Name: detection and visualization of dna damage-induced protein complexes in suspension cell cultures using the proximity ligation assay
Uploaded: 2017-06-09T15:00:00
Description: Watch this Scientific Journal Video about Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay at JoVE.com

Research in the Guikema laboratory is funded by the Innovational Research Incentives Scheme from The Netherlands Organization for Scientific Research (VIDI grant 016126355) and the 'Stichting Kinderen Kankervrij' KiKA (project 252).

1. Treatment of Cells and DNA Damage Induction
<ol>
	<li>Culture the human BCR-ABL+ B-cell acute lymphoblastic cell lines BV173 or SUP-B15 in IMDM supplemented with 20% FCS, 50 &#956;M &#946;-mercaptoethanol, 2 mM L-glutamine, 100 U/mL penicillin and 100 &#956;g/mL streptomycin at 37 &#176;C in an atmosphere of 5% CO2. Count cells and plate at 2 x 106 cell/mL in 6-wells plates, at 5 mL/well. 
	NOTE: Optionally, suspension cell lines of various origin can used.</li>
	<li>To arrest the ...

<ol>
	<li>Mar&#233;chal, A., Zou, L. <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+Sensing+by+the+ATM+and+ATR+Kinases.">DNA Damage Sensing by the ATM and ATR Kinases.</a> Cold Spring Harb Perspect Biol. 5 (9), a012716 (2013).</li><li>Huen, M. S. Y., 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=Assembly+of+checkpoint+and+repair+machineries+at+DNA+damage+sites.">Assembly of checkpoint and repair machineries at DNA damage sites.</a> Trends Biochem Sci. 35 (2), 101-108 (2010).</li><li>Bekker-Jensen, S., Mailand, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+function+of+DNA+double-strand+break+repair+foci+in+mammalian+cells.">Assembly and function of DNA double-strand break repair foci in mammalian cells.</a> DNA Repair. 9 (12), 1219-1228 (2010).</li><li>Matsuoka, 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=ATM+and+ATR+substrate+analysis+reveals+extensive+protein+networks+responsive+to+DNA+damage.">ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage.</a> Science. 316 (5828), 1160-1166 (2007).</li><li>Barlow, C., Brown, K. D., Deng, C. X., Tagle, D. A., Wynshaw-Boris, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atm+selectively+regulates+distinct+p53-dependent+cell-cycle+checkpoint+and+apoptotic+pathways.">Atm selectively regulates distinct p53-dependent cell-cycle checkpoint and apoptotic pathways.</a> Nature Genet. 17 (4), 453-456 (1997).</li><li>Tibbetts, R. 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=A+role+for+ATR+in+the+DNA+damage-induced+phosphorylation+of+p53.">A role for ATR in the DNA damage-induced phosphorylation of p53.</a> Genes Dev. 13 (2), 152-157 (1999).</li><li>Schermelleh, 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=Subdiffraction+Multicolor+Imaging+of+the+Nuclear+Periphery+with+3D+Structured+Illumination+Microscopy.">Subdiffraction Multicolor Imaging of the Nuclear Periphery with 3D Structured Illumination Microscopy.</a> Science. 320 (5881), 1332-1336 (2008).</li><li>Chapman, J. R., Sossick, A. J., Boulton, S. J., 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=BRCA1-associated+exclusion+of+53BP1+from+DNA+damage+sites+underlies+temporal+control+of+DNA+repair.">BRCA1-associated exclusion of 53BP1 from DNA damage sites underlies temporal control of DNA repair.</a> J Cell Sci. 125 (Pt 15), 3529-3534 (2012).</li><li>Gullberg, 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+sense+of+closeness:+protein+detection+by+proximity+ligation.">A sense of closeness: protein detection by proximity ligation.</a> Curr Opin Biotechnol. 14 (1), 82-86 (2003).</li><li>S&#246;derberg, O., 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=Characterizing+proteins+and+their+interactions+in+cells+and+tissues+using+the+in+situ+proximity+ligation+assay.">Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay.</a> Methods. 45 (3), 227-232 (2008).</li><li>Dong, Y., Shannon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+immunosensing+using+antigen+and+antibody+monolayers+on+gold+surfaces+with+electrochemical+and+scanning+probe+detection.">Heterogeneous immunosensing using antigen and antibody monolayers on gold surfaces with electrochemical and scanning probe detection.</a> Anal Chem. 72 (11), 2371-2376 (2000).</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 (7267), 1071-1078 (2009).</li><li>Economopoulou, P., Pappa, V., Papageorgiou, S., Dervenoulas, J., Economopoulos, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormalities+of+DNA+repair+mechanisms+in+common+hematological+malignancies.">Abnormalities of DNA repair mechanisms in common hematological malignancies.</a> Leuk Lymphoma. 52 (4), 567-582 (2011).</li><li>Rendleman, 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=Genetic+variation+in+DNA+repair+pathways+and+risk+of+non-Hodgkin's+lymphoma.">Genetic variation in DNA repair pathways and risk of non-Hodgkin's lymphoma.</a> PloS One. 9 (7), e101685 (2014).</li><li>Ochodnicka-Mackovicova, K., 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=The+DNA+Damage+Response+Regulates+RAG1/2+Expression+in+Pre-B+Cells+through+ATM-FOXO1+Signaling.">The DNA Damage Response Regulates RAG1/2 Expression in Pre-B Cells through ATM-FOXO1 Signaling.</a> J Immunol. , (2016).</li><li>Banin, 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=Enhanced+phosphorylation+of+p53+by+ATM+in+response+to+DNA+damage.">Enhanced phosphorylation of p53 by ATM in response to DNA damage.</a> Science. 281 (5383), 1674-1677 (1998).</li></ol>

In this report, it is demonstrated that PLA can be used to determine and visualize the specific interaction between proteins in suspension cell cultures. Of note, the protocol described here is not restricted to the study of DNA repair complexes but also applies to visualize and quantify other protein-protein interactions in suspension cell cultures. It is shown that the ATM kinase interacts with phosphorylated p53 in G1-arrested BCR-ABL+ B-ALL cells when exposed to a DNA damage-inducing agent. Previously, the PLA techni...

DNA damage triggers a highly regulated series of events involving protein-protein interactions and post-translational modifications that ensures the efficient and rapid repair of DNA, thereby safeguarding genomic integrity1. Typically, DNA repair is studied in cells exposed to ionizing radiation by monitoring the formation of so-called ionizing radiation-induced foci (IRIF) by (confocal) fluorescence microscopy. Many DNA repair and DNA damage-sensing proteins form IRIFs, which represent protein complexes that nucleate at chromatin sites sustaining DNA damage2,3. The location and resolution of IRIFs over time gives important insight into spatiotemporal organization of DNA repair, and may indicate the involvement of different DNA repair pathways. The nature of the DNA damage and the cell-cycle stage in which the damage is attained determines which DNA repair pathway is activated. For instance, in cells actively engaged in DNA replication (S-phase), homologous recombination (HR) is the dominant DNA repair pathway, whereas in cells in the G1- or G2/M-phase of the cell-cycle, the non-homologous end-joining (NHEJ) repair pathway predominates. One of the earliest events following DNA damage is the activation of the DNA damage-sensing kinases Ataxia Telangiectasia-Mutated protein (ATM), which is mostly active in the G1- and G2/M-phases of the cell-cycle and regulates NHEJ, and Ataxia Telangiectasia and Rad3-related protein (ATR), which acts in S-phase by activating HR. Both ATM and ATR are very pleiotropic kinases that phosphorylate many proteins that are involved in DNA repair, cell death and survival4. Both kinases have been shown to phosphorylate and activate the tumor-suppressor protein p53 following the exposure to genotoxic stress, indicating that these kinases are upstream mediators of a pivotal tumor suppressive signaling axis5,6.
The formation and composition of IRIFs is typically assessed by determining co-localization of different proteins using dual-color immunofluorescence staining and microscopy, however, not all proteins that are part of repair protein complexes form IRIFs, which limits the applicability of this approach. Moreover, (confocal) immunofluorescence microscopy is limited by the diffraction properties of light, resulting in a rather poor spatial resolution of about 200-300 nm, exceeding the size of most subcellular structures, which essentially prohibits the direct interrogation of protein-protein interactions at the molecular level. As such, the co-localization of immunofluorescence staining patterns as detected by (confocal) fluorescence microscopy is not necessarily indicative for direct protein-protein interactions. Recently, new super-resolution technologies have been developed, such as three-dimensional structured illumination microscopy (3D-SIM)7, which was successfully used to study 53BP1 and BRCA1 IRIF formation at nano-scale detail, revealing the spatial distribution characteristics of these proteins that could not detected by confocal laser scanning microscopy8.
Several other methods can be used to detect protein-protein interactions in vivo, such as co-immunoprecipitation, pull-down methods and yeast two-hybrid screening approaches. However, these techniques are rather cumbersome, require large amounts of cells or proteins or involve overexpression of proteins, which introduces experimental artifacts. More recently, a novel technique has been developed that allows the visualization and quantification of protein-protein interactions in situ (i.e. in cells and in tissues), which is termed Proximity Ligation Assay (PLA)9,10. Primary antibodies that recognize two proteins of interest are detected by secondary antibodies that are conjugated to oligonucleotides (so-called PLA probes). If the two different secondary antibodies are sufficiently close due to interactions between the proteins recognized by the primary antibodies, the conjugated oligonucleotides hybridize and can be ligated to form a closed circular DNA substrate. This circular substrate is subsequently amplified by rolling circle amplification, and visualized with fluorochrome-conjugated complementary oligonucleotides. Using PLA, the subcellular localization of the protein-protein interaction is preserved as the fluorescently labeled rolling circle amplification-product remains attached to the PLA probes. The resolution of this assay is &#60;50 nm, based on the finding that the diameter of an antibody is approximately 7-10 nm11. Rolling circle amplification can only take place in case two pairs of antibodies (primary + secondary) physically interact within the perimeter that is defined by their size (10 + 10 + 10 + 10 = 40 nm). The signal amplification step increases the sensitivity of the PLA assay and enables the detection of interactions of scarcely expressed proteins. PLA generates punctate, foci-like signals patterns that can be quantified on a per cell basis, by which the intra- and inter-cellular variation in protein-protein interactions can be assessed.
The formation and composition of DNA repair complexes and IRIFs is mostly studied in adherent cell lines such as the human bone osteosarcoma epithelial cell line U2OS, the human embryonic kidney cell line HEK293 and the retinal pigment epithelial cell line RPE-1, which are fast-growing and easy to transfect. Suspension cell cultures such a lymphoid and myeloid cell lines are used less frequently, as these are less amenable to transfection and generally do not adhere to coverslips, thus requiring additional/alternative steps for imaging. The resolution of DNA damage is however very relevant in the context of lymphoid and myeloid malignancies, as the DNA damage response is frequently affected by genomic (driver) aberrations in these tumors, playing a pivotal role in the malignant transformation of normal lymphoid and myeloid (progenitor) cells12,13,14.
This protocol describes how PLA can be used to assess and quantify protein-protein interactions following the induction of DNA damage in suspension cell cultures. Here, PLA is performed to determine and visualize the interactions between ATM and p53 upon DNA damage in human B-cell leukemia cells that are induced to undergo a G1-phase cell-cycle arrest. Of note, the protocol presented here is not restricted to studying ATM and p53 interactions in G1-arrested leukemia cells, but can also be used to visualize other protein-protein interactions in various cell types and suspension cell cultures.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BV173 cell line</td>
			<td>DSMZ</td>
			<td>AC-20</td>
			<td>BCR-ABL+ B-ALL cell line</td>
		</tr>
		<tr>
			<td>SUP-B15 cell line</td>
			<td>DSMZ</td>
			<td>ACC-389</td>
			<td>BCR-ABL+ B-ALL cell line</td>
		</tr>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td>Gibco (Life Technologies)</td>
			<td>21980-032</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Sigma Aldrich</td>
			<td>F7524</td>
			<td>lot #: 064M3396</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco (Life Technologies)</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>Gibco (Life Technologies)</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>imatinib methanesulfonate</td>
			<td>LC Laboratories</td>
			<td>I-5508</td>
			<td>Dissolve in DMSO, prepare 10 mM stock solution</td>
		</tr>
		<tr>
			<td>neocarzinostatin</td>
			<td>Sigma Aldrich</td>
			<td>N9162</td>
			<td>Mutagenic/teratogenic, handle with care</td>
		</tr>
		<tr>
			<td>KU55933</td>
			<td>Selleckchem</td>
			<td>S1092</td>
			<td>Dissolve in DMSO, prepare 5 mM stock solution</td>
		</tr>
		<tr>
			<td>Starfrost Microscopy Slides</td>
			<td>Waldemar Knittel</td>
			<td>VA11200 003FKB</td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen liquid blocker</td>
			<td>Sigma Aldrich</td>
			<td>Z377821-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytospin funnel</td>
			<td>Q Path Labonord SAS</td>
			<td>003411324</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink In Situ Red Starter Kit Goat/Rabbit</td>
			<td>Sigma Aldrich</td>
			<td>DUO92105</td>
			<td>Available for different species/combinations, also available in FarRED, Orange and Green</td>
		</tr>
		<tr>
			<td>goat-anti-ATM</td>
			<td>Bethyl Laboratories</td>
			<td>A300-136A</td>
			<td>PLA-grade; we succesfully used lot#A300-136A-1 in our studies</td>
		</tr>
		<tr>
			<td>rabbit-anti-phospho-Ser15-p53</td>
			<td>Cell Signaling Technology</td>
			<td>9284</td>
			<td>We succesfully used lot #9284-4 in our studies</td>
		</tr>
		<tr>
			<td>Vectashield antifading mounting medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield antifading mounting medium</td>
			<td>Vector Labs</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde in PBS</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-281692</td>
			<td>Also available from various other vendors</td>
		</tr>
	</tbody>
</table>

detection and visualization of dna damage-induced protein complexes in suspension cell cultures using the proximity ligation assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Phosphorylation of p53 at residue Ser15 was shown to be dependent on ATM kinase activity16. To demonstrate and confirm the specificity of the PLA technique on cytospin preparations of suspension cell cultures, it is shown that induction of DNA damage by 2 h NCS treatment of BCR-ABL+ B-ALL cells arrested in the G1-phase of the cell-cycle resulted in the specific interaction between ATM and phospho-Ser15-p53, as expected. Punctate PLA signals were observed in the nuc...

Watch this Scientific Journal Video about Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay at JoVE.com

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

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