Name: a faster, high resolution, mtpa-gfp-based mitochondrial fusion assay acquiring kinetic data of multiple cells in parallel using confocal microscopy
Uploaded: 2012-07-20T12:00:00
Duration: 10 min 45 s
Description: Watch this Scientific Journal Video about A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy at JoVE.co

Vi takker Tufts Center for Neuroscience Research, P30 NS047243 (Jackson), mitokondrierne Affinity Research Collaborative (mtARC) støttet af Evans Center for Interdisciplinary Biomedical Research ved Boston University Medical Campus, Link Medicine Corporation, og Zeiss for at støtte dette arbejde.

1. Billede Plate Forberedelse <ol><li> Kultur INS1 celler i RPMI-medier indeholdende 10% standard føtalt bovint serum, 1% penicillin-streptomycin, 2 mM L-glutamin, 50 uM 2-mercaptoethanol, 5 mM NaHCO3, 2 mM HEPES, 2 mM pyrodruesyre og 11 mM glucose til 80% konfluens. </li><li> Trypsinisér de INS1 cellekulturer med 0,05% trypsin og pladen dem på poly-D-lysin-overtrukne dækglas bund billeddannende plader (30-40% konfluens). </li><li> Mulighed for pladerne at nå 60-80% konfluens (ca. 2 dage), og der tilsættes mitokondriematriksen målrettet (COXVIII) adenoviral PA-GFP i 24 timer (MOI = 5). Udvekslingsmediet og tillader celler at vokse i yderligere 2 dage før billeddannelse. </li><li> På dagen for billeddannelse tilsættes 7-15 nM TMRE til billeddannende pladerne, og ækvilibrere i mindst 45 min. </li><li> I løbet af denne tid sving på inkubatoren (trin top inkubator er blevet anvendt her) og tillade mikroskop for at ækvilibrere til 37 ° C i omkring 1 time. Tænde 5% CO2. </li&gt; </ol> 2. Imaging Parametre for Zeiss LSM 710 konfokal mikroskop <ol><li> Efter mikroskop har ligevægt under Acquisition fanen, klik på &quot;Vis manuelle værktøjer&quot;, kan åbne Imaging oprette panel, og vælg &quot;kanal mode&quot; og skiftespor hver &quot;frame&quot;. </li><li> I lyset stien panelet, vælge LSM og kanal-mode. Arbejde fra prøven ikonet opad, skal du vælge &quot;bagside&quot;, MBS 690 + og MBS 488. Ved bunden af ​​panelet, vælg &quot;T-PMT&quot; at muliggøre visualisering af lyse område eller DIC kanal. </li><li> Slut tilpasning lysbanen parametre ved at intervallet for GFP farvestoffet til 490-540 nm og 580-700 nm for TMRE farvestof. </li><li> I købet tilstand, skal du bruge en 512 × 512 pixel scannings felt, gennemsnitlige 4x, og vælg en zoom faktor (som du måske ønsker at holde konsekvent til kalibreringsformål). </li></ol> 3. Optimering af Imaging Parametre <ol><li> Brug af planen apochromat 100x (1,4 NA) objektiv, FOCos om dine celler med halogen lys, ikke fluorescerende lys, for at beskytte mitokondrier fra fototoksicitet. </li><li> Screening for PA-GFP-udtrykkende celler under anvendelse af en maksimalt åbent hul og scanning for at finde de celler, der er lysere grøn. </li><li> Finde den laveste kraft 2-foton laser for at aktivere PA-GFP og optimere de billeddannende parametre, der sikrer, at signalet ikke mætte detektoren. Kontroller, at PA-GFP signal colocalizes med TMRE signal for et par z-serie. Tab af TMRE signalet angiver mitokondrisk depolarisering eller fototoksicitet, og cellerne udviser disse karakteristika bør ikke anvendes til analyse. </li><li> Find en PA-GFP-udtrykkende celler og angive en zoomfaktor. </li><li> Indstilles z-serien området at indsamle 6 skiver (dette interval skal tilfredsstille alle 10 celler med mindre der specifikt z-fokus er indstillet ved hver position). </li><li> Gem billeddannende metode for hver celle (celle 1, celle 2 osv.). </li></ol> 4. Juster Automatiserd del af programmet og udpege de 10 celler, du vil følge for 1 time Mitokondriel Fusion Assay <ol><li> På venstre panel af multitime vinduet, skal du vælge &quot;besparelse&quot; panel, og angiv den placering, hvor filerne skal gemmes. </li><li> I &quot;Erhvervelsen&quot; panel, indlæse overtagelsesmetoden gemt til celle 1 i scanningskonfigurationen og kontrollere z stak kassen. Vælg også &quot;mærket Z-midten af ​​z stakken&quot;. </li><li> I &quot;blokke&quot; panel, vælg &quot;enkelt blok på hvert sted&quot;. Klik på &quot;Tilføj blokke&quot; for hvert interval, der skal måles. </li><li> I &quot;timing&quot; panel, vælg &quot;vent interval&quot; og skriv &quot;0&quot; i &quot;vente interval før blok først beliggenhed kun&quot;. Blokke 2-4 vil have &quot;15 minutter&quot; i dette afsnit. </li><li> I stedet panelet, vælg &quot;flytte fokus for at indlæse position mellem steder&quot; og &quot;load scanne config når &#39;flytter til loc&#39; eller &#39;Næste LOC&quot; klikkede. Under &quot;Rediger placeringer liste&quot; vælge &quot;Slet alt&quot; og derefter vælge &quot;multiple steder motoriseret fase &quot;. </li><li> Undis den &quot;Bleach&quot; panel, skal du klikke på &quot;blege&quot; boks, og udpege konfigurationsfilen i &quot;config&quot; pull down menu, som angivet i vinduet af de vigtigste software. Så i &quot;blegning&quot; vinduet, skal du gemme den relevante fotoaktivering metode. I regioner panelet, vælge ROI for netop denne celle, og tilføje dette ROI til multitime ved at vælge &quot;add aktuelle område til ROI liste&quot; vinduet. Sørg for at vælge den samme placering i &quot;ROI&quot; pull down menuen. </li></ol> For timingen kunne fungere, og for hver celle har en 15 minutters interval, to metoder skal gemmes. På listen over blokke, vil den første har den &quot;rigtige&quot; fotoaktivering konfiguration, og resten vil have en &quot;mock&quot; konfiguration, der ikke bruger to-foton laser. Den første blok er også den eneste blok, der ikke har en forsinkelse (BKIntv = 0). Derfor fremgangsmåden begynder med en baselinescanning, efterfulgt af en fotoaktivering scanning. Resten af ​​blokkene har en 900 sek BkIntv, ogved hvert tidspunkt er der to skanninger lige så på tidspunktet 0 sek at opretholde timing konsistens. <ol start="7"><li> For hver af de 10 celler, der skal følges med tiden udfører denne sekvens: Find en PAGFP udtrykker celle-redde sit imaging metode Placering panel-markere scenen position, skal du angive billeddannende metode Main ROI panel-vælg et område af interesse at være fotoaktiveret Bleach panel-gemme specifikke ROI og også lægge det i fokusområdet: Slet ROI og nulstille scanningen zoom til 1 Find den næste celle, og indstille zoom Gentag processen for de næste 9 celler </li></ol> Kontroller, at hvert trin placering og alle blokke have passende billeddannende metode (scanningskonfigurationen) tilknyttet. Sørg også for, at den første blok på hvert sted svarer til den relevante fotoaktivering metode, mens resten indeholder en &quot;mock&quot;-metoden. Endelig skal du vælge &quot;kør&quot;. 5. Analyser PA-GFP signalintensitet <ol><li&gt; For de celler, hvor fotoaktiverbare-GFP-signal colocalizes med det røde TMRE signal, baggrunden i PAGFP billederne (her bruger vi metamorph) trækker. </li><li> Så eksportere data til Excel og beregne den gennemsnitlige intensitet for z-stakke på hvert tidspunkt. Skille optiske sektioner, der intet signal. </li><li> Måling af signalet fortynding i hvert z-stak ved at beregne procentdelen af ​​den oprindelige fotoaktiveret signal. </li></ol> Hvert datapunkt er scannet og registreret to gange, hvilket resulterer i duplikat z-stakken information for hvert tidspunkt. Kontroller, at z-stak værdier enige. Hvis ikke, hvilket er indikativt for et problem (f.eks fokus, cellebevægelse). 6. Repræsentative resultater Når en fusion begivenhed indtræffer mellem en aktiveret og ikke-aktiverede PA-GFP mitochondriet er PAGFP i mitokondriematriksen blandes med den ikke-mærkede matrix og bliver fortyndet over et større område, faldende signalIntensiteten (figur 1A). I INS1 cellen forekommer et signifikant fald i signalintensitet hvert 15. minut, indtil ækvilibrering af mitokondriel fusion er nået (ca. 1 time). Bemærk, at cellen i figur 1B viser næsten fuldstændig colokalisering af PA-GFP og TMRE signaler. I disse assays en meget lav koncentration af TMRE (15 nM) bruges til at hjælpe målrette fotoaktivering af PAGFP, og også at overvåge celle sundhed. Celler med en overflod af depolariseret mitokondrier vil have ufuldstændig colokalisering af PAGFP og TMRE og skal ikke analyseres, da dette indikerer enten fototoksicitet, eller celler i en døende tilstand. Ligevægtstidspunktet for mitokondrie fusion er sædvanligvis en time i INS1 celler, når ~ 15% af den mitokondrielle volumen aktiveret. Tider, selv om en lille område belyst fleste af mitokondrier blive fotoaktiveret grund til at være meget netværk, i hvilket tilfælde yderligere fusion er vanskeligt at detektere. Andre celletyper kan udvise forskellige ækvilibreringstider og skal testes med kortere intervaller, og over en længere periode til at karakterisere mitokondrie dynamik. At inhibere mitokondrie fusion, kan cellerne anbringes i et lipotoxic miljø. Det er tidligere blevet påvist, at 0,4 mM palmitat fragmenter mitokondrier, og inhiberer mitokondriel fusion 9. Denne virkning kan ses i figur 2, hvor mitokondrier er fragmenteret, men signalet intensitet mtPAGFP ikke ændrer så meget som under normale betingelser (figur 1). Derfor er fortynding af PA-GFP signal sandsynligvis skyldes mitochondrial fusionsprotein end fission. I andre celletyper, foreslår vi at anvende andre kendte måder til at inducere mitochondrial fragmentering, såsom silencing OPA1 som er nødvendig for mitokondrie fusion 14. <img alt="Figur 1" src="/files/ftp_upload/3991/3991fig1.jpg" /> Figur 1. </strong&gt; Typisk fortynding af PA-GFP signal efter fotoaktivering i et mitokondrie fusion assay. A. PA-GFP bliver gradvist svagere grund mitochondriale fusionsproteiner begivenheder, der fører til fortynding af protein over en større areal som det kan ses i disse projicerede billeder 6 optiske sektioner kvantificeres hver 15 minutter. B. TMRE med PA-GFP viser, at mitokondrier er aktive og ikke depolariseret. <a href="http://www.jove.com/files/ftp_upload/3991/3991fig1large.jpg" target="_blank">Klik her for at se større figur</a> . <img alt="Figur 2" src="/files/ftp_upload/3991/3991fig2.jpg" /> Figur 2. Mitochondrial fusion inhiberes med 0,4 mM palmitat nedsætter fortynding af PA-GFP. A. projektioner af 6 optiske sektioner viser kort mitokondrier med et uforanderligt signalintensitet over tid. B. colokalisering af PA-GFP med TMRE viser, at mitokondrier er ikke depolariseret. <a href="http://www.jove.com/files/ftp_upload/3991/3991fig2large.jpg" target="_blank">Klik her for at se større figur</a> .

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Denne fremgangsmåde giver mulighed for billeddannelse på omkring 10 celler ad gangen, hvis erhvervelsen sker hvert 15. minut efter fotoaktivering. Det nøjagtige antal celler vil afhænge af, hvor hurtigt man er i stand til at finde og markere mtPAGFP udtrykker cellerne i kultur fad, og hvor hurtigt man kan oprette alle de software parametre. For at gøre automatisering køre gnidningsløst et jævnt lag af celler, bør anvendes, fordi de udpegede z-stak margener vil gælde for alle celler. Størrelsen af ​​dette første fotoaktivering område vil styre ekvilibreringstid. At kunne måle mitochondrial fusion, er det vigtigt at photoactivate kun 10-20% af netværket, således at spredningen af ​​signalet til resten af ​​netværket kan overvåges over tid. Hvis for meget af netværket fotoaktiveret, er det muligt at fuldstændig sammensmeltning vil ske for hurtigt, og arrangementet vil ikke blive fanget. Den yderste forsigtighed skal træffesjustere lasereffekt af to-foton laser samt TMRE koncentrationen at undgå fototoksicitet, hvilket fører til mitokondriel depolarisering. Sikring af, at mtPAGFP signal colocalizes med TMRE signalet kan hjælpe med at vurdere fototoksicitet og generelle celle sundhed 8,15. Belysning med epifluoerescent lys bør undgås. Når du søger efter celler, der udtrykker mtPAGFP, bør pinhole være maksimalt åben mens den scanner med et lavt effektforbrug 488 nm excitation. Justering af to-foton lasereffekt på photoactivate nok PA-GFP til at måle signalet i løbet af 1 time, men ikke til oversaturate nogen af cellerne kan være vanskeligt 8. Dog skal tiden bruges på denne optimering trin, fordi når automatiseret program er startet, er det trættende at stoppe det, for at vælge flere celler, og genoptage. For kvalitetskontrol, billede (eller transmitteret lys) at overvåge fokus på cellerne erhvervelse af en differentieret interferens kontrast (DIC), kan være megethjælpsomme og også en god måde at opdage bobler i fordybelse olien under scanning, og dette undertiden sker fra bevægelser på scenen. Selv med denne mtPAGFP fremgangsmåde indsamler data på den ensrettede bevægelse af mitochondriale matrixproteiner fra fotoaktiveret mitokondrier til dem, der ikke er mærket, er det tænkeligt at anvende denne teknik til at undersøge andre processer. For eksempel kan specifikke fluorochromer fastgøres til membranproteiner at observere deres specifikke bevægelse under fusionsbegivenheder, som det er blevet vist for ABC-me, fusion som forekommer på en anden skala fra en blanding af opløselige matrixproteiner 15.

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> COXIII-adenoviral PA-GFP </td><td> Dr. Lippincott-Schwartz </td><td></td></tr><tr><td> TMRE </td><td> Invitrogen </td><td> T669 </td></tr><tr><td> Zeiss LSM 710 konfokalt </td><td> Zeiss </td><td></td></tr></tbody></table>

a faster, high resolution, mtpa-gfp-based mitochondrial fusion assay acquiring kinetic data of multiple cells in parallel using confocal microscopy

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment.
Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis1,2,3,13. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks4,10,13. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function18. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust14. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis9. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary.
Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay7, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay1,5. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process 4,5. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment.
A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP)6,11. Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal6. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE9,15. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE.
The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity8,15,16,17.
In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15nM TMRE8 in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.

Watch this Scientific Journal Video about A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy at JoVE.co

A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

Mitokondrie fusion blev målt ved at spore ækvilibrering af photoconverted matrix-målrettet GFP over mitokondriske netværket over tid. Hidtil kunne kun en celle udsættes for en time lange kinetisk analyse ad gangen. Vi præsenterer en metode, der samtidigt måler flere celler, og dermed fremskynde indsamlingen af ​​data.

En hurtigere, høj opløsning, MTPA-GFP-baserede Mitokondriel Fusion Assay Erhvervelse Kinetiske data i flere celler i parallel anvendelse af konfokal mikroskopi

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in ...

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