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Improvements and Applications of in situ Proximity Ligation Assays
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. (Söderberg)ORCID iD: 0000-0002-1053-5856
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cells building up the human body is in constant communication with each other. This communication is done through large complex networks of signaling pathways for inter- and intracellular signal transduction. The signaling activity regulates many important processes, for example cell death, proliferation and differentiation. Information within the signaling networks is communicated over the cell membrane, through the cytoplasm and entering the nucleus by protein activities such as protein-protein interactions (PPIs) and post translation modifications (PTMs). The cells adapts to their own environment, responding to multiple stimuli from their surroundings. This in combination with memory of previous responses, difference in cell cycles stages and sometimes altered genetic background generates heterogeneous cell populations in which every cell is slightly different from its neighbor. This calls for methods to study the activity of endogenous proteins in individual cells within a population.

In situ proximity ligation assay (in situ PLA) was originally developed to visualize interaction between endogenous proteins in fixed cells and tissue and can also be applied to detect PTMs. This thesis describe the application of in situ PLA to study PPIs in signaling pathways and the work to further develop and improve techniques for proximity dependent detection. 

In paper I in situ PLA is used to study cross talk between the Hippo and the TGFβ signaling pathways. The study shows the complex formation by the transcription co-factors of the Hippo pathway, Yap and Taz, and the main effectors of the TGFβ pathway Smad2/3. Furthermore the density dependent localization of the interaction is described.

Paper II presents a new version of the in situ PLA probes for simultaneous detection of multiple complexes. Visualization of various complexes involving EGFR, Her2 and Her3 is presented as a proof of concept.

The efficiency of in situ PLA is limited by several factors, one being the design of PLA probes and oligonucleotide systems. Even upon proximal binding of the probes there is a risk of formation of non-circular ligation products, which cannot be amplified and detected. In Paper III two new PLA probes are presented aiming to reduce the formation of non-circular ligation product and hence increase the detection efficiency of in situ PLA.

Paper IV presents a new method for detection of protein complexes and phosphorylation; proxHCR. ProxHCR combines signal amplification by enzyme free hybridization chain reaction (HCR) with the requirement of proximal binding of two affinity probes. As a proof of principle the method is used to detect multiple complexes and protein phosphorylation in fixed cells and tissue.  

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. , 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1099
National Category
Cell and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biomedical Laboratory Science/Technology
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-248876ISBN: 978-91-554-9233-5 (print)OAI: oai:DiVA.org:uu-248876DiVA: diva2:801280
Public defence
2015-05-29, Biomedicinskt centrum (BMC), B/B42, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Funder
Swedish Foundation for Strategic Research
Available from: 2015-05-07 Created: 2015-04-08 Last updated: 2015-07-07
List of papers
1. Crosstalk between Hippo and TGFβ: Subcellular localization of YAP/TAZ complexes
Open this publication in new window or tab >>Crosstalk between Hippo and TGFβ: Subcellular localization of YAP/TAZ complexes
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(English)Manuscript (preprint) (Other academic)
Keyword
Hippo, interaction, in situ PLA, Smad, TAZ, TGFβ, YAP
National Category
Biomedical Laboratory Science/Technology Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-248873 (URN)
Funder
Swedish Foundation for Strategic Research EU, FP7, Seventh Framework Programme
Available from: 2015-04-08 Created: 2015-04-08 Last updated: 2015-07-07
2. 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
Identifiers
urn:nbn:se:uu:diva-203549 (URN)10.1074/mcp.O112.023374 (DOI)000319865000007 ()
Note

De två första författarna delar förstaförfattarskapet.

Available from: 2013-07-15 Created: 2013-07-15 Last updated: 2017-12-06Bibliographically approved
3. Enhanced in situ proximity ligation assays via Unfolding PLA probes
Open this publication in new window or tab >>Enhanced in situ proximity ligation assays via Unfolding PLA probes
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-248874 (URN)
Funder
Swedish Foundation for Strategic Research EU, FP7, Seventh Framework Programme, 278568EU, FP7, Seventh Framework Programme, 264737
Available from: 2015-04-08 Created: 2015-04-08 Last updated: 2017-05-04
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
Identifiers
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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