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Flow cytometric in situ proximity ligation analyses of protein interactions and post-translational modification of the epidermal growth factor receptor family
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för onkologi, radiologi och klinisk immunologi, Enheten för biomedicinsk strålningsvetenskap.
Visa övriga samt affilieringar
2009 (Engelska)Ingår i: Cytometry: Part A, ISSN 1552-4922, Vol. 75A, nr 10, s. 833-839Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
2009. Vol. 75A, nr 10, s. 833-839
Nyckelord [en]
proximity ligation, flow cytometry, protein interactions, epidermal growth factor receptor, HER2
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
URN: urn:nbn:se:uu:diva-112106DOI: 10.1002/cyto.a.20771ISI: 000270419700004PubMedID: 19650109OAI: oai:DiVA.org:uu-112106DiVA, id: diva2:284920
Tillgänglig från: 2010-01-08 Skapad: 2010-01-08 Senast uppdaterad: 2018-12-04
Ingår i avhandling
1. High Content Analysis of Proteins and Protein Interactions by Proximity Ligation
Öppna denna publikation i ny flik eller fönster >>High Content Analysis of Proteins and Protein Interactions by Proximity Ligation
2010 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.



Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2010. s. 56
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 530
Nyckelord
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
Nationell ämneskategori
Cell- och molekylärbiologi
Forskningsämne
Molekylär medicin
Identifikatorer
urn:nbn:se:uu:diva-119530 (URN)978-91-554-7739-4 (ISBN)
Disputation
2010-04-16, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (Engelska)
Opponent
Handledare
Tillgänglig från: 2010-03-25 Skapad: 2010-02-26 Senast uppdaterad: 2018-01-12Bibliografiskt granskad
2. Visualizing Interacting Biomolecules In Situ
Öppna denna publikation i ny flik eller fönster >>Visualizing Interacting Biomolecules In Situ
2011 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Intra- and intercellular information is communicated by posttranslational modifications (PTMs) and protein-protein interactions, transducing information over cell membranes and to the nucleus. A cells capability to respond to stimuli by several highly complex and dynamic signaling networks provides the basis for rapid responses and is fundamental for the cellular collaborations required in a multicellular organism. Having received diverse stimuli, being positioned at various stages of the cell cycle or, for the case of cancer, containing altered genetic background, each cell in a population is slightly different from its neighbor. However, bulk analyses of interactions will only reveal an average, but not the true variation within a population. Thus studies of interacting endogenous biomolecules in situ are essential to acquire a comprehensive view of cellular functions and communication.

In situ proximity ligation assay (in situ PLA) was developed to investigate individual endogenous protein-protein interactions in fixed cells and tissues and was later applied for detection for PTMs. Progression of signals in a pathway can branch out in different directions and induce expression of different target genes. Hence simultaneous measurement of protein activity and gene expression provides a tool to determine the balance and progression of these signaling events. To obtain this in situ PLA was combined with padlock probes, providing an assay that can interrogate both PTMs and mRNA expression at a single cell level. Thereby different nodes of the signaling pathway as well as drug effects on different types of molecules could be investigated simultaneously.

In addition to regulation of gene expression, protein-DNA interactions present a mechanism to manage accessibility of the genomic DNA in an inheritable manner, providing the basis for lineage commitment, via e.g. histone PTMs. To enable analyses of protein-DNA interactions in situ we developed a method that utilizes the proximity dependence of PLA and the sequence selectivity of padlock probes.

This thesis presents new methods providing researchers with a set of tools to address cellular functions and communication in complex microenvironments, to improve disease diagnostics and to contribute to hopefully finding cures.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2011. s. 55
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 674
Nyckelord
proximity ligation, in situ PLA, padlock probe, rolling circle amplification, flow cytometry, in situ, single cell, single molecule, protein interaction, protein-DNA interaction, posttranslational modification
Nationell ämneskategori
Cell- och molekylärbiologi
Forskningsämne
Molekylär medicin
Identifikatorer
urn:nbn:se:uu:diva-151579 (URN)978-91-554-8078-3 (ISBN)
Disputation
2011-06-01, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (Engelska)
Opponent
Handledare
Tillgänglig från: 2011-05-11 Skapad: 2011-04-14 Senast uppdaterad: 2018-01-12Bibliografiskt granskad

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Landegren, UlfGedda, LarsSöderberg, Ola

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