Questo lavoro è stato supportato dall'Agenzia della Repubblica Ceca, progetto P302-12-G157 Grant. Esperimenti sono stati supportati anche dalla CZ09 di programma di ricerca ceco-norvegese, che è sorvegliato da fondi norvegesi e dal Ministero della pubblica istruzione, gioventù e Sport della Repubblica Ceca (concessione numero: 7F14369).

<ol>
	<li>Cadet, J., Mouret, S., Ravanat, J. L., Douki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoinduced+damage+to+cellular+DNA:+direct+and+photosensitized+reactions.">Photoinduced damage to cellular DNA: direct and photosensitized reactions.</a> Photochem Photobiol. 88 (5), 1048-1065 (2012).</li><li>Cooke, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunochemical+detection+of+UV-induced+DNA+damage+and+repair.">Immunochemical detection of UV-induced DNA damage and repair.</a> J Immunol Methods. 280 (1-2), 125-133 (2003).</li><li>Lahtz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma+irradiation+does+not+induce+detectable+changes+in+DNA+methylation+directly+following+exposure+of+human+cells.">Gamma irradiation does not induce detectable changes in DNA methylation directly following exposure of human cells.</a> PLoS One. 7 (9), e44858 (2012).</li><li>Cremer, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+chromosome+aberrations+in+the+human+interphase+nucleus+by+visualization+of+specific+target+DNAs+with+radioactive+and+non-radioactive+in+situ+hybridization+techniques:+diagnosis+of+trisomy+18+with+probe+L1.84.">Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L1.84.</a> Hum Genet. 74 (4), 346-352 (1986).</li><li>Zorn, C., Cremer, T., Cremer, C., Zimmer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+UV+microirradiation+of+interphase+nuclei+and+post-treatment+with+caffeine.+A+new+approach+to+establish+the+arrangement+of+interphase+chromosomes.">Laser UV microirradiation of interphase nuclei and post-treatment with caffeine. A new approach to establish the arrangement of interphase chromosomes.</a> Hum Genet. 35 (1), 83-89 (1976).</li><li>Bartova, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+of+Oct4+protein+to+UV-damaged+chromatin+in+embryonic+stem+cells.">Recruitment of Oct4 protein to UV-damaged chromatin in embryonic stem cells.</a> PLoS One. 6 (12), e27281 (2011).</li><li>Stixova, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HP1beta-dependent+recruitment+of+UBF1+to+irradiated+chromatin+occurs+simultaneously+with+CPDs.">HP1beta-dependent recruitment of UBF1 to irradiated chromatin occurs simultaneously with CPDs.</a> Epigenetics Chromatin. 7 (1), 39 (2014).</li><li>Stixova, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+microscopy+techniques+used+for+comparison+of+UVA-+and+gamma-irradiation-induced+DNA+damage+in+the+cell+nucleus+and+nucleolus.">Advanced microscopy techniques used for comparison of UVA- and gamma-irradiation-induced DNA damage in the cell nucleus and nucleolus.</a> Folia Biol (Praha). 60, 76-84 (2014).</li><li>Luijsterburg, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochromatin+protein+1+is+recruited+to+various+types+of+DNA+damage.">Heterochromatin protein 1 is recruited to various types of DNA damage.</a> J Cell Biol. 185 (4), 577-586 (2009).</li><li>Bartova, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coilin+is+rapidly+recruited+to+UVA-induced+DNA+lesions+and+gamma-radiation+affects+localized+movement+of+Cajal+bodies.">Coilin is rapidly recruited to UVA-induced DNA lesions and gamma-radiation affects localized movement of Cajal bodies.</a> Nucleus. 5 (3), 460-468 (2014).</li><li>Franek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+Image+Acquisition+and+Analytical+Techniques+for+Studies+of+Living+Cells+and+Tissue+Sections.">Advanced Image Acquisition and Analytical Techniques for Studies of Living Cells and Tissue Sections.</a> Microsc Microanal. 22 (2), 326-341 (2016).</li><li>Lemmer, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+conventional+fluorescent+markers+for+far-field+fluorescence+localization+nanoscopy+allows+resolution+in+the+10-nm+range.">Using conventional fluorescent markers for far-field fluorescence localization nanoscopy allows resolution in the 10-nm range.</a> J Microsc. 235 (2), 163-171 (2009).</li><li>Reits, E. A., Neefjes, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+fixed+to+FRAP:+measuring+protein+mobility+and+activity+in+living+cells.">From fixed to FRAP: measuring protein mobility and activity in living cells.</a> Nat Cell Biol. 3 (6), E145-E147 (2001).</li><li>Lakowitz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Fluorescence Spectroscopy. , (2006).</li><li>Piston, D. W., Kremers, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein+FRET:+the+good,+the+bad+and+the+ugly.">Fluorescent protein FRET: the good, the bad and the ugly.</a> Trends Biochem Sci. 32 (9), 407-414 (2007).</li><li>Levitt, J. A., Matthews, D. R., Ameer-Beg, S. M., Suhling, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+lifetime+and+polarization-resolved+imaging+in+cell+biology.">Fluorescence lifetime and polarization-resolved imaging in cell biology.</a> Curr Opin Biotechnol. 20 (1), 28-36 (2009).</li><li>Shimomura, O., Johnson, F. H., Saiga, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction,+purification+and+properties+of+aequorin,+a+bioluminescent+protein+from+the+luminous+hydromedusan,+Aequorea.">Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea.</a> J Cell Comp Physiol. 59, 223-239 (1962).</li><li>Iwabuchi, K., Bartel, P. L., Li, B., Marraccino, R., Fields, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+cellular+proteins+that+bind+to+wild-type+but+not+mutant+p53.">Two cellular proteins that bind to wild-type but not mutant p53.</a> Proc Natl Acad Sci U S A. 91 (13), 6098-6102 (1994).</li><li>Ward, I. M., Minn, K., van Deursen, J., Chen, J. <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+53BP1+is+required+for+DNA+damage+responses+and+tumor+suppression+in+mice.">p53 Binding protein 53BP1 is required for DNA damage responses and tumor suppression in mice.</a> Mol Cell Biol. 23 (7), 2556-2563 (2003).</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 (7), 377-385 (1998).</li><li>Walter, J., Schermelleh, L., Cremer, M., Tashiro, S., Cremer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+order+in+HeLa+cells+changes+during+mitosis+and+early+G1,+but+is+stably+maintained+during+subsequent+interphase+stages.">Chromosome order in HeLa cells changes during mitosis and early G1, but is stably maintained during subsequent interphase stages.</a> J Cell Biol. 160 (5), 685-697 (2003).</li><li>Sakaue-Sawano, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+the+silhouette+of+individual+cells+in+S/G2/M+phases+with+fluorescence.">Tracing the silhouette of individual cells in S/G2/M phases with fluorescence.</a> Chem Biol. 15 (12), 1243-1248 (2008).</li><li>Sorokin, D. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+movement+and+morphology+of+UBF1-positive+nucleolar+regions+are+changed+by+gamma-irradiation+in+G2+phase+of+the+cell+cycle.">Localized movement and morphology of UBF1-positive nucleolar regions are changed by gamma-irradiation in G2 phase of the cell cycle.</a> Nucleus. 6 (4), 301-313 (2015).</li><li>Vogel, S. S., Nguyen, T. A., van der Meer, B. W., Blank, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+heterogeneity+and+dark+acceptor+states+on+FRET:+implications+for+using+fluorescent+protein+donors+and+acceptors.">The impact of heterogeneity and dark acceptor states on FRET: implications for using fluorescent protein donors and acceptors.</a> PLoS One. 7 (11), e49593 (2012).</li><li>Foltankova, V., Matula, P., Sorokin, D., Kozubek, S., Bartova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+detectors+improved+time-lapse+confocal+microscopy+of+PML+and+53BP1+nuclear+body+colocalization+in+DNA+lesions.">Hybrid detectors improved time-lapse confocal microscopy of PML and 53BP1 nuclear body colocalization in DNA lesions.</a> Microsc Microanal. 19 (2), 360-369 (2013).</li><li>Suchankova, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+kinetics+of+DNA+repair+protein+accumulation+at+DNA+lesions+and+cell+cycle-dependent+formation+of+gammaH2AX-+and+NBS1-positive+repair+foci.">Distinct kinetics of DNA repair protein accumulation at DNA lesions and cell cycle-dependent formation of gammaH2AX- and NBS1-positive repair foci.</a> Biol Cell. 107 (12), 440-454 (2015).</li><li>B&#225;rtov&#225;, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCNA+is+recruited+to+irradiated+chromatin+in+late+S+phase+and+is+most+pronounced+in+G2+phase+of+the+cell+cycle.">PCNA is recruited to irradiated chromatin in late S phase and is most pronounced in G2 phase of the cell cycle.</a> Protoplasma. , (2017).</li><li>Krejci, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-Translational+Modifications+of+Histones+in+Human+Sperm.">Post-Translational Modifications of Histones in Human Sperm.</a> J Cell Biochem. 116 (10), 2195-2209 (2015).</li><li>Chou, D. 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Gli autori dichiarano che non esistono senza conflitti di interesse.

Tecniche di microscopia rappresentano strumenti fondamentali in laboratori di ricerca. Qui, una breve descrizione dei metodi utilizzati per lo studio del reclutamento di proteine e cinetica alle lesioni del DNA è presentato. Abbiamo notato soprattutto la nostra esperienza sperimentale nel campo della locale microirradiation di cellule viventi, e discutiamo lo studio della cinetica della proteina di interazione proteina-proteina e FRAP alle lesioni del DNA di candeggio accettore FRET28 e sua avanz...

Danno del DNA porta alla comparsa di lesioni del DNA che consiste della pirimidina del cyclobutane (CPDs), 8-oxo-7,8-dihydro-2'-deoxyguanosine e single-strand o double-strand breaks1,2. Γ-raggi sono la forma di radiazione ionizzante con la più alta energia e alta penetranza, così questa fonte di radiazione è ampiamente usata in radioterapia3. D'altra parte, sperimentalmente indotto danni al DNA causati da UV laser imita naturale esposizione ai raggi UV. Microirradiation UVA, come un metodo microscopico, rappresenta uno strumento sperimentale per lo studio di danno del DNA in cellule viventi individuali. Microirradiation è stato usato per la prima volta 40 anni fa per rivelare l'organizzazione delle regioni del cromosoma4,5. Questa tecnica è altamente dipendente le proprietà funzionali di microscopi confocali o i limiti tecnici di nanoscopia moderno. Per indurre lesioni del DNA, le cellule possono essere presensitized da 5' bromodeossiuridina (BrdU) o Hoechst 33342 prima dell'irradiazione UV. Bártová et al. 6 precedentemente descritto il passo presensitization e recentemente abbiamo ottimizzato questa tecnica microirradiation al fine di evitare la morte cellulare o apoptosi. Ad esempio, l'uso di un laser UV 405 nm (senza Hoechst 33342 presensitization) porta all'induzione del doppio-filo di 53BP1-positivi (DSBs) a scapito di dimeri di pirimidina cyclobutane (CPDs). D'altra parte, presensitization passi combinati con UV microirradiation inducono livelli molto elevati di CPDs e DSBs contemporaneamente7,8. Questa metodologia è difficile da applicare allo studio di una singola via di riparazione del DNA.
Con microirradiation, è possibile analizzare l'assunzione di proteine, cinetica e interazione alle lesioni del DNA in cellule viventi. Un esempio di questo metodo è stato pubblicato da Luijsterburg et al. 9 per eterocromatina proteina 1 β e ci ha mostrato recentemente per la prima volta che il fattore di pluripotenza Oct4 e una proteina connessa con corpi di Cajal, coilina, sono reclutati per le lesioni del DNA indotto da UV6,10. Cinetica della proteina in queste lesioni del DNA possono anche essere studiati utilizzando il FRAP (Fluorescence Recovery After Photobleaching)11,12,13 o FRET (Fluorescence Resonance Energy Transfer) tecniche14 ,15. Questi metodi hanno il potenziale per rivelare semplice diffusione delle proteine alle lesioni del DNA o interazioni proteina-proteina. Uno strumento utile per ulteriore caratterizzazione delle proteine è FLIM (fluorescenza Lifetime Imaging microscopia) o la sua combinazione con FRET tecnologia (FRET-FLIM)16. Questi metodi consentono lo studio dei processi in cellule che sono stabilmente viventi o transitoriamente esprimenti la proteina di interesse contrassegnati da una molecola fluorescente17. Qui, è riportato un esempio del tempo di decadimento esponenziale (τ) per proteina GFP-etichettato p53 e il suo partner di interazione, mCherry-etichetta 53BP1, giocando un ruolo importante nel DNA danni risposta18,19 . Il parametro τ, la durata del fluorocromo fornito da calcoli di FLIM, è specifico per una tintura di fluorescenza determinata, le sue abilità di associazione e il suo ambiente cellulare. Pertanto, questo metodo può mostrarci distinzioni tra sottopopolazioni di proteina, le loro capacità di associazione e le loro proprietà funzionali dopo, ad esempio, il danno del DNA.
Qui, un contorno di approcci metodologici delle tecniche di microscopia avanzata che viene utilizzato nel nostro laboratorio per studiare il reclutamento di proteine specifiche del tempo, cinetica, diffusione e interazioni proteina-proteina nel sito della cromatina microirradiated è presentato. La metodologia dettagliata per l'induzione di lesioni locali del DNA in cellule viventi e una descrizione delle metodologie utili per studi di eventi relativi a DNA-danno alle lesioni del DNA localmente indotte causate da laser UV sono forniti.

<table>
	<tbody>
		<tr>
			<td>Cell cultivation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa</td>
			<td>ATCC</td>
			<td>CCL-2TM</td>
			<td></td>
		</tr>
		<tr>
			<td>ES-D3 [D3]</td>
			<td>ATCC</td>
			<td>CRL-11632TM</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa -Fucci cells</td>
			<td></td>
			<td></td>
			<td>http://ruo.mbl.co.jp/bio/e/product/flprotein/fucci.html</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>PAN-Biotech</td>
			<td>P03-0710</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D6429-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>HyClone</td>
			<td>SV30180.03</td>
			<td></td>
		</tr>
		<tr>
			<td>ES Cell FBS</td>
			<td>Gibco</td>
			<td>16141-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids (NEAA)</td>
			<td>Gibco</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr>
			<td>mLIF (mouse Leukemia Inhibitor Factor)</td>
			<td>Merck Millipore</td>
			<td>ESG1107</td>
			<td></td>
		</tr>
		<tr>
			<td>MTG (1-Thioglycerol)</td>
			<td>Sigma-Aldrich</td>
			<td>M6145-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>Biosera</td>
			<td>XC-A4122/100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin - EDTA</td>
			<td>Biosera</td>
			<td>XC-T1717/100</td>
			<td>dilute with 1 &#215; PBS in ratio 1:6</td>
		</tr>
		<tr>
			<td>Nunclon cell culture dishes</td>
			<td>Sigma-Aldrich</td>
			<td>P7866</td>
			<td>cultivation of mESCs D3 cells</td>
		</tr>
		<tr>
			<td>&#181;-Dish 35m+A15:I35m Grid-500</td>
			<td>Ibidi GmbH</td>
			<td>81166</td>
			<td>microscopic dish</td>
		</tr>
		<tr>
			<td>0.2% Gelatine</td>
			<td>Sigma-Aldrich</td>
			<td>G1890-100G</td>
			<td>dilute in destille water and autoclaved</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell transfection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GFP-p53 plasmid</td>
			<td>Addgene</td>
			<td>12091</td>
			<td></td>
		</tr>
		<tr>
			<td>mCherry-PCNA plasmid</td>
			<td></td>
			<td></td>
			<td>generous gift from Cristina Cardoso, Technische Universit&#228;t Darmstadt</td>
		</tr>
		<tr>
			<td>mCherry-53BP1 plasmid</td>
			<td>Addgene</td>
			<td>19835</td>
			<td></td>
		</tr>
		<tr>
			<td>Metafecetene</td>
			<td>Biontex Laboratories GmbH</td>
			<td>T020&#8211;2.0</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#215; PBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9625</td>
			<td>for transfection use 1 &#215; PBS diluted in nuclease-free water</td>
		</tr>
		<tr>
			<td>5-bromo-2&#8217;-deoxy-uridine</td>
			<td>Sigma-Aldrich</td>
			<td>11296736001</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Confocal microscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Leica TCS SP5</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Leica TCS SP8</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>White-light laser</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>355-nm laser</td>
			<td>Coherent Inc.</td>
			<td></td>
			<td>laser power 80 mW</td>
		</tr>
		<tr>
			<td>405-nm laser</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>laser power 50 mW</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Immunofluorescence staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>VWR International Ltd</td>
			<td>631-1580</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Affymetrix</td>
			<td>19943 1 LT</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X100</td>
			<td>MP Biomedicals</td>
			<td>2194854</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin from quillaja bark</td>
			<td>Sigma-Aldrich</td>
			<td>S4521</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>53BP1</td>
			<td>Abcam</td>
			<td>ab21083</td>
			<td>primary antibody</td>
		</tr>
		<tr>
			<td>AlexaFluore 647</td>
			<td>Thermo Fisher Scientific</td>
			<td>A27040</td>
			<td>secondary antibody</td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Laboratories Ltd</td>
			<td>H-1000</td>
			<td>mounting medium</td>
		</tr>
	</tbody>
</table>

advanced confocal microscopy techniques to study protein-protein interactions and kinetics at dna lesions

Microirradiation locale con laser rappresenta uno strumento utile per gli studi del DNA-riparazione-processi in cellule vive. Qui, descriviamo un approccio metodologico per l'analisi cinetica della proteina alle lesioni del DNA nel corso del tempo o proteine interazioni su localmente microirradiated della cromatina. Mostriamo anche come riconoscere le singole fasi del ciclo cellulare usando il sistema cellulare di Fucci per studiare la cinetica di cellula-ciclo-dipendente della proteina alle lesioni del DNA. Una descrizione metodologica dell'uso di due laser UV (355 nm e 405 nm) per indurre diversi tipi di danno del DNA inoltre è presentata. Solo il microirradiated di cellule dal laser del diodo di 405 nanometro procedeva normalmente attraverso mitosi ed erano privi di dimeri di pirimidina cyclobutane (CPDs). Mostriamo anche come cellule di microirradiated possono essere risolto in un punto determinato momento per eseguire immunodetection di proteine endogene di interesse. Per gli studi di riparazione del DNA, descriviamo inoltre l'uso di metodi biofisici compreso FRAP (Fluorescence Recovery After Photobleaching) e FLIM (fluorescenza Lifetime Imaging microscopia) nelle cellule con spontaneamente d'avvenimento i fuochi di danni del DNA. Mostriamo anche un'applicazione di FLIM-FRET (Fluorescence Resonance Energy Transfer) negli studi sperimentali delle interazioni proteina-proteina.

Usando la microscopia confocal avanzata, abbiamo osservato un accumulo di proteine 53BP1 e mCherry-PCNA mCherry-etichettate alle lesioni del DNA. Analisi sono state eseguite da microirradiation locale di cellule viventi. Per riconoscere i modelli di distribuzione nucleare delle proteine correlate a DNA-repair in fasi del ciclo cellulare individuale, abbiamo usato il sistema cellulare di Fucci, mediante il quale è possibile determinare il G1, primi S, e G2 fasi della cella ciclo (<strong ...

Watch this Scientific Journal Video about Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions at JoVE.com

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Microirradiation laser è uno strumento utile per gli studi della riparazione del DNA in cellule viventi. È indicato un approccio metodologico per l'utilizzo del laser UVA per indurre varie lesioni del DNA. Abbiamo ottimizzato un metodo per microirradiation locale che mantiene il normale ciclo cellulare; quindi, cellule irradiate procedono attraverso mitosi.

Avanzate tecniche di microscopia confocale per studiare le interazioni proteina-proteina e cinetica alle lesioni del DNA

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

The overall goal of these experiments is to induce DNA lesions using local micro irradiation of cells and to observe the recruitment of proteins involved in DNA repair processes.This method can help us to understand a very complex and profound question such as, how normal and cancer cells respond to DNA injury.The main advantage of this technique is that we are able to study the DNA damage response in individual live cells.Demonstrating the procedure will be by Sona Legartova, a post-doc and Jana Suchankova, a PhD student from my laboratory.To begin, cultivate HeLa cells in DMAM supplemented with 10%FBS and appropriate antibiotics.Incubate the cells at 37

advanced-confocal-microscopy-techniques-to-study-protein-protein

Research

JoVE Journal

Biology

10.8K Views.  Institute of Biophysics of the Czech Academy of Sciences. The overall goal of these experiments is to induce DNA lesions using local micro irradiation of cells and to observe the recruitment of proteins involved in DNA repair processes.This method can help us to understand a very complex and profound question such as, how normal and cancer cells respond to DNA injury.The main advantage of this technique is that we are able to study the DNA damage response in individual live cells.Demonstrating the procedure will be by Sona Legartova, a post-doc and ...

Video: Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Imaging interazioni proteina-proteina In vivo</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Ricerca

Biologia

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Basato sulla fluorescenza Misura Store-Entry operato di calcio in cellule vive: dal Cancer Cell coltura per fibra muscolare scheletrica

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Il MultiBac Protein Complex Piattaforma di produzione al EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

L&#39;analisi citofluorimetrica di bimolecular Complementation Fluorescenza: A High Throughput Metodo quantitativo per studiare l&#39;interazione proteina-proteina

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Analizzando craniofacciale morfogenesi in Zebrafish Uso 4D Microscopia confocale

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

Combinando singola molecola di manipolazione e di imaging per lo studio delle interazioni proteina-DNA

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

Probing ad alta densità di microarrays della proteina funzionali per rilevare le interazioni proteina-proteina

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

Studio delle interazioni proteina-proteina nella ricerca di autofagia

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations. We propose here a detailed protocol for FLIM-FRET measurements that we applied to monitor PPIs in live Pseudomonas aeruginosa in the particular case of two interacting proteins expressed with highly different copy numbers to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. This protocol describes in detail all the necessary steps for PPI characterization - starting from bacterial mutant constructions up to the final analysis using recently developed tools providing advanced visualization possibilities for a straightforward interpretation of complex FLIM-FRET data.

We describe here a protocol to characterize protein-protein interactions between two highly-differently expressed proteins in live Pseudomonas aeruginosa using FLIM-FRET measurements. The protocol includes bacteria strain constructions, bacteria immobilization, imaging and post-imaging data analysis routines.

Misurazioni FLIM-FRET delle interazioni proteina-proteina nei batteri vivi.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster ...

Autofluorescence Imaging to Evaluate Red Algae Physiology

Red algae (Rhodophyta) contain phycobiliproteins and colonize habitats with dim light, however some (e.g., some Chroothece species) can also develop in full sunshine. Most rhodophytes are red, however some can appear bluish, depending on the proportion of blue and red biliproteins (phycocyanin and phycoerythrin). Different phycobiliproteins can capture light at diverse wavelengths and transmit it to chlorophyll a, which makes photosynthesis under very different light conditions possible. These pigments respond to habitat changes in light, and their autofluorescence can help to study biological processes. Using Chroothece mobilis as a model organism and the spectral lambda scan mode in a confocal microscope, the adaptation of photosynthetic pigments to different monochromatic lights was studied at the cellular level to guess the species' optimal growth conditions. The results showed that, even when the studied strain was isolated from a cave, it adapted to both dim and medium light intensities. The presented method is especially useful for studying photosynthetic organisms that do not grow or grow very slowly under laboratory conditions, which is usually the case for those living in extreme habitats.

The present protocol describes step-by-step autofluorescence imaging and evaluation of phycobiliprotein changes in red algae based on spectral analysis. This is a label-free and non-destructive method to evaluate cellular adaptation to extreme habitats, when only scarce material is available and cells grow slowly, or not at all, under laboratory conditions.

Autofluorescenza per valutare la fisiologia delle alghe rosse

Red algae (Rhodophyta) contain phycobiliproteins and colonize habitats with dim light, however some (e.g., some Chroothece species) can also develop in ...