Name: study of protein-protein interactions in autophagy research
Uploaded: 2017-09-09T13:00:00
Description: Watch this Scientific Journal Video about Study of Protein-protein Interactions in Autophagy Research at JoVE.com

This work was supported by Scientific and Technological Research Council of Turkey (TUBITAK) 1001 Grant 107T153, the Sabanci University. SEB and NMK are supported by a TUBITAK BIDEB 2211 Scholarship for Ph.D. studies.

1. Immunofluorescence
<ol>
	<li>Maintain HEK293T human embryonic kidney cells in DMEM high glucose medium, and N2A mouse neuroblast cells in DMEM low glucose medium, in a 5% CO2-humidified incubator at 37 &#176;C. Supplement the culture media with 10% heat-inactivated fetal bovine (FBS) serum, antibiotics (50 U/mL penicillin, 50 &#181;g/mL streptomycin), and L-glutamine (2 mM).</li>
	<li>Detach the cells by using 0.25% trypsin. First remove the media of the cell culture then wash the cells with 10 mL sterile...

<ol>
	<li>Oral, O., Akkoc, Y., Bayraktar, O., Gozuacik, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+and+pathological+significance+of+the+molecular+cross-talk+between+autophagy+and+apoptosis.">Physiological and pathological significance of the molecular cross-talk between autophagy and apoptosis.</a> Histol Histopathol. 31, 479-498 (2016).</li><li>Korkmaz, G., Tekirdag, K. A., Ozturk, D. G., Kosar, A., Sezerman, O. U., Gozuacik, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MIR376A+is+a+regulator+of+starvation-induced+autophagy.">MIR376A is a regulator of starvation-induced autophagy.</a> PLoS One. 8, e82556 (2013).</li><li>Itah, Z., 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=Hydrodynamic+cavitation+kills+prostate+cells+and+ablates+benign+prostatic+hyperplasia+tissue.">Hydrodynamic cavitation kills prostate cells and ablates benign prostatic hyperplasia tissue.</a> Exp Biol Med (Maywood). 238, 1242-1250 (2013).</li><li>Gozuacik, D., Kimchi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+as+a+cell+death+and+tumor+suppressor+mechanism).">Autophagy as a cell death and tumor suppressor mechanism).</a> Oncogene. 23, 2891-2906 (2004).</li><li>Erzurumlu, Y., Kose, F. A., Gozen, O., Gozuacik, D., Toth, E. A., Ballar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+IBMPFD-related+P97/VCP+mutation+with+differential+binding+pattern+and+subcellular+localization.">A unique IBMPFD-related P97/VCP mutation with differential binding pattern and subcellular localization.</a> Int J Biochem Cell Biol. 45, 773-782 (2013).</li><li>Kuzuoglu-Ozturk, D., 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=Autophagy-related+gene,+TdAtg8,+in+wild+emmer+wheat+plays+a+role+in+drought+and+osmotic+stress+response.">Autophagy-related gene, TdAtg8, in wild emmer wheat plays a role in drought and osmotic stress response.</a> Planta. 236, 1081-1092 (2012).</li><li>Longatti, A., Tooze, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+trafficking+and+autophagosome+formation.">Vesicular trafficking and autophagosome formation.</a> Cell Death Differ. 16, 956-965 (2009).</li><li>Erbil, 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=RACK1+Is+an+Interaction+Partner+of+ATG5+and+a+Novel+Regulator+of+Autophagy.">RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy.</a> J Biol Chem. 291, 16753-16765 (2016).</li><li>Gozuacik, D., Yagci-Acar, H. F., Akkoc, Y., Kosar, A., Dogan-Ekici, A. I., Ekici, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticancer+use+of+nanoparticles+as+nucleic+acid+carriers.">Anticancer use of nanoparticles as nucleic acid carriers.</a> J Biomed Nanotechnol. 10, 1751-1783 (2014).</li><li>Phizicky, E. M., 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=Protein-protein+interactions:+methods+for+detection+and+analysis.">Protein-protein interactions: methods for detection and analysis.</a> Microbiol Rev. 59, 94-123 (1995).</li><li>Uetz, 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=A+comprehensive+analysis+of+protein-protein+interactions+in+Saccharomyces+cerevisiae.">A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae.</a> Nature. 403, 623-627 (2000).</li></ol>

Immunoprecipitation and immunofluorescence techniques are crucial for the study of protein-protein interactions. Although these two techniques are commonly used and well-established, several criteria should be considered to define the quality of experiments while using these techniques.
First, primary antibodies that are used in these tests should be specific to the proteins of interest. To ensure this, use shRNA knockdowns or knockout cells to test the specificity of the antibody in question....

Macroautophagy (autophagy, herein) is a cellular stress mechanism that is characterized by the sequestration of bulk cytoplasm, proteins, and organelles in double membrane vesicles called autophagic vesicles. Through fusion of the outer layer of the double membrane, autophagic vesicles and their cargo are delivered to lysosomes and degraded therein1. Autophagy occurs at low basal levels in all cell types and in all organisms, performing homeostatic functions such as protein degradation and organelle (e.g., mitochondria) turnover. Under conditions leading to cellular stress, such as starvation, autophagy is rapidly upregulated and allows the cell to maintain energy levels and basic metabolism1,2,3.
Around 30 autophagy genes have been cloned from the yeast and their protein products were shown to play a role in various stages of the autophagic process, including vesicle nucleation, expansion, vesicle fusion to late endosome/lysosome, and cargo degradation4,5. Orthologs of the majority of these genes have been identified and studies in various organisms confirmed preservation of their cellular functions6. Studies in the last decade showed that several autophagy-related protein complexes and protein-protein interactions exist and that they govern autophagy pathways in an intricate and controlled manner. Intersections, backups, feedback, and feedforward mechanisms exist, and they allow the cell to coordinate autophagy with other related events (such as vesicular secretion, lysosome biogenesis, endosomal sorting and transport7, etc.) In an unbiased yeast-two hybrid screen using the autophagy protein ATG5 as a bait, (ATG5 is a key autophagy protein involved in the E2-like conjugating system that mediates LC3 lipidation in starvation induced autophagy), we have identified Receptor Activated C-Kinase 1 (RACK1; GNB2L1) as a strong interactor and a novel autophagy component8. Importantly, the screen showed that the ATG5-RACK1 interaction was indispensable for autophagy induction by classical autophagy inducers (i.e., starvation and mTOR inhibition).
Immunofluorescence-based methods are commonly used to monitor protein-protein interactions. These techniques are mainly antibody-based, and help to visualize interactions and confirm cellular localizations. In this technique, fluorescent tag conjugated antibodies that are specific to proteins of interest are generally used for specific staining. Each protein may be labeled with antibodies coupled to different fluorescent dyes. Using protein-specific antibodies, an overlap in the signal when images are merged indicates the co-localization of proteins under confocal microscopy. The technique is applicable to cells or even tissues. Immunofluorescence techniques provide clues about interaction dynamics, and help identify the size and distribution of protein complexes, while tracking general changes in cellular morphology under different conditions9. Immunoprecipitation is another commonly used antibody-based technique that allows for the analysis of interactions between given proteins10. Using this technique, proteins of interest are isolated from cells or tissue extracts using specific antibodies, resulting in the precipitation of proteins that are in a complex or in contact with a protein of interest. Co-immunoprecipitation, where the protein and its co-interactor are detected, reveals not only the interaction between the two proteins, but can measure its strength of interaction under different circumstances11.
This protocol describes in detail key techniques that were used to confirm and characterize ATG5-RACK1 and RACK1-LC3 interaction. The focus is on immunofluorescence and immunoprecipitation techniques, with emphasis of critical steps and pitfalls for autophagy research, as well as troubleshooting suggestions.

<table border="0" cellpadding="0" cellspacing="0" width="716">
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		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Trypsin EDTA Solution A</td>
			<td style="width:181px;">Biological Industries&#160;</td>
			<td style="width:133px;">BI03-050-1A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">PBS</td>
			<td style="width:181px;">GE Healthcare</td>
			<td style="width:133px;">SH-30256.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">DMEM (high glucose)</td>
			<td style="width:181px;">Sigma&#160;</td>
			<td style="width:133px;">5671</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">DMEM (low glucose)</td>
			<td style="width:181px;">Sigma</td>
			<td>5546</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Trypan Blue</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:133px;">T8154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Hemocytometer</td>
			<td style="width:181px;">Sigma</td>
			<td>Z359629-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">coverslides</td>
			<td>Jena Bioscience</td>
			<td>CSL-103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">slides</td>
			<td style="width:181px;">Isolab</td>
			<td>I.075.02.005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Poly-L-Lysine</td>
			<td style="width:181px;">Sigma&#160;</td>
			<td style="width:133px;">P8920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Torin</td>
			<td>Tocris</td>
			<td style="width:133px;">4247</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">DMSO</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:133px;">VWRSAD2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">EBSS</td>
			<td style="width:181px;">Biological Industries&#160;</td>
			<td style="width:133px;">BI02-010-1A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Paraformaldehyde (PFA)</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:133px;">15812-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">BSA</td>
			<td>Sigma</td>
			<td style="width:133px;">&#160;A4503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Saponin</td>
			<td>Sigma</td>
			<td style="width:133px;">84510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">LC3 Antibody</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:133px;">L7543</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Anti-Rabbit IgG Alexa Fluor 568</td>
			<td style="width:181px;">&#160;Invitrogen</td>
			<td style="width:133px;">A11011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">RACK1 Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td style="width:133px;">&#160;sc-17754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse IgG Alexa Fluor 488</td>
			<td style="width:181px;">&#160;Invitrogen</td>
			<td style="width:133px;">A11001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">NP-40</td>
			<td style="width:181px;">Applichem</td>
			<td style="width:133px;">A16694.0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Sodium Chloride</td>
			<td style="width:181px;">Applichem</td>
			<td style="width:133px;">A9242.5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium deoxycholate</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:133px;">30970</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium dodecyl sulphate (SDS)</td>
			<td style="width:181px;">Biochemika</td>
			<td style="width:133px;">A2572</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Trizma Base</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:133px;">T1503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton-X&#160;</td>
			<td style="width:181px;">Applichem</td>
			<td style="width:133px;">4975</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium orthovanadate</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:133px;">450243</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">ATG5 Antibody</td>
			<td>Sigma</td>
			<td style="width:133px;">A0856</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td style="width:181px;">Applichem</td>
			<td style="width:133px;">A4453</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-Mercaptoethanol&#160;</td>
			<td style="width:181px;">Applichem</td>
			<td style="width:133px;">A1108.0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromophenol blue&#160;</td>
			<td style="width:181px;">Applichem</td>
			<td style="width:133px;">A3640.0005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Non-Fat milk</td>
			<td style="width:181px;">Applichem</td>
			<td style="width:133px;">A0830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Tween 20</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:133px;">P5927</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Sodium Azide</td>
			<td style="width:181px;">Riedel de Haen</td>
			<td style="width:133px;">13412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol red&#160;</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:133px;">114537-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">anti-rabbit IgG , HRP conjugated</td>
			<td>Jackson Immuno.&#160;</td>
			<td style="width:133px;">1110305144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Luminol</td>
			<td style="width:181px;">Fluka</td>
			<td style="width:133px;">9253</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Coumeric Acid</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:133px;">C9008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Hydrogen Peroxide</td>
			<td style="width:181px;">Merck</td>
			<td style="width:133px;">K35522500604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti mouse IgG, HRP conjugated</td>
			<td style="width:181px;">Jackson Immuno.</td>
			<td style="width:133px;">115035003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-Actin Antibody</td>
			<td style="width:181px;">Sigma&#160;</td>
			<td style="width:133px;">A5441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal rabbit serum</td>
			<td style="width:181px;">Santa Cruz Biotechnology</td>
			<td style="width:133px;">sc-2027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Rapamycin</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:133px;">&#160;R0395</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Protein A-Agarose Beads</td>
			<td style="width:181px;">Santa Cruz Biotechnology</td>
			<td style="width:133px;">sc-2001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal bovine serum&#160;</td>
			<td style="width:181px;">Biowest</td>
			<td>S1810-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">penicillin/streptomycin solution</td>
			<td style="width:181px;">Biological Industries&#160;</td>
			<td>03-031-1B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine&#160;</td>
			<td style="width:181px;">Biological Industries&#160;</td>
			<td>BI03-020-1B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Bradford Solution</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:133px;">6916</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Nitocellulose membrane</td>
			<td style="width:181px;">GE Healthcare</td>
			<td style="width:133px;">A10083108</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:235px;">X-ray Films</td>
			<td style="width:181px;">Fujifilm</td>
			<td style="width:133px;">47410 19289</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Protease inhibitor</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:133px;">P8340</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In Figure 1 an example of a co-localization result obtained using this protocol is shown. A figure from our recent paper is presented8. Here, endogenous RACK1 protein was stained in green, while endogenous LC3 was stained in red. The yellow dots that are observed in the merged pictures indicate sites of overlap between the green and red signals. Thus the yellow dots represent the partial co-localization of these two proteins.
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Watch this Scientific Journal Video about Study of Protein-protein Interactions in Autophagy Research at JoVE.com

Study of Protein-protein Interactions in Autophagy Research

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.

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

Research

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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 ...