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Application of Proximity Ligation Assay for Multidirectional Studies on Transforming Growth Factor-β Pathway
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. (Ola Söderberg)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A comprehensive understanding of how the body and all its components function is essential when this knowledge is exploited for medical purposes. The achievements in biological and medical research during last decades has provided us with the complete human genome and identified signaling pathways that governs the cellular processes that facilitates the development and maintenance of higher order organisms. This has brought about the realization that diseases such as cancer is a consequence of genomic aberrations that effects these signaling pathways, endowing cancer cells with the capacity to circumvent homeostasis by acquiring features like self-sustained proliferation and insensitivity to apoptosis. The increased understanding of biology and medicine has been made possible by the development of advanced methods to carry out biological and clinical analyses. The demands of a method often differ regarding in what context it will be applied. It may be acceptable for method to be laborious and time consuming if it is used in basic research, but for medical purposes molecular methods need to be fast and straightforward to perform. Innovative technologies should preferentially address the demands of both researchers and clinicians and provide data not possible to obtain by other methods. An example of such a method is the in situ proximity ligation assay (in situ PLA). In this thesis I have used this method to determine the activity status, at the single-cell level, of the transforming growth factor-β (TGF-β) signaling pathway and activating protein-1 (AP-1) family of transcription factors.  Both of these pathways are frequently involved in cancer development and progression. In addition to this research I herein also present further modifications of in situ PLA, and analyses thereof, to increase the utility and resolution of this assay.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. , 43 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 763
Keyword [en]
proximity ligation, TGF-β signaling, Smad, single cell analysis, cell cycle, context-dependent signaling, CRC
National Category
Cell and Molecular Biology Cancer and Oncology Biomedical Laboratory Science/Technology
Research subject
Molecular Cellbiology; Medicinal Chemistry; Molecular Biology
URN: urn:nbn:se:uu:diva-171952ISBN: 978-91-554-8336-4OAI: diva2:513327
Public defence
2012-05-25, Rudbecksalen, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Available from: 2012-05-02 Created: 2012-03-29 Last updated: 2013-09-02Bibliographically approved
List of papers
1. Intercellular variation in signaling through the TGF-β pathway and its relation to cell densityand cell cycle phase
Open this publication in new window or tab >>Intercellular variation in signaling through the TGF-β pathway and its relation to cell densityand cell cycle phase
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2012 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, no 7, M111.013482Article in journal (Refereed) Published
Abstract [en]

Fundamental open questions in signal transduction remain concerning the sequence and distribution of molecular signaling events among individual cells. In this work we have characterized the intercellular variability of transforming growth factor β-induced Smad interactions, providing essential information about TGF-β signaling and its dependence on the density of cell populations and the cell-cycle phase. By employing the recently developed in situ proximity ligation assay, we investigated the dynamics of interactions and modifications of Smad proteins and their partners under native and physiological conditions. We analyzed the kinetics of assembly of Smad complexes and the influence of cellular environment and relation to mitosis. We report rapid kinetics of formation of Smad complexes, including native Smad2-Smad3-Smad4 trimeric complexes, in a manner influenced by the rate of proteasomal degradation of these proteins, and we found a striking cell to cell variation of signaling complexes. The single-cell analysis of TGF-β signaling in genetically unmodified cells revealed previously unknown aspects of regulation of this pathway, and it provided a basis for analysis of these signaling events to diagnose pathological perturbations in patient samples, and to evaluate their susceptibility to drug treatment.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2012
National Category
Cell Biology
Research subject
Molecular Cellbiology
urn:nbn:se:uu:diva-171949 (URN)10.1074/mcp.M111.013482 (DOI)000306411300017 ()22442258 (PubMedID)
Available from: 2012-03-29 Created: 2012-03-29 Last updated: 2015-05-20Bibliographically approved
2. Specific interactions between Smad proteins and AP-1 components determine TGFβ-induced breast cancer cell invasion
Open this publication in new window or tab >>Specific interactions between Smad proteins and AP-1 components determine TGFβ-induced breast cancer cell invasion
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2013 (English)In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 32, no 31, 3606-3615 p.Article in journal (Refereed) Published
Abstract [en]

Deregulation of the transforming growth factor β (TGFβ) signal transduction cascade is functionally linked to cancer. In early phases, TGFβ acts as a tumor suppressor by inhibiting tumor cell proliferation, whereas in late phases, it can act as a tumor promoter by stimulating tumor cell invasion and metastasis. Smad transcriptional effectors mediate TGFβ responses, but relatively little is known about the Smad-containing complexes that are important for epithelial-mesenchymal transition and invasion. In this study, we have tested the hypothesis that specific members of the AP-1 transcription factor family determine TGFβ signaling specificity in breast cancer cell invasion. Using a 3D model of collagen-embedded spheroids of MCF10A-MII premalignant human breast cancer cells, we identified the AP-1 transcription factor components c-Jun, JunB, c-Fos and Fra1 as essential factors for TGFβ-induced invasion and found that various mesenchymal and invasion-associated TGFβ-induced genes are co-regulated by these proteins. In situ proximity ligation assays showed that TGFβ signaling not only induces complexes between Smad3 and Smad4 in the nucleus but also complexes between Smad2/3 and Fra1, whereas complexes between Smad3, c-Jun and JunB could already be detected before TGFβ stimulation. Finally, chromatin immunoprecipitations showed that c-Jun, JunB and Fra1, but not c-Fos, are required for TGFβ-induced binding of Smad2/3 to the mmp-10 and pai-1 promoters. Together these results suggest that in particular formation of Smad2/3-Fra1 complexes may reflect activation of the Smad/AP-1-dependent TGFβ-induced invasion program.

National Category
Medical and Health Sciences
urn:nbn:se:uu:diva-180124 (URN)10.1038/onc.2012.370 (DOI)000322638400005 ()22926518 (PubMedID)

Agata Zieba & Eleftheria Vasilaki contributed equally to this work.

Available from: 2012-08-30 Created: 2012-08-30 Last updated: 2013-11-19Bibliographically approved
3. A detailed analysis of 3D subcellular signal localization
Open this publication in new window or tab >>A detailed analysis of 3D subcellular signal localization
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2009 (English)In: Cytometry Part A, ISSN 1552-4922, Vol. 75A, no 4, 319-328 p.Article in journal (Refereed) Published
Abstract [en]

Detection and localization of fluorescent signals in relation to other subcellular structures is an important task in various biological studies. Many methods for analysis of fluorescence microscopy image data are limited to 2D. As cells are in fact 3D structures, there is a growing need for robust methods for analysis of 3D data. This article presents an approach for detecting point-like fluorescent signals and analyzing their subnuclear position. Cell nuclei are delineated using marker-controlled (seeded) 3D watershed segmentation. User-defined object and background seeds are given as input, and gradient information defines merging and splitting criteria. Point-like signals are detected using a modified stable wave detector and localized in relation to the nuclear membrane using distance shells. The method was applied to a set of biological data studying the localization of Smad2-Smad4 protein complexes in relation to the nuclear membrane. Smad complexes appear as early as 1 min after stimulation while the highest signal concentration is observed 45 min after stimulation, followed by a concentration decrease. The robust 3D signal detection and concentration measures obtained using the proposed method agree with previous observations while also revealing new information regarding the complex formation.

3D image analysis, fluorescence signal segmentation, subcellular positioning, Smad detection
National Category
Computer and Information Science
urn:nbn:se:uu:diva-98014 (URN)10.1002/cyto.a.20663 (DOI)000264513800006 ()
Available from: 2009-02-05 Created: 2009-02-05 Last updated: 2012-05-09Bibliographically approved
4. Bright-Field Microscopy Visualization of Proteins and Protein Complexes by In Situ Proximity Ligation with Peroxidase Detection
Open this publication in new window or tab >>Bright-Field Microscopy Visualization of Proteins and Protein Complexes by In Situ Proximity Ligation with Peroxidase Detection
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2010 (English)In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 56, no 1, 99-110 p.Article in journal (Refereed) Published
Abstract [en]


The in situ proximity ligation assay (PLA) allows a protein or protein complex to be represented as an amplifiable DNA molecule. Recognition is mediated by proximity probes consisting of antibodies coupled with oligonucleotides. Upon dual binding of the proximity probes, the oligonucleotides direct the formation of a circular DNA molecule, which is then amplified by rolling-circle replication. The localized concatemeric product is then detected with fluorescent probes. The in situ PLA enables localized detection of individual native proteins or interacting protein pairs in fixed cells or tissue sections, thus providing an important tool for basic and clinical research.


We used horseradish peroxidase (HRP)conjugated oligonucleotides to couple in situ PLA with enzymatic visualization of the localized detection event.


We demonstrate the detection of protein complexes, both in cells and in tissue sections, and show that we can quantify the complexes with image-analysis software specially developed for recognizing HRP signals in bright-field microscopy images. We show that fluorescence and HRP signals produce equivalent results, both ill cultured cells and in tissue samples.


The combination of in situ PLA with bright-field detection and automated image analysis allows the signals present to be Counted in an automated fashion and thus provides a sensitive and specific method for quantification of proteins and protein complexes with bright-field microscopy. With this approach, in situ PLA can be used without the requirement for expensive fluorescence microscopes, thereby avoiding problems with nonspecific fluorescence while maintaining compatibility with conventional histologic staining.

National Category
Medical Genetics Bioinformatics and Systems Biology
Research subject
Clinical Genetics; Computerized Image Analysis
urn:nbn:se:uu:diva-111499 (URN)10.1373/clinchem.2009.134452 (DOI)000273466300016 ()
Available from: 2009-12-15 Created: 2009-12-15 Last updated: 2012-05-09Bibliographically approved

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