex+form+of+attack.">Pore formation: an ancient yet complex form of attack.</a> Biochim. Biophys. Acta. 1778, 1611-1623 (2008).</li><li>Morgan, B. P., Campbell, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+recovery+of+human+polymorphonuclear+leucocytes+from+sublytic+complement+attack+is+mediated+by+changes+in+intracellular+free+calcium.">The recovery of human polymorphonuclear leucocytes from sublytic complement attack is mediated by changes in intracellular free calcium.</a> Biochem. 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Cell Biol. 180, 905-914 (2008).</li><li>Bansal, 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=Defective+membrane+repair+in+dysferlin-deficient+muscular+dystrophy.">Defective membrane repair in dysferlin-deficient muscular dystrophy.</a> Nature. 423, 168-172 (2003).</li><li>McNeil, P. L., Miyake, K., Vogel, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endomembrane+requirement+for+cell+surface+repair.">The endomembrane requirement for cell surface repair.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 4592-4597 (2003).</li><li>Tam, 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=Exocytosis+of+acid+sphingomyelinase+by+wounded+cells+promotes+endocytosis+and+plasma+membrane+repair.">Exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair.</a> J. Cell Biol. 189, 1027-1038 (2010).</li><li>Weisleder, 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=Visualization+of+MG53-mediated+cell+membrane+repair+using+in+vivo+and+in+vitro+systems.">Visualization of MG53-mediated cell membrane repair using in vivo and in vitro systems.</a> J. Vis. Exp. (52), e2717 (2011).</li><li>Tweten, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol-dependent+cytolysins,+a+family+of+versatile+pore-forming+toxins.">Cholesterol-dependent cytolysins, a family of versatile pore-forming toxins.</a> Infect. Immun. 73, 6199-6209 (2005).</li><li>Zha, X., 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=Sphingomyelinase+treatment+induces+ATP-independent+endocytosis.">Sphingomyelinase treatment induces ATP-independent endocytosis.</a> J. Cell Biol. 140, 39-47 (1998).</li><li>Buckley, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossing+three+membranes.+Channel+formation+by+aerolysin.">Crossing three membranes. Channel formation by aerolysin.</a> FEBS Lett. 307, 30-33 (1992).</li><li>Parker, M. W., 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=Structure+of+the+Aeromonas+toxin+proaerolysin+in+its+water-soluble+and+membrane-channel+states.">Structure of the Aeromonas toxin proaerolysin in its water-soluble and membrane-channel states.</a> Nature. 367, 292-295 .</li><li>Yamaji-Hasegawa, 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=Oligomerization+and+pore+formation+of+a+sphingomyelin-specific+toxin,+lysenin.">Oligomerization and pore formation of a sphingomyelin-specific toxin, lysenin.</a> J. Biol. Chem. 278, 22762-22770 (2003).</li></ol>

Tidligere undersøgelser af mekanisk skade og plasmamembranen reparation påberåbt sig forskellige mekanismer for at skade celler, der spænder fra slippe glasperler, micropipetting, klipning, ridser eller skrabning. Udlæsningen af ​​alle disse analyser var kvalitative snarere end kvantitative, og ikke give præcise oplysninger om kinetikken af ​​reparation mekanisme. Cellernes evne til at genforsegle deres plasmamembranen efter disse former for skade blev målt ved tilstedeværelse eller fravær af sporstoffe...

Det har været kendt i flere årtier, at plasmamembran reparation efter mekanisk skade er en Ca2 +-afhængig proces 1,2. Senere undersøgelser viste, at Ca2 + indstrømning gennem såret udløser en kraftig proces exocytose af intracellulære vesikler på læsionsstedet, hvilket er påkrævet for genlukning 3,4. Satsen for tab af et fluorescerende farvestof indlæst i cytoplasmaet i celler blev brugt til at vurdere hastigheden af reparation, og konkluderede, at genlukning blev afsluttet inden &lt;30 sek efter skade 5.. To modeller blev oprindeligt foreslået at forklare kravet om exocytose i plasmamembranen reparation: 1) &quot;patch&quot;-modellen, som foreslog, at Ca2 + tilstrømning gennem læsionen udløser oprindeligt homotypisk fusion af intracellulære vesikler, der udgør en stor &quot;patch&quot;, der ville derefter anvendes på plasmamembranen at forsegle såret 6 og 2) spændinger reduktion model, som foreslog, at membran-Added af Ca2 +-afhængig exocytose i nærheden af såret vil reducere plasma membranspænding lette genlukning af dobbeltlaget 7. Den rolle, Ca 2 +-udløst exocytose i plasmamembranen reparation blev yderligere forstærket af undersøgelser, der viser, at lysosomer indeholder en Ca 2 +-sensor molekyle, synaptotagmin VII, hvilket letter deres exocytose og plasma membran reparation i skadede celler 8,9,10,11 . Imidlertid har yderligere dokumentation tilkendegivet, at exocytose alene ikke var tilstrækkelig til at fremme plasmamembran reparation. Ud over mekaniske tårer på plasmamembranen, en hyppig form for cellebeskadigelse er permeabilisering af poredannende toksiner produceret af bakterier 12,13 eller immunceller 14,15. I modsætning til mekaniske tårer, poredannende proteiner indsætte sig på plasmamembranen danne en stabil, protein-foret porestørrelse, som ikke kan lukkes igen blot ved at anvende en membran &quot;patch&quot; eller ved reduktionning membranspænding. Interessant undersøgelser viste, at pattedyrceller har en effektiv mekanisme til at reparere deres plasmamembranen efter permeabilisering med pore-dannende proteiner, og denne proces kræver også tilstedeværelsen af ekstracellulær Ca2 + 12. Dette fund rejst spørgsmålet om, hvorvidt transmembrane pore fjernelse fra celleoverfladen var også en hurtig proces, som observeret med mekaniske sår 5. Overraskende vores nylige undersøgelser viste, at genlukning af cellerne permeabiliseret med poredannende toksiner har meget lignende egenskaber til reparation af mekaniske sår: processen kræver ekstracellulær Ca2 + og er afsluttet inden for ~ 30 sek. Undersøgelse denne proces mere detaljeret, for nylig har vi lært, at ud over Ca 2 +-reguleret exocytose af lysosomer plasmamembranen reparation medfører en hurtig form for endocytose, som udløses ved frigivelse af det lysosomale enzym acid sphingomyelinase (ASM) og er afgørende ikke kun for the fjernelse af transmembrane porer, men også til reparation af mekaniske sår 16. For at bestemme kinetikken af celle genlukning efter permeabilisering med pore-dannende proteiner i vores laboratorium vi tilpasset en levende billedbehandling metode, der var blevet brugt tidligere til at vurdere genlukning af laser-såret isolerede muskelfibre 17. Dette assay er baseret på egenskaberne af den lipofile farvestof FM1-43, som stabilt interkalerer ind i det ydre blad af lipiddobbeltlag stigende i fluorescensintensitet. Når plasma membrandobbeltlaget er forstyrret ekstracellulære farvestof får adgang til intracellulære membraner, hvilket giver en følsom metode til påvisning af plasmamembranen skade og reparation 18,16,19,20. At tilpasse dette assay til vurdering af celle genlukning efter permeabilisering med poredannende proteiner, vi præinkuberes celler ved 4 ° C med det bakterielle toksin streptolysin O (SLO), som binder til membran cholesterol 21. Synkron cellepermeabilisering kan derefter let opnås ved at bevæge cellerne fra isen til at varme medium i en opvarmet mikroskopobjektbord, som aktiverer oligomerisering og ændring i konformation, som fører til transmembranprotein poredannelse. En fordel ved denne fremgangsmåde i de tidligere publicerede assays ved hjælp af laser sårdannelse, er, at et større antal celler kan analyseres samtidigt i en mikroskopisk felt, der giver bedre prøvetagning af cellepopulationen. I betragtning af de mekanistiske ligheder mellem cellen genlukning proces ses efter mekanisk skade og permeabilisering med poredannende toksiner assayet beskrevet her giver en meget alsidig og effektiv metode til at dissekere faktorer involveret i den grundlæggende proces med plasmamembranen reparation. Som et eksempel viser vi, at det er muligt at anvende dette assay til at identificere Ca2 + afhængige og uafhængige trin i reparationsprocessen.

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		 </tr>
		 <tr>
			<td>HeLa 229 cell line</td>
			<td>ATCC</td>
			<td>CCL2-1</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM High Glucose </td>
			<td>Invitrogen</td>
			<td>11965</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM High Glucose No Calcium</td>
			<td>Invitrogen</td>
			<td>20168</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FM1-43</td>
			<td>Invitrogen</td>
			<td>T3163</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Optimem Reduced Serum</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lipofectamine RNAiMax</td>
			<td>Invitrogen</td>
			<td>13778</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Control medium GC content RNAi oligo</td>
			<td>Invitrogen</td>
			<td>12935300</td>
			<td></td>
		 </tr>
		 <tr>
			<td>SMPD1 RNAi oligo</td>
			<td>Invitrogen</td>
			<td>HSS143988</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal Bovine Serum Heat-inactivated</td>
			<td>Gemini-Bioproducts</td>
			<td>100-106</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sphingomyelinase from Bacillus cereus</td>
			<td>Sigma</td>
			<td>S7651</td>
			<td></td>
		 </tr>
		 <tr>
			<td>35 mm-glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-0-14</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Material</td>
		 </tr>
		 <tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Camera</td>
			<td>Hamamatsu Photonics</td>
			<td>C9100-50</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Spinning disk confocal microscope</td>
			<td>PerkinElmer</td>
			<td>UltraViewVoX</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software analysis software</td>
			<td>PerkinElmer</td>
			<td>Volocity Suite</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Environmental chamber</td>
			<td>Pathology Devices</td>
			<td>LiveCell System Chamber</td>
		 </tr>
		</tbody>
 </table>

live imaging assay for assessing the roles of ca2+ and sphingomyelinase in the repair of pore-forming toxin wounds

Plasma membran skade er en hyppig begivenhed, og sår skal hurtigt repareret for at sikre cellulær overlevelse. Tilgangen af Ca2 + er en vigtig signalering hændelse, der udløser reparation af mekaniske sår på plasmamembranen i ~ 30 sek. Nylige undersøgelser har vist, at pattedyrceller også tillukkes deres plasmamembranen efter permeabilisering med poredannende toksiner i en Ca2 +-afhængig proces, der involverer exocytose af lysosomale enzym acid sphingomyelinase efterfulgt af pore endocytose. Her beskriver vi den metode, der anvendes til at påvise, at genlukning af celler permeabiliserede af giftstoffet streptolysin O er også hurtig og afhængig af Ca 2 + tilstrømning. Assay design tillader synkronisering af skaden begivenhed og en præcis kinetisk måling af cellernes evne til at genoprette plasmamembran integritet ved billeddannelse og kvantificere det omfang, hvormed den liphophilic farvestof FM1-43 når intracellulære membraner. Denne levende assay also tillader en følsom vurdering af evnen af ​​eksogent tilsat opløselige faktorer såsom sphingomyelinase at inhibere FM1-43 tilstrømning, hvilket afspejler cellernes evne til at reparere deres plasmamembran. Dette assay tilladt os at vise for første gang, at sphingomyelinase virker nedstrøms for Ca2 +-afhængig exocytose, da ekstracellulær tilsætning af enzymet fremmer genlukning af cellerne permeabiliseret i fravær af Ca2 +.

Lave koncentrationer af bakteriel sphingomyelinase (SM) rednings plasmamembran reparation i celler udtømte i lysosomale syre sphingomyelinase. Brug FM1-43 imaging assay vi tidligere viste, at celler, der mangler for lysosomale enzym ASM og udsat for SLO sårdannelse i nærvær af Ca2 + har en plasmamembran defekt reddes af ekstracellulær tilsætning af det rekombinante humane enzym 19. Da den menneskelige lysosomale ASM har en lav pH-optimum, undersøgte vi, om genluknin...

Watch this Scientific Journal Video about Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds at JoVE.com

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Levende billeddannelse af celler udsat for den lipofile farvestof FM1-43 muliggør en præcis bestemmelse af kinetikken, som poredannende toksiner er fjernet fra plasmamembranen. Det er et følsomt assay, der kan bruges til at vurdere kravene til Ca 2 +, sphingomyelinase og andre faktorer på plasma membran reparation.

Lev Imaging Assay for at vurdere de roller Ca<sup&gt; 2 +</sup&gt; Og sphingomyelinase i reparation af poredannende Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

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Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

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The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

Tomat / GFP-FLP / FRT Metode til Live Imaging af Mosaic Voksen<em&gt; Drosophila</em&gt; fotoreceptorceller

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipo-klar: En væv Clearing metode til tredimensional billeddannelse af fedtvæv

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...