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Direct observation of individual endogenous protein complexes in situ by proximity ligation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
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2006 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 3, no 12, 995-1000 p.Article 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.

Place, publisher, year, edition, pages
2006. Vol. 3, no 12, 995-1000 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-16092DOI: 10.1038/nmeth947ISI: 000242213900018PubMedID: 17072308OAI: oai:DiVA.org:uu-16092DiVA: diva2:43863
Available from: 2008-06-25 Created: 2008-06-25 Last updated: 2017-12-08Bibliographically approved
In thesis
1. High Content Analysis of Proteins and Protein Interactions by Proximity Ligation
Open this publication in new window or tab >>High Content Analysis of Proteins and Protein Interactions by Proximity Ligation
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. 56 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 530
Keyword
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:nbn:se:uu:diva-119530 (URN)978-91-554-7739-4 (ISBN)
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: 2010-03-25Bibliographically approved
2. Visualization of Protein Activity Status in situ Using Proximity Ligation Assays
Open this publication in new window or tab >>Visualization of Protein Activity Status in situ Using Proximity Ligation Assays
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In 2001 the human proteome organization (HUPO) was created with the ambition to identify and characterize all proteins encoded in the human genome according to several criteria; their expression levels in different tissues and under different conditions; the sub-cellular localization; post-translational modifications; interactions, and if possible also the relationship between their structure and function.When the knowledge of different proteins and their potential interactions increases, so does the need for methods able to unravel the nature of molecular processes in cells and organized tissues, and ultimately for clinical use in samples obtained from patients.

The in situ proximity ligation assay (in situ PLA) was developed to provide localized detection of proteins, post-translational modifications and protein-protein interactions in fixed cells and tissues. Dual recognition of the target or interacting targets is a prerequisite for the creation of a circular reporter DNA molecule, which subsequently is locally amplified for visualization of individual protein molecules in single cells. These features offer the high sensitivity and selectivity required for detection of even rare target molecules.

Herein in situ PLA was first established and then employed as a tool for detection of both interactions and post-translational modifications in cultured cells and tissue samples. In situ PLA was also adapted to high content screening (HCS) for therapeutic effects, where it was applied for cell-based drug screening of inhibitors influencing post-translational modifications. This was performed using primary cells, paving the way for evaluation of drug effects on cells from patient as a diagnostic tool in personalized medicine.

In conclusion, this thesis describes the development and applications of in situ PLA as a tool to study proteins, post-translational modifications and protein-protein interactions in genetically unmodified cells and tissues, and for clinical interactomics.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 44 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 609
Keyword
proximity ligation, in situ, high content screening, platelet-derived growth factor receptor, rolling circle amplification, protein interactions, in situ PLA, post-translational modifications, drug screening
National Category
Cell and Molecular Biology Medical Genetics
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-131934 (URN)978-91-554-7919-0 (ISBN)
Public defence
2010-12-04, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2010-11-11 Created: 2010-10-11 Last updated: 2011-01-13Bibliographically approved

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Söderberg, OlaJarvius, MalinLeuchowius, Karl-JohanJarvius, JonasWester, KennethBahram, FuadLandegren, Ulf

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