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Making Visible the Proximity Between Proteins
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. (Söderberg)
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Genomic DNA is the template of life - the entity which is characterized by a self-sustaining anatomical development, regulated signaling processes, the ability to reproduce and to respond to stimuli. Through what is classically known as the central dogma, the genome is transcribed into mRNA, which in turn is translated into proteins. The proteins take part in most, if not all, cellular processes, and it is by unraveling these processes that we can begin to understand life and disease-causing mechanisms.

In vitro and in vivo assays are two levels at which protein communication may be studied, and which permit manipulation and control over the proteins under investigation. But in order to retrieve a representation of the processes as close to reality as possible, in situ analysis may instead be applied as a complement to the other two levels of study. In situ PLA offers the ability to survey protein activity in tissue samples and primary cell lines, at a single cell level, detecting single targets in their natural unperturbed environment.  

In this thesis new developments of the in situ PLA are described, along with a new technique offering in situ enzyme-free detection of proximity between biomolecules.

The dynamic range of in situ PLA has now been increased by several orders of magnitude to cover analogous ranges of protein expression; the output signals have been modified to offer a greater signal-to-noise ratio and to limit false-positive-rates while also extending the dynamic range further; simultaneous detection of multiple protein complexes is now possible; proximity-HCR is presented as a robust and inexpensive enzyme-free assay for protein complex detection.

The thesis also covers descriptions on how the techniques may be simultaneously applied, also together with other techniques, for the multiple data-point acquisition required by the emerging realm of systems biology. A future perspective is presented for how much more information may be simultaneously acquired from tissue samples to describe biomolecular interactions in a new manner. This will allow new types of biomarkers and drugs to be discovered, and a new holistic understanding of life.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. , 48 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 973
Keyword [en]
Proximity ligation assay, In situ PLA, rolling circle amplification, protein interaction, protein-protein interaction, in situ, single cell, single molecule, protein complex, antibody, cancer, tissue section, microscopy, image analysis, system biology, multiplex, dynamic range, methods development, systems biology
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biomedical Laboratory Science/Technology Biochemistry and Molecular Biology
Research subject
Molecular Medicine
URN: urn:nbn:se:uu:diva-217772ISBN: 978-91-554-8878-9OAI: oai:DiVA.org:uu-217772DiVA: diva2:695615
Public defence
2014-03-28, B8, Biomedicinskt centrum, Husargatan 3, SE-75123 Uppsala, 09:00 (English)
Knut and Alice Wallenberg FoundationEU, FP7, Seventh Framework Programme, 259796EU, FP7, Seventh Framework Programme, 278568
Available from: 2014-03-06 Created: 2014-02-04 Last updated: 2014-04-29Bibliographically approved
List of papers
1. Increasing the dynamic range of in situ PLA
Open this publication in new window or tab >>Increasing the dynamic range of in situ PLA
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2011 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 8, no 11, 892-893 p.Article in journal, Editorial material (Refereed) Published
National Category
Biological Sciences
urn:nbn:se:uu:diva-159199 (URN)10.1038/nmeth.1743 (DOI)000296891800004 ()
Available from: 2011-09-25 Created: 2011-09-25 Last updated: 2014-03-21Bibliographically approved
2. Compaction of rolling circle amplification products increases signal strength and integrity
Open this publication in new window or tab >>Compaction of rolling circle amplification products increases signal strength and integrity
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology
urn:nbn:se:uu:diva-217748 (URN)
Available from: 2014-02-04 Created: 2014-02-04 Last updated: 2014-03-21Bibliographically approved
3. Parallel Visualization of Multiple Protein Complexes in Individual Cells in Tumor Tissue
Open this publication in new window or tab >>Parallel Visualization of Multiple Protein Complexes in Individual Cells in Tumor Tissue
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2013 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, no 6, 1563-1571 p.Article in journal (Refereed) Published
Abstract [en]

Cellular functions are regulated and executed by complex protein interaction networks. Accordingly, it is essential to understand the interplay between proteins in determining the activity status of signaling cascades. New methods are therefore required to provide information on different protein interaction events at the single cell level in heterogeneous cell populations such as in tissue sections. Here, we describe a multiplex proximity ligation assay for simultaneous visualization of multiple protein complexes in situ. The assay is an enhancement of the original proximity ligation assay, and it is based on using proximity probes labeled with unique tag sequences that can be used to read out which probes, from a pool of probes, have bound a certain protein complex. Using this approach, it is possible to gain information on the constituents of different protein complexes, the subcellular location of the complexes, and how the balance between different complex constituents can change between normal and malignant cells, for example. As a proof of concept, we used the assay to simultaneously visualize multiple protein complexes involving EGFR, HER2, and HER3 homo- and heterodimers on a single-cell level in breast cancer tissue sections. The ability to study several protein complex formations concurrently at single cell resolution could be of great potential for a systems understanding, paving the way for improved disease diagnostics and possibilities for drug development.

National Category
Medical and Health Sciences
urn:nbn:se:uu:diva-203549 (URN)10.1074/mcp.O112.023374 (DOI)000319865000007 ()

De två första författarna delar förstaförfattarskapet.

Available from: 2013-07-15 Created: 2013-07-15 Last updated: 2015-07-07Bibliographically approved
4. Proximity Depended Initiation of Hybridization Chain Reaction
Open this publication in new window or tab >>Proximity Depended Initiation of Hybridization Chain Reaction
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point of care devices should be cheap and robust.

Results: Building on hybridization chain reaction, we designed a four hairpin system which is metastable in solution at 37°C for several hours and undergoes rapid signal amplification upon introduction of an initiator oligonucleotide. When the proximity hairpins are conjugated to antibodies these proximity probes in combination with the HCR hairpins and the initiator oligonucleotide provide a specific, enzyme free method to detect HIF-1α/HIF-1β and potentially other protein interactions and PTMs in situ. Furthermore it was possible to detect single proteins in the different compartments of the cell, further proving the specificity of this technique.

Conclusion: In this study we present proximity dependent HCR, which is a cheap and robust method to detect protein interactions and post-translational modifications. Because of its independence from enzymes the technique has only low demands on storage and handling which makes it interesting for point of care devices.

National Category
Cell and Molecular Biology
urn:nbn:se:uu:diva-218249 (URN)
Available from: 2014-02-10 Created: 2014-02-10 Last updated: 2015-07-07Bibliographically approved

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