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Weibrecht, Irene
Publications (4 of 4) Show all publications
Weibrecht, I., Grundberg, I., Nilsson, M. & Söderberg, O. (2011). Simultaneous visualization of both signaling cascade activity and end-point gene expression in single cells. PLoS ONE, 6(5), e20148
Open this publication in new window or tab >>Simultaneous visualization of both signaling cascade activity and end-point gene expression in single cells
2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 5, p. e20148-Article in journal (Refereed) Published
Abstract [en]

We have developed an approach for simultaneous detection of individual endogenous protein modifications and mRNA molecules in single cells in situ. For this purpose we combined two methods previously developed in our lab: in situ proximity ligation assay for the detection of individual protein interactions and -modifications and in situ detection of single mRNA molecules using padlock probes. As proof-of-principle, we demonstrated the utility of the method for simultaneous detection of phosphorylated PDGFRβ and DUSP6/MKP-3 mRNA molecules in individual human fibroblasts upon PDGF-BB stimulation. Further we applied drugs disrupting the PDGFRβ signaling pathway at various sites to show that this combined method can concurrently monitor the molecular effect of the drugs, i.e. inhibition of downstream signaling from the targeted node in the signaling pathway. Due to its ability to detect different types of molecules in single cells in situ the method presented here can contribute to a deeper understanding of cell-to-cell variations and can be applied to e.g. pinpoint effector sites of drugs in a signaling pathway.

Keywords
in situ PLA, padlock probe, rolling circle amplification, single molecule, single cell
National Category
Medical Genetics
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-149877 (URN)10.1371/journal.pone.0020148 (DOI)000291006500029 ()21647446 (PubMedID)
Note

De 2 första författarna delar förstaförfattarskapet.

Available from: 2011-03-24 Created: 2011-03-24 Last updated: 2018-01-12Bibliographically approved
Weibrecht, I. (2011). Visualizing Interacting Biomolecules In Situ. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Visualizing Interacting Biomolecules In Situ
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. p. 55
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 674
Keywords
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
National Category
Cell and Molecular Biology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-151579 (URN)978-91-554-8078-3 (ISBN)
Public defence
2011-06-01, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2011-05-11 Created: 2011-04-14 Last updated: 2018-01-12Bibliographically approved
Jarvius, M., Paulsson, J., Weibrecht, I., Leuchowius, K.-J., Andersson, A.-C., Wählby, C., . . . Söderberg, O. (2007). In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method. Molecular & Cellular Proteomics, 6(9), 1500-1509
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
Weibrecht, I., Gavrilovic, M., Lindbom, L., Landegren, U., Wählby, C. & Söderberg, O.Visualizing individual sequence-specific protein-DNA interactions in situ.
Open this publication in new window or tab >>Visualizing individual sequence-specific protein-DNA interactions in situ
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Gene expression – a key feature for modulating cell fate – is regulated in part by histone modifications, which modulate accessibility of the chromatin to transcription factors. Until now, protein-DNA interactions have mostly been studied in bulk without retrieving spatial information from the sample or with poor sequence resolution. New tools are needed to reveal proteins interacting with specific DNA sequences in situ for further understanding of the orchestration of transcription control within the nucleus.

We present herein an approach to visualize individual protein-DNA interactions within cells, based on the in situ proximity ligation assay (PLA). This assay, previously used for detection of protein-protein interactions in situ, was adapted for analysis of target protein-DNA interactions, using padlock probes to identify unique DNA sequences in complex genomes. As a proof-of-principle we detected histone H3 interacting with a 26 bp consensus sequence of the Alu-repeat abundantly expressed in the human genome, but absent in mice. However, the mouse genome contains a highly similar sequence, providing a model system to analyze the selectivity of the developed methods. Although efficiency of detection currently is limiting, we conclude that in situ PLA can be used to achieve a highly selective analysis of protein-DNA interactions in single cells.

Keywords
protein-DNA interaction, in situ, fluorescence microscopy, proximity ligation assay, padlock probe
National Category
Medical Genetics
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-151578 (URN)
Available from: 2011-04-14 Created: 2011-04-14 Last updated: 2018-01-12
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