Name: advanced confocal microscopy techniques to study protein-protein interactions and kinetics at dna lesions
Uploaded: 2017-11-12T13:00:00
Description: Watch this Scientific Journal Video about Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions at JoVE.com

This work was supported by the Grant Agency of the Czech Republic, project P302-12-G157. Experiments were also supported by the Czech-Norwegian Research Programme CZ09, which is supervised by Norwegian funds, and by the Ministry of Education, Youth and Sport of the Czech Republic (grant number: 7F14369).

1. Cultivation of Cell Lines
<ol>
	<li>HeLa-derived cell lines 
	NOTE: Use HeLa cervical carcinoma cells: either HeLa cells stably expressing histone H2B tagged with GFP or HeLa-Fucci cells expressing RFP-Cdt1 in the G1 and early S phases and GFP-geminin in the S/G2-M phases (Figure 1).
	<ol>
		<li>For cultivation of all HeLa-derived cell lines, use Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum and appropriate antibiotics a...

<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. 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=A+chromatin+localization+screen+reveals+poly+(ADP+ribose)-regulated+recruitment+of+the+repressive+polycomb+and+NuRD+complexes+to+sites+of+DNA+damage.">A chromatin localization screen reveals poly (ADP ribose)-regulated recruitment of the repressive polycomb and NuRD complexes to sites of DNA damage.</a> Proc Natl Acad Sci U S A. 107 (43), 18475-18480 (2010).</li><li>Sustackova, G., 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=Acetylation-dependent+nuclear+arrangement+and+recruitment+of+BMI1+protein+to+UV-damaged+chromatin.">Acetylation-dependent nuclear arrangement and recruitment of BMI1 protein to UV-damaged chromatin.</a> J Cell Physiol. 227 (5), 1838-1850 (2012).</li><li>Smirnov, 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=Separation+of+replication+and+transcription+domains+in+nucleoli.">Separation of replication and transcription domains in nucleoli.</a> J Struct Biol. 188 (3), 259-266 (2014).</li><li>Bastiaens, P. I., Squire, A. <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+imaging+microscopy:+spatial+resolution+of+biochemical+processes+in+the+cell.">Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell.</a> Trends Cell Biol. 9 (2), 48-52 (1999).</li><li>Day, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+protein+interactions+using+Forster+resonance+energy+transfer+and+fluorescence+lifetime+imaging+microscopy.">Measuring protein interactions using Forster resonance energy transfer and fluorescence lifetime imaging microscopy.</a> Methods. 66 (2), 200-207 (2014).</li><li>Konig, K., Uchugonova, A., Breunig, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+multiphoton+cryomicroscopy.">High-resolution multiphoton cryomicroscopy.</a> Methods. 66 (2), 230-236 (2014).</li></ol>

Microscopy techniques represent basic tools in research laboratories. Here, a brief description of the methods used for the study of protein recruitment and kinetics at DNA lesions is presented. We especially noted our experimental experience in the field of local microirradiation of living cells, and we discuss the study of protein kinetics by FRAP and protein-protein interaction at DNA lesions by acceptor-bleaching FRET28 and its advanced modification FRET-FLIM (Figure 4A</s...

DNA damage leads to the appearance of DNA lesions consisting of cyclobutane pyrimidine dimers (CPDs), 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine, and single-strand or double-strand breaks1,2. &#947;-rays are the form of ionizing radiation with the highest energy and high penetrance, thus this source of radiation is widely used in radiotherapy3. On the other hand, experimentally induced DNA damage caused by UV lasers mimics natural exposure to UV light. UVA microirradiation, as a microscopic method, represents an experimental tool for studying DNA damage in individual living cells. Microirradiation was used for the first time 40 years ago in order to reveal the organization of chromosome regions4,5. This technique is highly dependent on either the functional properties of confocal microscopes or the technical limits of modern nanoscopy. To induce DNA lesions, cells can be presensitized by 5&#39; bromodeoxyuridine (BrdU) or Hoechst 33342 prior to UV irradiation. B&#225;rtov&#225; et al.6 previously described the presensitization step, and recently we optimized this microirradiation technique in order to avoid cell death, or apoptosis. For example, the use of a 405-nm UV laser (without Hoechst 33342 presensitization) leads to the induction of 53BP1-positive double-strand breaks (DSBs) at the expense of cyclobutane pyrimidine dimers (CPDs). On the other hand, presensitization steps combined with UV microirradiation induce very high levels of CPDs and DSBs simultaneously7,8. This methodology is difficult to apply to the study of a single DNA repair pathway.
With microirradiation, it is possible to analyze protein recruitment, kinetics, and interaction at DNA lesions in living cells. An example of this method was published by Luijsterburg et al.9 for heterochromatin protein 1&#946;, and we recently showed for the first time that the pluripotency factor Oct4 and a protein associated with Cajal bodies, coilin, are recruited to UV-induced DNA lesions6,10. Protein kinetics at these DNA lesions can also be studied using the FRAP (Fluorescence Recovery After Photobleaching)11,12,13 or FRET (Fluorescence Resonance Energy Transfer) techniques14,15. These methods have the potential to reveal simple diffusion of proteins at DNA lesions or protein-protein interactions. A useful tool for additional characterization of proteins is FLIM (Fluorescence Lifetime Imaging Microscopy) or its combination with FRET technology (FRET-FLIM)16. These methods enable the study of processes in living cells that are stably or transiently expressing the protein of interest tagged by a fluorescent molecule17. Here, an example of exponential decay time (&#964;) for GFP-tagged p53 protein and its interaction partner, mCherry-tagged 53BP1, playing an important role in DNA damage response18,19 is shown. The parameter &#964;, the lifetime of the fluorochrome provided by FLIM calculations, is specific for a given fluorescence dye, its binding abilities, and its cellular environment. Therefore, this method can show us distinctions between protein subpopulations, their binding abilities, and their functional properties after, for example, DNA damage.
Here, an outline of the methodological approaches of the advanced microscopy techniques that is used in our laboratory to study time-specific protein recruitment, kinetics, diffusion, and protein-protein interactions at the site of microirradiated chromatin is presented. The step-by-step methodology for the induction of local DNA lesions in living cells, and a description of methodologies useful for studies of DNA-damage-related events at locally induced DNA lesions caused by UV lasers are provided.

<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

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Using advanced confocal microscopy, we observed an accumulation of mCherry-tagged 53BP1 and mCherry-PCNA proteins at DNA lesions. Analyses were performed by local microirradiation of living cells. To recognize the nuclear distribution patterns of DNA-repair-related proteins in individual cell cycle phases, we used the Fucci cellular system, by which it is possible to determine the G1, early S, and G2 phases of the cell cycle (Figure 1). The biological applica...

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

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

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

Research

JoVE Journal

Biology

Imaging Protein-protein Interactions in vivo

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, ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

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. ...

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 ...