Detection of SMAD complex formation by Duolink™ provides qualitative information on the activation status of TGF-ß signaling pathway on single molecular interactions in unmodified cells.
Detection of SMAD complex formation with Duolink identifies activated TGF-β signaling pathways.
Duolink goes further by visualizing the subcellular localization of complexes.
Duolink provides a detailed and qualitative means
of detecting interactions of SMAD proteins with other
signaling components.
Studies of SMAD protein interactions are limited by the fact that traditional
technologies neither offer the means to work on unmodified cells nor the sensitivity to visualize individual interactions in modified cells. Duolink
™ resolves these limitations and thereby provides an excellent method
for detailed and qualitative studies of SMAD protein interactions. As SMAD complex formation predefines an active TGF-ß signaling pathway, we can assess the activation status of the pathway by observing SMAD complexes.
TGF-ß is a growth factor that controls a number of cellular processes,
and is implicated in a number of pathological situations, among others cancer and fibrosis. After the ligand binds to its receptors, it activates downstream effectors of the SMAD protein
family. SMAD proteins are transcription factors that once phosphorylated by the TGF-ß receptors become activated, translocate to the nucleus where they hetero-oligomerize and regulate transcription of target genes (for an extensive review see Feng, et al. 2005).
Although the TGF-ß signaling pathway has been extensively studied, several questions about the regulation of the pathway remain unanswered, due to the lack of methods sensitive and specific enough to detect endogenous protein interactions. Furthermore, detecting an activated TGF-ß signaling pathway in unmodified tissue samples is essential in a number of pathological
conditions.
The in situ Proximity Ligation Assay
and the Duolink detection kit
Traditional cell imaging methods like immunohistochemistry (IHC) can detect the presence of specific proteins, but observations are semi-quantitative at best and are limited to detecting presence of proteins but not protein interactions.
There has long been a need for better methods to quantify the interaction
of proteins objectively as well as to visualize interactions of proteins in situ at naturally occurring levels.
The in situ Proximity Ligation Assay (in situ PLA) recently described by Söderberg
et al. (Nature Methods, 2006) resolves these limitations by providing a sensitive means to detect and visualize proteins as well as individual protein-protein interactions in situ. Importantly, in situ PLA allows point detection of individual endogenous proteins which reveals both their subcellular locations and the frequency of occurrences. This opens completely new opportunities to accurately quantify proteins and protein interactions in unmodified single cells and tissues.
Duolink™ from Olink Bioscience provides a generic approach to using the in situ Proximity Ligation Assay with any pair of antibodies, based on secondary antibody labelling. Duolink is based on the recognition of proteins by the binding
of two secondary antibodies to two primary antibodies, which in turn are bound to selected epitopes on the target protein(s). This is followed by DNA amplification that occurs when the secondary antibodies are in close proximity and fluorescent labeling of the DNA product (for a detailed technical description,
see the Principle of Duolink note). No modification of cells or tissues is required beyond fixation using standard methods.
Evaluation of the primary antibodies
used for in situ PLA
A pre-study was carried out to confirm that the primary antibodies used for in situ PLA showed sufficient affinities for their respective proteins, SMAD1/2/3 (Figure 1) and SMAD4 (Figure 2) in the cell system studied. Figure 1 shows a translocation of SMAD2 and SMAD3 proteins to the cell nucleus after 45 minutes
stimulation with TGF-ß. Similarly, Figure 2 shows a translocation of SMAD4 proteins to the nucleus after stimulation. Neither of these experiments can, however, demonstrate the formation of complexes. This pre-study was useful
in several respects. It confirmed that the fixation/blocking steps in sample preparation made the protein epitopes available, that the antibodies showed affinity for the epitopes, and that the cells responded to TGF-ß stimulation, thereby verifying the activation of the pathway by TGF-ß.
Analysis of Heterodimerization of SMAD1/2/3 and SMAD4 by Duolink
in situ PLA enables point detection, i.e. it generates a discrete signal for each detected event. The discrete nature of the assay facilitates an objective approach
to image analysis and comparisons of SMAD complex numbers in unstimulated
cells to TGF-ß stimulated cells.
After stimulation, a higher frequency of events is observed in the cytoplasm, and events localized in the nucleus are clearly discernable. This confirmed that SMAD1/2/3-SMAD4 complexes are formed after TGF-ß stimulation as well as that complexes are translocated to the nucleus (Figures 3 and 5).
Methodological considerations for the design
of in situ PLA assays
As for any method used to detect specific molecules in cells and tissues, special
considerations must be taken that may be unique to your particular material
and analyte to make them amenable to treatment and detection. You may need to optimize analyte-specific aspects such as the permeability of cellular compartments and the availability of epitopes through fixation, and the elimination
of non-specific background by the use of special blocking agents. We have found that assay design is greatly improved by testing different combinations of antibodies, and by having access to appropriate control samples. Depending on the protein or interaction being assayed, it may be important to evaluate the choice of epitopes that are available for labeling.
Methods
Sample preparation
Mouse embryonic fibroblasts (MEF) were seeded on glass slides in regular culture medium (DMEM supplemented with 10% FBS in the presence of antibiotics)
and allowed to adhere overnight. The TGF-ß signaling pathway was inhibited using 5 µM of the GW6604 low molecular weight inhibitor for 2 h prior to stimulation. Then the cells were rinsed twice with DMEM and either were stimulated with 20 ng/ml TGF-ß or remained unstimulated for 45 min. Then the cells were rinsed briefly with PBS and fixed with freshly prepared 3% PFA for 30 min, rinsed with PBS, and the cells were permeabilized with 0.5 % TritonX-100 in PBS for 10 min, again rinsed with PBS and 70% ethanol, air dried and stored at -20 0C until analyzed with the Duolink assay according to the manufacturer’s instructions (available at www.olink.com).
Imaging
Image acquisition was made at 20X magnification (Zeiss Plan-Apochromat, 20X/0.8NA dry) with the Zeiss Axio Imager M1 fluorescence microscope and the AxioVision 4.5 software (Carl Zeiss). Images were captured with an AxioCam
MRm-camera with an extension tube of 1X (Zeiss 60N-C 1” 1x). Filters used were 575/605 nm (in situ PLA signal) and 350/461 nm (nuclear stain). A Z-stack of 20 images was taken at 20X magnification of the in situ PLA image channel. We acquired one image of nuclei in the Hoechst/DAPI channel by auto-exposure and auto-focus. Typically, an image field contained 25- 30 cells.
Image Analysis
Cell images acquired using the AxioVision software were exported in TIFF format
for subsequent analysis in the BlobFinder image analysis software. The analysis program first performs a signal enhancement of the stack, upon which the rest of the image analysis is based. To perform batch analysis of image stacks, one representative stack of images was selected and a configuration file was created and applied on the batch. Output from the analysis contained data showing both the dots per single cell as well as a maximum intensity projection
of the raw image data side-by-side with the segmented image showing identified nuclei and the Duolink assay signals.
References and footnotes
Allalou A. and Wählby C. BlobFinder image analysis software:
www.cb.uu.se/~amin/BlobFinder
Söderberg O, Gullberg M, Jarvius M, Ridderstråle K, Leuchowius KJ, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson LG, Landegren U. Direct observation of individual endogenous protein complexes in situ by proximity ligation.
Nature Methods. 2006 Dec;3(12):995-1000. Epub 2006 Oct 29.
Feng X. H., Derynck R. Specificity and versatility in TGF-ß signaling through SMADs. Annu Rev Cell Dev Biol. 2005. 21: 659-93).
Olink Bioscience is a privately held company based in Uppsala, Sweden, founded
in 2004 by Prof. Ulf Landegren and partners. Olink focuses on innovative methods for detecting endogenous proteins and protein complexes for basic research and high-content screening to deliver accurate information on disease mechanisms and drug response. Our mission is to contribute to a better understanding
of the interactome. For more information, visit www.olink.com
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