Tæthedsgradient ultracentrifugering til undersøgelse af endoytisk genanvendelse i pattedyrceller
Summary
Dette papir har til formål at præsentere en protokol til forberedelse af genanvendelse endosomes fra pattedyr celler ved hjælp af saccharosetæthed gradient ultracentrifugering.
Abstract
Endosomal handel er en vigtig cellulær proces, der regulerer en bred vifte af biologiske begivenheder. Proteiner internaliseres fra plasmamembranen og transporteres derefter til de tidlige endoomer. De internaliserede proteiner kan overføres til lysosomet til nedbrydning eller genbruges tilbage til plasmamembranen. En robust endoytisk genbrugsvej er nødvendig for at afbalancere fjernelsen af membranmaterialer fra endokytose. Forskellige proteiner rapporteres at regulere vejen, herunder ADP-ribosylation faktor 6 (ARF6). Tæthedsgradient ultracentrifugering er en klassisk metode til cellefraktionering. Efter centrifugeringen er organeller sedimenteret på deres isopykiske overflade. Brøkene indsamles og bruges til andre downstream-applikationer. Beskrevet her er en protokol for at opnå en genanvendelse endosome-holdige fraktion fra transfected pattedyr celler ved hjælp af tæthed gradient ultracentrifugering. De isolerede fraktioner blev udsat for standard vestlige blotting for at analysere deres proteinindhold. Ved at anvende denne metode, identificerede vi, at plasmamembran målretning af opslugt og celle motilitet 1 (ELMO1), en Ras-relaterede C3 botulinum toksin substrat 1 (Rac1) guanine nukleotid udveksling faktor, er gennem ARF6-medieret endokritisk genanvendelse.
Introduction
Endosomal handel er en vigtig fysiologisk proces, der implicerer forskellige biologiske hændelser1, for eksempel transport af signalering receptorer, ion kanaler, og vedhæftning molekyler. Proteiner lokaliseret på plasmamembranen internaliseres ved endokytose2. De internaliserede proteiner sorteres derefter efter den tidlige endosome3. Nogle af proteinerne er målrettet mod lysosomer til nedbrydning4. Men en betydelig mængde proteiner genbruges tilbage til celleoverfladen ved hurtig genanvendelse og langsomme genanvendelsesprocesser. Ved hurtig genanvendelse forlader proteiner de tidlige endoomer og vender direkte tilbage til plasmamembranen. Omvendt sorteres proteiner først i langsom genanvendelse til det endoytiske genbrugsrum og transporteres derefter tilbage til plasmamembranen. Forskellige lastproteiner, for eksempel clathrin, retromer kompleks, retriever kompleks og Wiskott-Aldrich syndrom protein, og SCAR Homologue (WASH) kompleks, deltage i sådanne membran genanvendelse processer4,5,6,7,8,9. Balancen i endokytose og genanvendelse begivenhed er afgørende for celle overlevelse og bidrager til forskellige cellulære begivenheder10, for eksempel, celle vedhæftning, celle migration, celle polaritet, og signal transduktion.
ARF6, en lille GTPase, er en rapporteret regulator for endokritisk handel7,11,12. Af interesse har forskellige forskergrupper illustreret betydningen af ARF6 i endokritisk genanvendelse13,14,15,16,17. Undersøgelsen har til formål at undersøge forholdet mellem ARF6-medieret neurit udvækst og endokritisk genanvendelse. Den tidligere rapport tyder på, at aktiveringen af ARF6 er opstrøms til Rac1 aktivitet ved at handle på ELMO1-dedicator af cytokinesis 1 (DOCK180) kompleks18. Det er dog fortsat uklart, hvordan ARF6 udløser ELMO1-DOCK180-medieret Rac1-signalering. Density gradient ultracentrifugering blev anvendt til at undersøge den rolle, ARF6-medieret endokritisk genanvendelse i en sådan proces. Ved at bruge dette blev genanvendelses-endosome-holdige fraktion opnået fra cellelytter19. Fraktionen blev udsat for vestlig blotting for proteinindhold analyse. Immunoblot-resultaterne viste, at under tilstedeværelse af FE65, et hjerneberiget adapterprotein, øgede aktiv ARF6 betydeligt elmo1-niveauet i genanvendelses-endosomeholdig fraktion. Følgende protokol omfatter procedurerne for (1) transfecting pattedyrceller; 2) udarbejdelse af prøverne og densitetsgradientkolonnerne og (3) opnåelse af den genanvendelses-endosomeholdige fraktion.
Protocol
Representative Results
Discussion
Ovenstående protokol skitserer procedurerne for isolering af genanvendelsesenomer fra dyrkede celler ved ultracentrifugering. Pålideligheden af denne metode er blevet påvist ved den seneste publikation22, der beviser, at genanvendelse endosomes er med succes isoleret fra andre organeller (figur 1), såsom Golgi apparatet og mitokondrier. Nogle kritiske skridt skal være opmærksomme på for at opnå et godt adskillelsesresultat. Under tilberedningen af saccharoseop…
Declarações
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af midler fra Research Grants Council Hong Kong, CUHK direkte tilskudsordning, United College begavelse fond, og TUYF Charitable Trust. Tallene i dette arbejde er tilpasset fra vores tidligere publikation, “ARF6-Rac1 signalmedieret neurit udvækst er potentiated af neuronal adapter FE65 gennem orkestrering ARF6 og ELMO1” offentliggjort i FASEB Journal i oktober 2020.
Materials
| 1 mL, Open-Top Thickwall Polypropylene Tube, 11 x 34 mm | Beckman Coulter | 347287 | |
| 100 mm tissue culture dish | SPL | 20100 | |
| 13.2 mL, Certified Free Open-Top Thinwall Ultra-Clear Tube, 14 x 89 mm | Beckman Coulter | C14277 | |
| 5x Sample Buffer | GenScript | MB01015 | |
| cOmplete, EDTA-free Protease Inhibitor Cocktail | Roche | 11873580001 | |
| COX IV (3E11) Rabbit mAb | Cell Signaling Technology | 4850S | Rabbit monoclonal antibody for detecting COX IV. |
| Cycloheximide | Sigma-Aldrich | C1988 | |
| Dounce Tissue Grinder, 7 mL | DWK Life Sciences | 357542 | |
| Dulbecco's Modified Eagle Medium (DMEM) with low glucose | HyClone | SH30021.01 | |
| ELMO1 antibody (B-7) | Santa Cruz Biotechnology | SC-271519 | Mouse monoclonal antibody for detecting ELMO1. |
| EndoFree Plasmid Maxi Kit | QIAGEN | 12362 | |
| FE65 antibody (E-20) | Santa Cruz Biotechnology | SC-19751 | Goat polyclonal antibody for detecting FE65. |
| Fetal Bovine Serum, Research Grade | HyClone | SV30160.03 | |
| GAPDH Monoclonal Antibody (6C5) | Ambion | AM4300 | Mouse monoclonal antibody for detecting GAPDH. |
| ImageLab Software | Bio-Rad | Measurement of band intensity | |
| Imidazole | Sigma-Aldrich | I2399 | |
| Lipofectamine 2000 Transfection Reagent | Invitrogen | 11668019 | |
| Monoclonal Anti-β-COP antibody | Sigma | G6160 | Mouse monoclonal antibody for detecting β-COP. |
| Myc-tag (9B11) mouse mAb | Cell Signaling Technology | 2276S | Mouse monoclonal antibody for detecting myc tagged proteins. |
| OmniPur EDTA, Disodium Salt, Dihydrate | Calbiochem | 4010-OP | |
| Optima L-100 XP | Beckman Coulter | 392050 | |
| Optima MAX-TL | Beckman Coulter | A95761 | |
| Opti-MEM I Reduced Serum Media | Gibco | 31985070 | |
| PBS Tablets | Gibco | 18912014 | |
| PhosSTOP | Roche | 4906845001 | |
| RAB11A-Specific Polyclonal antibody | Proteintech | 20229-1-AP | Rabbit polyclonal antibody for detecting Rab11. |
| Sucrose | Affymetrix | AAJ21931A4 | |
| SW 41 Ti Swinging-Bucket Rotor | Beckman Coulter | 331362 | |
| TLA-120.2 Fixed-Angle Rotor | Beckman Coulter | 362046 | |
| Trypsin-EDTA (0.05%), phenol red | Gibco | 25300062 |
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