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. 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.
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.
1. Immunofluorescence
2. Immunoprecipitation
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.
Figure 1: Representative immunofluorescence results. HEK293T cells were cultured on coverslides. Cells were treated or not treated with rapamycin (Rapa, 200 nM, 16 h) or Torin 1 (Torin,250 nM, 3 h), or starved in EBSS (Stv,2 h). Then endogenous proteins were immunostained by using anti-RACK1 and anti-LC3 primary antibodies. Cells were analyzed under a confocal microscope. CNT,non-treated cells; Merge, overlay of greenand redsignals. White arrowsshow yellowcytoplasmic dots with RACK1 and LC3 co-localization. This research was originally published by Erbil et al.8 Please click here to view a larger version of this figure.
In Figure 2, an example of an endogenous immunoprecipitation result is presented. In this figure, ATG5 protein was precipitated and RACK1 co-immunoprecipitation was detected under different conditions. Normal rabbit serum was used as a negative control.
Figure 2: Representative immunoprecipitation results. HEK293T cells were treated or not treated with rapamycin (Rapa ,200 nM, 16 h) or Torin 1 (Torin, 250 nM, 3 h), or starved in EBSS (2 h). Endogenous ATG5 protein was immunoprecipitated from cell extracts using anti-ATG5 antibodies that were coupled to protein A Plus beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Serum,control rabbit serum. This research was originally published by Erbil et al.8. Please click here to view a larger version of this figure.
These results indicate that RACK1 protein is involved in autophagy pathways. RACK1 protein co-localized with the LC3 protein on the autophagosome-like structures during autophagy induction. Moreover, co-immunoprecipitation results showed that RACK1 and ATG5 proteins were interacting even at basal conditions, and the interaction levels increased under autophagy activating conditions. We previously confirmed using p62 degradation and LC3-II accumulation tests in the presence or absence of lysosomal inhibitors (Bafilomycin A, E64D/Pepstatin A) that autophagic flux was not inhibited under the experimental conditions8. Therefore, the results presented here and elsewhere showed that RACK1-ATG5 interaction is important for the initial stages of autophagy.
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. Additionally, use positive controls, or treat cells with a known inducer of the protein of interest. Batch to batch variations of antibodies exist as well. Moreover, some polyclonal antibodies or sera might have high background and non-specific bands. Although some of these bands may be alternative forms of the protein of interest, in general they result from cross-reactivity of polyclonal antibodies with related proteins or they may be non-specific interactors. Monoclonal antibodies are in general more specific in this sense, although signals generated can be weaker. The confirmation of the antibody specificity with tagged proteins and established anti-tag antibodies may be useful, but endogenous interactions are still more valuable and essential.
Co-localization or co-immunoprecipitation tests will not definitively establish whether protein-protein interactions are direct. It is possible that additional partners can mediate the observed interactions. Indeed, in Figure 1, a partial co-localization of RACK1 and LC3 proteins was observed, which could be a sign of their interaction; however, direct binding tests such as in vitro pull-down assays using recombinant proteins or Fluorescence Resonance Energy Transfer (FRET) microscopy may be used to establish whether the observed interactions are direct or not. On the other hand, techniques such as gel filtration will help to uncover the complex protein interactions involving more than two proteins in a more convincing manner. A more rigorous analysis of the immunofluorescence results from an autophagy perspective would be to monitor autophagy induction by counting the number of dots that are positive for the LC3 autophagy marker in every cell, since an increase of the number of LC3 positive dots correlates with the number of autophagosomes. Usage of the Pearson's coefficient, Li's method, or Manders' coefficients tests provide better quantitative and comparative assessment of the experimental results.
Another important point is the validation of interactions using independent techniques and different cell lines. Some interactions may be artifacts that are related to the stickiness of proteins or protein overexpression. If not carefully washed, the ATG5 protein is also prone to stick to the beads that are used in immunoprecipitations (both agarose or sepharose beads). Additionally, in immunofluorescence tests, protein overexpression can form aggregates that look like autophagosomes or autolysosomes. Therefore, while performing transient transfections using co-transfected fluorescent proteins as the transfection efficacy control, in immunoblots, expression levels of proteins of interest can be compared to fluorescent protein levels in the extracts using specific antibodies, providing a method of normalization.
In all these tests, reproducibility of results is of utmost importance. We prefer to repeat each experiment at least 3-4 times to be convinced of obtained results. We also perform similar experiments in at least two different cell lines to prove that our observations are not cell type-specific. Additionally, correct positive and negative controls (e.g., beads alone or control antibodies plus serum for immunoprecipitation tests) should be planned to reach correct conclusions. During optimization of immunofluorescence techniques, it is useful to have a secondary antibody only control where primary antibody is omitted. This control make sure that observed signals originate from the primary antibody binding to the protein of interest, and not from a non-specific binding of the secondary antibody.
The number of protein complexes regulating autophagy and other biological events are increasing in discovery, yet the research is far from having a complete picture. Although high-throughput techniques such as mass spectrometry or bioinformatics-based predictions continue to reveal networks of interaction for several autophagy-related proteins, confirmation of these interactions using classical biochemistry and microscopy methods is important in order to uncover functional and dynamic properties of this basic and important cellular pathway.
The authors have nothing to disclose.
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.
Trypsin EDTA Solution A | Biological Industries | BI03-050-1A | |
PBS | GE Healthcare | SH-30256.01 | |
DMEM (high glucose) | Sigma | 5671 | |
DMEM (low glucose) | Sigma | 5546 | |
Trypan Blue | Sigma | T8154 | |
Hemocytometer | Sigma | Z359629-1EA | |
coverslides | Jena Bioscience | CSL-103 | |
slides | Isolab | I.075.02.005 | |
Poly-L-Lysine | Sigma | P8920 | |
Torin | Tocris | 4247 | |
DMSO | Sigma | VWRSAD2650 | |
EBSS | Biological Industries | BI02-010-1A | |
Paraformaldehyde (PFA) | Sigma | 15812-7 | |
BSA | Sigma | A4503 | |
Saponin | Sigma | 84510 | |
LC3 Antibody | Sigma | L7543 | |
Anti-Rabbit IgG Alexa Fluor 568 | Invitrogen | A11011 | |
RACK1 Antibody | Santa Cruz Biotechnology | sc-17754 | |
Anti-Mouse IgG Alexa Fluor 488 | Invitrogen | A11001 | |
NP-40 | Applichem | A16694.0250 | |
Sodium Chloride | Applichem | A9242.5000 | |
Sodium deoxycholate | Sigma | 30970 | |
Sodium dodecyl sulphate (SDS) | Biochemika | A2572 | |
Trizma Base | Sigma | T1503 | |
Triton-X | Applichem | 4975 | |
Sodium orthovanadate | Sigma | 450243 | |
ATG5 Antibody | Sigma | A0856 | |
Glycerol | Applichem | A4453 | |
β-Mercaptoethanol | Applichem | A1108.0250 | |
Bromophenol blue | Applichem | A3640.0005 | |
Non-Fat milk | Applichem | A0830 | |
Tween 20 | Sigma | P5927 | |
Sodium Azide | Riedel de Haen | 13412 | |
Phenol red | Sigma | 114537-5G | |
anti-rabbit IgG , HRP conjugated | Jackson Immuno. | 1110305144 | |
Luminol | Fluka | 9253 | |
Coumeric Acid | Sigma | C9008 | |
Hydrogen Peroxide | Merck | K35522500604 | |
anti mouse IgG, HRP conjugated | Jackson Immuno. | 115035003 | |
β-Actin Antibody | Sigma | A5441 | |
Normal rabbit serum | Santa Cruz Biotechnology | sc-2027 | |
Rapamycin | Sigma | R0395 | |
Protein A-Agarose Beads | Santa Cruz Biotechnology | sc-2001 | |
fetal bovine serum | Biowest | S1810-500 | |
penicillin/streptomycin solution | Biological Industries | 03-031-1B | |
L-glutamine | Biological Industries | BI03-020-1B | |
Bradford Solution | Sigma | 6916 | |
Nitocellulose membrane | GE Healthcare | A10083108 | |
X-ray Films | Fujifilm | 47410 19289 | |
Protease inhibitor | Sigma | P8340 |