En celle Gratis Assay til at studere Chromatin dekondensering i slutningen af Mitose
Summary
The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.
Abstract
During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.
Introduction
Xenopus laevis æg ekstrakt er en kraftfuld og almindeligt anvendt værktøj til at studere komplicerede cellulære begivenheder i enkelhed et cellefrit assay. Siden deres første beskrivelse af Lohka & Masui 1 de er blevet grundigt anvendt til at undersøge mitoseprocesser såsom chromatinkondensation 2, spindelenhed 3, opdeling 4 kernemembranen, men også nucleocytoplasmic transport 5 eller DNA-replikation 6. De begivenheder, der finder sted i slutningen af mitose, der er nødvendige for gendannelse af interphasic kerne såsom reformation kernemembranen og kerneporen kompleks samling er langt mindre forstået forhold til begyndelsen af mitotiske begivenheder, men kan på lignende måde undersøgt ved anvendelse af Xenopus æggeekstrakt 7. Vi har for nylig etableret en analyse baseret på Xenopus æg ekstrakt for at studere kromatin dekondensering i slutningen af mitose 8, en under-undersøgt proces, der venter its detaljeret karakterisering.
I metazoans, er kromatin meget kondenseret ved mitotisk post med henblik på at udføre trofast adskillelse af det genetiske materiale. For at sikre at kromatin er tilgængelig for genekspression og DNA-replikation under interfase, skal det de-komprimeres i slutningen af mitose. I hvirveldyr, chromatin er op til halvtreds gange mere komprimeret under mitose i forhold til interfase 9, i modsætning til gær, hvor mitotiske komprimering er normalt meget lavere, f.eks kun to gange i S. cerevisiae 10. Hvirveldyr chromatin dekondensering det meste har været undersøgt i forbindelse med sperm DNA-reorganisation efter ægbefrugtning. En molekylære mekanisme, ved hvilken nucleoplasmin, en rigelig oocyt protein, udveksler sperm-specifikke protaminer til histoner H2A og H2B er lagret i ægget. Denne proces blev også belyst ved hjælp af Xenopus æggeekstrakt 11, 12. Men udtrykketaf nucleoplasmin er begrænset til oocytter 13 og mitotiske kromatin indeholder ikke disse sperm-specifikke protaminer. Derfor chromatin dekondensering i slutningen af mitose er nucleoplasmin uafhængig 8.
Til in vitro dekondensering reaktion beskæftiger vi ekstrakt genereret fra aktiverede X. laevis æg og kromatin klynger isoleret fra synkroniserede HeLa-celler. Behandling af æg med en calciumionophor efterligner calcium udsætning i ægget genereret af sæd post under befrugtningen. Calcium bølge udløser cellecyklus genoptagelse og ægget, anholdt i den anden metafase af meiose, udvikler sig til den første interfase 14. Derfor æg ekstrakter fremstillet danner aktiverede æg repræsenterer mitotiske exit / interfase tilstand og er kompetente til at fremkalde begivenheder specifikke for mitotiske exit ligesom chromatin dekondensering, nuklear kuvert og pore komplekse reformation. Til isolering af mitotiske lmromatin klynger vi brugte en let modificeret version af protokollen udgivet af Gasser & Laemmli 15, hvor kromosom klynger frigives ved lysis fra HeLa-celler synkroniseret i mitose og isolerede i polyamin indeholdende buffere ved gradient centrifugeringer.
Protocol
Representative Results
Discussion
Xenopus laevis æg ekstrakter er et meget nyttigt redskab til at gengive cellulære processer in vitro, og dette system blev med succes anvendt i karakteriseringen af cellecyklus og celledeling begivenheder 2, 3,5,6,17. På grund af store lagre af nukleare komponenter afsondret i ægget under oogenese, æg ekstrakter er en glimrende kilde til cellulære komponenter. Sammenlignet med andre tilgange som RNAi om pattedyrvæv cellelinjer eller genetisk manipulation, det giver flere fo…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af den tyske Research Foundation og ERC (AN377 / 3-2 og 309.528 CHROMDECON til WA) og en ph.d.-Eventyret om Boehringer Ingelheim Fonds til AKS Figur 1 & 2 er genoptrykt fra Developmental Cell 31 (3), Magalska et al., RuvB-lignende ATPaser funktion i kromatin dekondensering i slutningen af mitose, 305-318, 2014, med venlig tilladelse fra Elsevier.
Materials
| spermine tetrahydrochloride | Fluka analytical | 85610-25G | |
| spermidine trihydrochloride | Sigma | S2501-5G | |
| high-purity digitonin | Millipore | 300410-1GM | toxic |
| PMSF | Applichem | A0999,0100 | toxic |
| thymidine | Calbiochem | 6060 | |
| nocodazole | Calbiochem | 487928 | toxic |
| 37 % formaldehyde solution | Roth | 7398-1 | toxic |
| trypan blue solution (0.4%) | Sigma | T8154 | toxic |
| 1,4-dithiothreitol (DTT) | Roth | 6908.2 | |
| AEBSF hydrochloride | Applichem | A1421,0001 | |
| pepstatin | Roth | 2936.1/2/3 | |
| leupeptin | Roth | CN334 | |
| aprotinin | Roth | A162.3 | |
| Percoll (colloidal silica particles solution) | GE Healthcare | 17-0891-01 | |
| glutamine | Gibco | 25030-024 | |
| Penicillin-Streptomycin | Gibco | 15140-122 | |
| 75 cm² tissue culture flasks | Greiner Bio-one | 658175 | |
| heat-inactivated fetal bovine serum (FBS) | Gibco | 10500-064 | |
| Homogenizer (40 mL tissue grinder) | Wheaton | 357546 | |
| Neubauer chamber | Assistent | 441/1 | |
| Oak Ridge Centrifuge Tubes, polycarbonate (50 ml) | Nalgene | 3118-0050 | |
| 100 µm cell strainer, nylon | BD Falcon | 352360 | |
| cytochalasin B | Applichem | A7657,0010 | toxic |
| cycloheximide | Roth | 8682.3 | toxic |
| L-cystein | Merck | 1,028,381,000 | |
| hCG available as Ovogest | MSD | 1431593 | |
| PMSG available as Intergonan | MSD | 1431015 | |
| A23187 (mixed calcium-magnesium-salt) | Enzo | ALX-450-002-M010 | toxic |
| syringe needles (1.20 x 40 mm, 18 G x 1 1/2") | Braun | 4665120 | |
| ATP | Serva | 10920.03 | |
| GTP, 2 Na x 3 H20 | Roth | K056.1/2/3/4 | |
| creatine phosphat disodium salt | Calbiochem | 2380 | |
| creatine phosphokinase | Sigma | C3755-35KU | |
| DMAP | Sigma | D2629-1G | |
| DAPI | Roche | 10236276001 | |
| PFA | Sigma | P-6148 | toxic |
| centrifugation tubes for TLA 100 (7 x 10 mm, 5/16 x 13/16 in.) | Beckman Coulter | 343775 | |
| "Cell-Saver" (tips with wide opening, 1000 µL) | Biozym | 729065 | |
| 50 % glutaraldehyde solution, grade I | Sigma alderich | G7651-10 mL | toxic |
| 0.1 % (w/v) poly-L-lysine solution | Sigma | P8920-100 mL | |
| flat-bottom tubes (6 mL, 16.0/55 mm) | Greiner Bio-one | 145211 | |
| Vectashield mounting medium | Vector laboratories | H1000 | |
| tubes for TLA120 (11 x 34 mm, 7/16 x 1 3/8 in.) | Beckman Coulter | 343778 | |
| "Cell-Saver" (tips with wide opening, 200 µL) | Biozym | 729055 | |
| 12 mm coverslips | Thermo Scientific | 0784 #1 |
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