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High Content Analysis of Proteins and Protein Interactions by Proximity Ligation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools. (Ulf Landegren)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fundamental to all biological processes is the interplay between biomolecules such as proteins and nucleic acids. Studies of interactions should therefore be more informative than mere detection of expressed proteins. Preferably, such studies should be performed in material that is as biologically and clinically relevant as possible, i.e. in primary cells and tissues. In addition, to be able to take into account the heterogeneity of such samples, the analyses should be performed in situ to retain information on the sub-cellular localization where the interactions occur, enabling determination of the activity status of individual cells and allowing discrimination between e.g. tumor cells and surrounding stroma. This requires assays with an utmost level of sensitivity and selectivity.

Taking these issues into consideration, the in situ proximity-ligation assay (in situ PLA) was developed, providing localized detection of proteins, protein-protein interactions and post-translational modifications in fixed cells and tissues. The high sensitivity and selectivity afforded by the assay's requirement for dual target recognition in combination with powerful signal amplification enables visualization of single protein molecules in intact single cells and tissue sections.

To further increase the usefulness and application of in situ PLA, the assay was adapted to high content analysis techniques such as flow cytometry and high content screening. The use of in situ PLA in flow cytometry offers the possibility for high-throughput analysis of cells in solution with the unique characteristics offered by the assay. For high content screening, it was demonstrated that in situ PLA can enable cell-based drug screening of compounds affecting post-translational modifications and protein-protein interactions in primary cells, offering superior abilities over current assays.

The methods presented in this thesis provide powerful new tools to study proteins in genetically unmodified cells and tissues, and should offer exciting new possibilities for molecular biology, diagnostics and drug discovery.



Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2010. , p. 56
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 530
Keywords [en]
in situ, proximity ligation, flow cytometry, high content screening, rolling circle amplification, in situ PLA, single-cell, single-molecule, protein interactions, drug screening, post-translational modifications
National Category
Cell and Molecular Biology
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-119530ISBN: 978-91-554-7739-4 (print)OAI: oai:DiVA.org:uu-119530DiVA, id: diva2:301950
Public defence
2010-04-16, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2010-03-25 Created: 2010-02-26 Last updated: 2018-01-12Bibliographically approved
List of papers
1. Direct observation of individual endogenous protein complexes in situ by proximity ligation
Open this publication in new window or tab >>Direct observation of individual endogenous protein complexes in situ by proximity ligation
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2006 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 3, no 12, p. 995-1000Article in journal (Refereed) Published
Abstract [en]

Cellular processes can only be understood as the dynamic interplay of molecules. There is a need for techniques to monitor interactions of endogenous proteins directly in individual cells and tissues to reveal the cellular and molecular architecture and its responses to perturbations. Here we report our adaptation of the recently developed proximity ligation method to examine the subcellular localization of protein-protein interactions at single-molecule resolution. Proximity probes-oligonucleotides attached to antibodies against the two target proteins-guided the formation of circular DNA strands when bound in close proximity. The DNA circles in turn served as templates for localized rolling-circle amplification (RCA), allowing individual interacting pairs of protein molecules to be visualized and counted in human cell lines and clinical specimens. We used this method to show specific regulation of protein-protein interactions between endogenous Myc and Max oncogenic transcription factors in response to interferon-gamma (IFN-gamma) signaling and low-molecular-weight inhibitors.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-16092 (URN)10.1038/nmeth947 (DOI)000242213900018 ()17072308 (PubMedID)
Available from: 2008-06-25 Created: 2008-06-25 Last updated: 2017-12-08Bibliographically approved
2. In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method
Open this publication in new window or tab >>In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method
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2007 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 6, no 9, p. 1500-1509Article in journal (Refereed) Published
Abstract [en]

Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor β (PDGFRβ) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRβ in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRβ in porcine aortic endothelial cells transfected with the β-receptor, but not in cells transfected with the α-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRβ. We furthermore visualized tyrosine phosphorylated PDGFRβ in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-12659 (URN)10.1074/mcp.M700166-MCP200 (DOI)000249237200004 ()17565975 (PubMedID)
Available from: 2008-05-28 Created: 2008-05-28 Last updated: 2017-12-11Bibliographically approved
3. Flow cytometric in situ proximity ligation analyses of protein interactions and post-translational modification of the epidermal growth factor receptor family
Open this publication in new window or tab >>Flow cytometric in situ proximity ligation analyses of protein interactions and post-translational modification of the epidermal growth factor receptor family
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2009 (English)In: Cytometry: Part A, ISSN 1552-4922, Vol. 75A, no 10, p. 833-839Article in journal (Refereed) Published
Abstract [en]

Interactions between members of the epidermal growth factor receptor (EGFR) family mediates cellular responses to ligand stimulation. Measurement of these interactions could provide important information and may prove useful as prognostic markers in malignancy. Therefore, to develop methods to study these interactions in genetically unmodified cells, such as clinical samples, in a sensitive and selective way, with good statistical accuracy, is important. The in situ proximity ligation assay (in situ PLA) was used to quantify homo- and heteromeric interactions between EGFR and HER2 in cultured cells, using flow cytometry as the readout method. Cells were monitored for changes in dimerization patterns and phosphorylation status upon stimulation. The different cell lines displayed varying amounts of interactions between EGFR and HER2, but the amount of dimerization was not found to be affected significantly upon stimulation by EGF. Activation of EGFR could be visualized by in situ PLA, but not by immunofluorescence staining. In situ PLA was successfully used to study receptor dimerization and activation of the EGF-receptor family with high selectivity and sensitivity. The combination of in situ PLA and flow cytometry provided a statistically powerful way of analyzing protein-protein interactions and post-translational modifications on a single-cell basis.

Keywords
proximity ligation, flow cytometry, protein interactions, epidermal growth factor receptor, HER2
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-112106 (URN)10.1002/cyto.a.20771 (DOI)000270419700004 ()19650109 (PubMedID)
Available from: 2010-01-08 Created: 2010-01-08 Last updated: 2018-12-04
4. High content screening for inhibitors of protein interactions and post-translational modifications in primary cells by proximity ligation
Open this publication in new window or tab >>High content screening for inhibitors of protein interactions and post-translational modifications in primary cells by proximity ligation
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2010 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, no 1, p. 178-183Article in journal (Refereed) Published
Abstract [en]

The cost of developing new drugs is a major obstacle for pharmaceutical companies and academia with many drugs identified in the drug discovery process failing approval for clinical use due to lack of intended effect or because of severe side effects. Since the early 1990 s, high throughput screening of drug compounds has increased enormously in capacity but has not resulted in a higher success rate of the identified drugs. Thus, there is a need for methods that can identify biologically relevant compounds and more accurately predict in vivo effects early in the drug discovery process. To address this, we developed a proximity ligation-based assay for high content screening of drug effects on signaling pathways. As a proof of concept, we used the assay to screen through a library of previously identified kinase inhibitors, including six clinically used tyrosine kinase inhibitors, to identify compounds that inhibited the platelet-derived growth factor (PDGF) receptor beta signaling pathway in stimulated primary human fibroblasts. Thirteen of the 80 compounds were identified as hits, and the dose responses of these compounds were measured. The assay exhibited a very high Z' factor (0.71) and signal to noise ratio (11.7), demonstrating excellent ability to identify compounds interfering with the specific signaling event. A comparison with regular immunofluorescence detection of phosphorylated PDGF receptor demonstrated a far superior ability by the in situ proximity ligation assay to reveal inhibition of receptor phosphorylation. In addition, inhibitor-induced perturbation of protein-protein interactions of the PDGF signaling pathway could be quantified, further demonstrating the usefulness of the assay in drug discovery.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-112102 (URN)10.1074/mcp.M900331-MCP200 (DOI)000275505900014 ()19864249 (PubMedID)
Available from: 2010-01-08 Created: 2010-01-08 Last updated: 2017-12-12Bibliographically approved

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