Name: two- and three-dimensional live cell imaging of dna damage response proteins
Uploaded: 2012-09-28T12:00:00
Description: Watch this Scientific Journal Video about Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins at JoVE.com

Delvist understøttet af R01NS064593 og R21ES016636 (KV). Mikroskopi blev udført ved VCU - Neurobiologisk Afdeling &amp; Anatomi Microscopy Facility, støttet til dels med støtte fra NIH-NINDS center kerne tilskud 5P30NS047463. Den spinning disk konfokal mikroskop blev købt med en NIH-NCRR prisen (1S10RR027957).

A. Cell Preparation <ol><li> Normale humane primære fibroblaster (GM02270) blev opnået fra Coriell Cell Repository, Camden, New Jersey, og immortaliseret med hTERT 6. Celler blev dyrket og ekspanderet i CellStar 6-cm skåle i mediet (4 ml) bestående af MEM suppleret med 20% føtalt bovint serum (GIBCO), non-essential/essential aminosyrer, vitaminer, natriumpyruvat, og penicillin / streptomycin (HyClone ). </li><li> Human embryonisk nyre 293 (HEK293) celler blev opnået fra American Type Culture Collectio...

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	<li>Botuyan, M. V., Lee, J., Ward, I. M., Kim, J. E., Thompson, J. R., Chen, J., Mer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+the+methylation+state-specific+recognition+of+histone+H4-K20+by+53BP1+and+Crb2+in+DNA+repair.">Structural basis for the methylation state-specific recognition of histone H4-K20 by 53BP1 and Crb2 in DNA repair.</a> Cell. 127, 1361-1373 (2006).</li><li>Dimitrova, N., Chen, Y. C., Spector, D. L., de Lange, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=53BP1+promotes+non-homologous+end+joining+of+telomeres+by+increasing+chromatin+mobility.">53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility.</a> Nature. 456, 524-528 (2008).</li><li>Feuerhahn, S., Egly, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+to+study+DNA+repair:+what's+in+the+box.">Tools to study DNA repair: what's in the box.</a> Trends Genet. 24, 467-474 (2008).</li><li>Giunta, S., Belotserkovskaya, R., Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+signaling+in+response+to+double-strand+breaks+during+mitosis.">DNA damage signaling in response to double-strand breaks during mitosis.</a> J. Cell Biol. 190, 197-207 (2010).</li><li>Giunta, S., Jackson, S. 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Ther. 8, 730-738 (2009).</li><li>Huyen, Y., Zgheib, O., Ditullio, R. A., Gorgoulis, V. G., Zacharatos, P., Petty, T. J., Sheston, E. A., Mellert, H. S., Stavridi, E. S., Halazonetis, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylated+lysine+79+of+histone+H3+targets+53BP1+to+DNA+double-strand+breaks.">Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks.</a> Nature. 432, 406-411 (2004).</li><li>Jackson, S. P., Bartek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA-damage+response+in+human+biology+and+disease.">The DNA-damage response in human biology and disease.</a> Nature. 461, 1071-1078 (2009).</li><li>Kanda, T., Sullivan, K. F., Wahl, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone-GFP+fusion+protein+enables+sensitive+analysis+of+chromosome+dynamics+in+living+mammalian+cells.">Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells.</a> Curr. Biol. 8, 377-385 (1998).</li><li>Massignani, M., Canton, I., Sun, T., Hearnden, V., Macneil, S., Blanazs, A., Armes, S. P., Lewis, A., Battaglia, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+fluorescence+imaging+of+live+cells+by+effective+cytosolic+delivery+of+probes.">Enhanced fluorescence imaging of live cells by effective cytosolic delivery of probes.</a> PLoS One. 5, e10459 (2010).</li><li>Nakamura, K., Sakai, W., Kawamoto, T., Bree, R. T., Lowndes, N. F., Takeda, S., Taniguchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+vertebrate+53BP1:+a+major+role+in+non-homologous+end+joining+of+DNA+double+strand+breaks.">Genetic dissection of vertebrate 53BP1: a major role in non-homologous end joining of DNA double strand breaks.</a> DNA Repair (Amst). 5, 741-749 (2006).</li><li>Schultz, L. B., Chehab, N. H., Malikzay, A., Halazonetis, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53+binding+protein+1+(53BP1)+is+an+early+participant+in+the+cellular+response+to+DNA+double-strand+breaks.">p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks.</a> J. Cell. Biol. 151, 1381-1390 (2000).</li><li>Valerie, K., Povirk, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+mechanisms+of+mammalian+double-strand+break+repair.">Regulation and mechanisms of mammalian double-strand break repair.</a> Oncogene. 22, 5792-5812 (2003).</li><li>Wang, B., Matsuoka, S., Carpenter, P. B., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=53BP1,+a+mediator+of+the+DNA+damage+checkpoint.">53BP1, a mediator of the DNA damage checkpoint.</a> Science. 298, 1435-1438 (2002).</li><li>Ward, I. M., Minn, K., Jorda, K. 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Opretholdelse af genomisk integritet er afgørende for celleoverlevelse. Manglende bevare genom resulterer i for tidlig aldring, carcinogenese, eller død 8. Der er en intens interesse for kræsne, hvordan DDR funktioner, der stammer fra dets betydning for både grundforskning og klinisk forskning. Mange teknikker er blevet udviklet gennem årene for at hjælpe til undersøgelse af, hvordan cellerne registrere og reparere DNA-skader. Traditionelle metoder såsom immunocytokemi og western blotting har været g...

<table border="1"><tbody><tr><td> Produkt </td><td> Firma </td></tr><tr><td> CellStar dyrkningsskåle </td><td> Greiner Bio-on </td></tr><tr><td> FluroDish glas bunden retter </td><td> Verden Precision Instruments, Inc. </td></tr><tr><td> MEM-medium </td><td> GIBCO </td></tr><tr><td> Ikke-essentielle aminosyrer </td><td> GIBCO </td></tr><tr><td> Aminosyrer </td><td> GIBCO </td></tr><tr><td> Vitaminer </td><td> GIBCO </td></tr><tr><td> Natriumpyruvat </td><td> Invitrogen </td></tr><tr><td> Penicillin / streptomycin </td><td> HyClone </td></tr><tr><td> Føtalt bovint serum </td><td> GIBCO </td></tr><tr><td> N-Myc-53BP1 WT pLPC-Puro; plasmid 19.836 </td><td> Addgene </td></tr><tr><td> pCLNR-H2BG; plasmid 17.735 </td><td> Addgene </td></tr><tr><td> Superfect </td><td> Qiagen </td></tr><tr><td> Zeiss Cell Observer SD Imaging-system </td><td> Zeiss </td></tr><tr><td> AxioVision (release 4.8.2) </td><td> Zeiss </td></tr><tr><td> Zeiss Immersol W Oil </td><td> Zeiss </td></tr><tr><td> Volocity software (version 6,0) </td><td> PerkinElmer </td></tr></tbody></table>

two- and three-dimensional live cell imaging of dna damage response proteins

Dobbelt-strengbrud (DSB&#39;er) er den mest skadelige DNA-læsioner en celle kan støde på. Hvis venstre udbedrede, at DSBs havn stort potentiale generere mutationer og kromosomafvigelser 1. For at forhindre dette traume fra at katalysere genomisk ustabilitet, er det afgørende for celler til at opdage DSBs, aktivere DNA skade responset (DDR), og reparere DNA. Når stimuleres, DDR arbejder for at bevare genomisk integritet ved at udløse cellecyklusstandsning at muliggøre reparation finde sted eller tvinge cellen til at undergå apoptose. De dominerende mekanismer i DSB-reparation ske gennem ikke-homolog ende-sammenføjning (NHEJ) og homolog rekombination reparation (HRR) (revideret i 2). Der er mange proteiner, hvis aktiviteter skal præcist orkestreret for DDR til at fungere ordentligt. Heri, beskriver vi en fremgangsmåde til 2 - og 3-dimensionelle (D) visualisering af et af disse proteiner, 53BP1. Den p53-bindende protein 1 (53BP1) lokaliseres til områder afDSB&#39;er ved binding til modificerede histoner 3,4 og danner foci løbet af 5-15 minutter 5. Histon modifikationer og rekruttering af 53BP1 og andre DDR proteiner til DSB sites menes at lette den strukturelle omlægning af kromatin omkring områder med skader og bidrage til DNA-reparation 6. Ud over direkte deltagelse i reparation, er yderligere roller blevet beskrevet til 53BP1 i DDR, såsom regulering af en intra-S checkpoint, en G2 / M checkpoint, og aktivering af downstream DDR-proteiner 7-9. For nylig blev det opdaget, at 53BP1 ikke danner foci som reaktion på DNA-skade induceret under mitose i stedet venter på celler for at indtaste G1 før lokalisering til i nærheden af DSB&#39;er 6. DDR proteiner, såsom 53BP1 har vist sig at associere med mitotiske strukturer (såsom kinetochores) under progression gennem mitose 10. I denne protokol beskrives anvendelsen af ​​2 - og 3-D levende celler til at visualiseredannelsen af ​​53BP1 foci som svar på DNA-ødelæggende middel camptothecin (CPT), og 53BP1 opførsel under mitose. Camptothecin er en topoisomerase I inhibitor, der primært bevirker DSB&#39;er under DNA-replikation. For at opnå dette, anvendte vi en tidligere beskrevet 53BP1-mCherry fluorescerende fusionsproteinkonstruktion består af en 53BP1 proteindomæne stand til at binde DSB&#39;er 11. Desuden blev der anvendt en histon H2B-GFP-fluorescerende fusionsproteinkonstruktion kunne følge chromatin dynamik gennem cellecyklussen, men især under mitosen 12. Live-cell imaging i flere dimensioner er et fremragende værktøj til at uddybe vores forståelse af funktionen af ​​DDR-proteiner i eukaryote celler.

Watch this Scientific Journal Video about Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins at JoVE.com

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Denne protokol beskriver en metode til at visualisere en DNA-dobbeltstrenget brud signalering protein aktiveres som reaktion på DNA-skade såvel som dens lokalisering under mitose.

To-og tre-dimensionelle Live-Cell Imaging af DNA skade responset Proteiner

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

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