Måling Protein Stabilitet i Living zebrafisk embryoner anvendelse af fluorescens Decay Efter fotoomdannelse (FDAP)
Summary
Protein niveauer i celler og væv er ofte stramt reguleret af balance proteinproduktion og clearance. Anvendelse af fluorescens Decay Efter fotoomdannelse (FDAP) kan clearance kinetik proteiner eksperimentelt målt in vivo.
Abstract
Protein stabilitet påvirker mange aspekter af biologi, og måling af clearance kinetik proteiner kan give vigtig indsigt i biologiske systemer. I FDAP eksperimenter, kan måles clearance af proteiner i levende organismer. Et protein af interesse er mærket med en photoconvertible fluorescerende protein udtrykkes in vivo og photoconverted, og faldet i photoconverted signal over tid overvåges. Dataene bliver derefter forsynet med et passende clearance model til at bestemme protein halveringstid. Vigtigt er det, kan clearance kinetikken for protein befolkninger i forskellige rum i organismen undersøges separat ved anvendelse af rumlige masker. Denne fremgangsmåde er blevet anvendt til at bestemme de intra- og ekstracellulære halveringstider af secernerede signalproteiner under zebrafisk udvikling. Her beskriver vi en protokol for FDAP eksperimenter i zebrafisk embryoner. Det bør være muligt at anvende FDAP at bestemme clearance kinetikenhver taggable protein i enhver optisk tilgængelig organisme.
Introduction
Niveauerne af proteiner i celler og organismer bestemmes af deres satser produktions- og clearance. Protein halveringstider kan variere fra minutter til dage 1-4. I mange biologiske systemer, stabilisering eller clearance af vigtige proteiner har vigtige virkninger på cellulær aktivitet. Modulation af intracellulær proteinstabilitet er påkrævet for cellecyklusprogression 5,6, udviklingsmæssige signalering 7-9, apoptose 10 og normal funktion og vedligeholdelse af neuroner 11,12. Ekstracellulært protein stabilitet påvirker fordelingen og tilgængeligheden af udskilte proteiner, såsom morfogener 13,14, inden et væv.
I de seneste årtier har proteinstabilitet primært blevet vurderet i cellekultur ved hjælp radioaktiv puls-mærkning eller cycloheximid chase eksperimenter 15. I sådanne puls-chase forsøg celler enten transient udsættes for en "puls" af radioaktivt aminosyreforløberne der er inkorporeret i nyligt syntetiserede proteiner, eller de udsættes for cycloheximid, hvilket inhiberer proteinsyntese. Dyrkede celler opsamles derefter på forskellige tidspunkter, og enten immunopræcipitation efterfulgt af autoradiografi (i radioaktive puls-chase forsøg) eller western blotting (i cycloheximid forsøg) anvendes til at kvantificere clearance af protein over tid.
Konventionelle proteinstabilitet assays har flere mangler. Først proteiner i disse assays er ofte ikke udtrykkes i deres endogene miljøer, men snarere i vævskultur og undertiden i celler fra forskellige arter. For proteiner, hvis stabilitet er kontekstafhængig, denne fremgangsmåde er problematisk. For det andet er det ikke muligt at følge protein clearance i individuelle celler eller organismer over tid, og dataene fra disse assays afspejler et gennemsnit af forskellige populationer af celler på forskellige tidspunkter. Da de enkelte celler kan have startetmed forskellige mængder af protein, kan have taget op det radioaktive etiket eller cycloheximid på forskellige tidspunkter, eller kan have forskellige clearance kinetik, sådanne aggregerede data kan være vildledende. Endelig, i tilfælde af cycloheximid chase eksperimenter tilsætning af proteinsynteseinhibitor kan have utilsigtede fysiologiske virkninger, som kan kunstigt ændre proteinstabilitet 16-18. Disse mangler kan undgås ved hjælp af fluorescens Decay Efter fotoomdannelse (FDAP), en teknik, der udnytter photoconvertible proteiner til at måle protein clearance dynamisk i levende organismer 19-25 (se Diskussion for begrænsninger i FDAP teknik).
Photoconvertible proteiner er fluorescerende proteiner, hvis excitation og emission egenskaber ændres efter udsættelse for bestemte bølgelængder af lys 26. Et almindeligt brugt variant er Dendra2, en "grøn-til-rød" photoconvertible protein, der i første omgang har excitation og emissions- egenskaber svarende til grønt fluorescerende proteiner, men efter eksponering for UV-lys- "fotoomdannelse"-dens excitation / emission egenskaber bliver ligner dem af røde fluorescerende proteiner 23,27. Vigtigt er det, vil nye protein produceret efter fotoomdannelse ikke har de samme excitation / emission egenskaber som den photoconverted protein, så afkobling af produktion og clearance ved fotoomdannelse og observation af kun en pulje af photoconverted protein. Tagging proteiner af interesse med photoconvertible proteiner således giver en bekvem måde at pulse-label proteiner i intakte, optisk tilgængelige levende organismer.
I FDAP assays (figur 1A), er et protein af interesse mærket med et photoconvertible protein og udtrykkes i en levende organisme (figur 1B). Fusionsproteinet er photoconverted, og faldet i photoconverted signal over tid overvåges af fluorescence mikroskopi (figur 1C). Dataene bliver derefter forsynet med en passende model for at bestemme halveringstiden af fusionsproteinet (figur 1D).
Den FDAP assay beskrevet her var designet til at bestemme de ekstracellulære halveringstider af secernerede signalproteiner i zebrafisk embryoner under tidlig embryogenese 19. Imidlertid kan denne fremgangsmåde tilpasses til en hvilken som helst transparent model organisme, som tolererer levende billeddannelse, og kan bruges til at overvåge clearance af enhver taggable intracellulært eller ekstracellulært protein. Variationer af den her beskrevne teknik er blevet udført i dyrkede celler 20,23 og Drosophila 22 og mus 21 embryoner.
Protocol
Representative Results
Discussion
Succesen med en FDAP eksperiment afhængig af generering af et funktionelt photoconvertible fusionsprotein. Mærkning af et protein kan påvirke dets biologiske aktivitet og / eller biofysiske egenskaber, herunder dens lokalisering, opløselighed og stabilitet 36-41. Vær forberedt på at teste aktiviteten af flere forskellige fusionskonstruktioner for at finde en, der er aktiv. Vi har fundet, at ændre positionen af photoconvertible protein i forhold til proteinet af interesse eller ved hjælp af læng…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne vil gerne takke Jeffrey Farrell, James Gagnon, og Jennifer Bergmann for kommentarer til manuskriptet. KWR blev støttet af National Science Foundation Graduate Research Fellowship Program under udviklingen af den FDAP analysen. Dette arbejde blev støttet af tilskud fra NIH til AFS og med tilskud fra den tyske Research Foundation (Emmy Noether Program), Max Planck Society, og Human Frontier Science Program (Career Development Award) til PM.
Materials
| PyFDAP (download from the following website: http://people.tuebingen.mpg.de/mueller-lab) | Install and operate using the instructions provided on the PyFDAP website; PyFDAP is compatible with Linux, Mac, and Windows operating systems. | ||
| mMessage mMachine Sp6 Transcription Kit (Life Technologies, AM1340) | To generate capped mRNA for injection into embryos | ||
| Alexa488-dextran conjugate, 3 kDa (Life Technologies, D34682) | Co-inject with mRNA to create intracellular and extracellular masks | ||
| 6-well plastic dish (BD Falcon) | Incubate embryos in agarose-coated wells until ready for mounting | ||
| Embryo medium | 250 mg/l Instant Ocean salt, 1 mg/l methylene blue in reverse osmosis water adjusted to pH 7 with NaHCO3 | ||
| Protease from Streptomyces griseus (Sigma, P5147) | Make a 5 mg/ml stock and use at 1 mg/ml to dechorionate embryos at the one-cell stage | ||
| 5 cm diameter glass petri dish | For embryo dechorionation | ||
| 200 ml glass beaker | For embryo dechorionation | ||
| Microinjection apparatus | For injection of mRNA and dye into embryos at the one-cell stage | ||
| Stereomicroscope | For injecting and mounting embryos | ||
| 1x Danieau's medium | Dilute low melting point agarose and perform imaging in this medium; recipe: 0.2 mm filtered solution of 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.3 mM CaCl2, 5 mM HEPES pH 7.2 | ||
| UltraPure low melting point agarose (Invitrogen, 16520-100) | For mounting embryos; use at a concentration of 1% in Danieau's medium: add 200 mg to 20 ml Danieau's medium, microwave until dissolved, then aliquot 1 ml into microcentrifuge tubes; aliquots can be stored at 4 °C, re-melted at 70 °C, and cooled to 40 – 42 °C when ready to use | ||
| Glass Pasteur pipette (Kimble Chase (via Fisher), 63A53WT) | For mounting embryos; flame the tip to prevent jagged edges from injuring embryos | ||
| Metal probe | For positioning embryos during mounting | ||
| Glass bottom dishes (MatTek, P35G-1.5-14-C) | Use the appropriate cover glass thickness for your objective; part number listed here is for cover glass No. 1.5 | ||
| 15 ml tube (BD Falcon) filled with ~5 ml embryo medium | For rinsing residual agarose from the Pasteur pipette | ||
| Inverted laser scanning confocal microscope | A mercury arc lamp, 488 nm laser, 543 nm laser, and the appropriate filter sets are required | ||
| Heated stage | To maintain embryos at the optimal temperature of 28 °C during the experiment | ||
| Confocal software capable of time-lapse imaging | Must be able to define multiple positions and automatically image them at defined intervals | ||
| 25x or 40x water objective | Objective for imaging | ||
| 10x air objective | Objective for photoconversion | ||
| Immersion oil | Immersion oil with the same refractive index as water |
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