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Simultaneous visualization of both signaling cascade activity and end-point gene expression in single cells
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab. (Molecular Medicine)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab. (Molekylär Diagnostik)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. 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. Uppsala University, Science for Life Laboratory, SciLifeLab. (Molecular Medicine)
2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 5, e20148- p.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.

Place, publisher, year, edition, pages
2011. Vol. 6, no 5, e20148- p.
Keyword [en]
in situ PLA, padlock probe, rolling circle amplification, single molecule, single cell
National Category
Medical Genetics
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-149877DOI: 10.1371/journal.pone.0020148ISI: 000291006500029PubMedID: 21647446OAI: oai:DiVA.org:uu-149877DiVA: diva2:405871
Note

De 2 första författarna delar förstaförfattarskapet.

Available from: 2011-03-24 Created: 2011-03-24 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Genotyping and Mutation Detection In Situ: Development and application of single-molecule techniques
Open this publication in new window or tab >>Genotyping and Mutation Detection In Situ: Development and application of single-molecule techniques
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The human body is composed of trillions of cells closely working together to maintain a functional organism. Every cell is unique in molecular composition and can acquire genetic variations that might cause it to turn pathological. It is essential to develop improved tools to better understand the development of normal and disease tissue, ideally enabling single-cell expression studies in preserved context of complex tissue with single-nucleotide resolution. This thesis presents the development and application of a new in situ method for localized detection and genotyping of individual transcripts directly in cells and tissues. The described technique utilizes padlock probes and target-primed rolling circle amplification and is highly suitable for sensitive in situ analysis.

First, a new strategy for directed cleavage of single stranded DNA was investigated, e.g. nucleic acid targets with extended 3´ ends, for successful initiation of rolling circle amplification. The presented cleavage strategy is simple and applicable for subsequent enzymatic reactions, e.g. ligation and polymerization. Specific cleavage of long target overhangs was demonstrated in synthetic oligonucleotides and in genomic DNA and the detection efficiency was substantially increased.

For multiplex detection and genotyping of individual transcripts in single cells, a new in situ method was developed. The technique showed a satisfactorily detection efficiency and was later applied as a general mutation analysis tool for detection of KRAS point mutations in complex tumor tissue sections, e.g. formalin-fixed, paraffin-embedded tumor tissues and cytologic tumor imprints. Mutation status was assessed in patient samples by in situ padlock probe detection and results were confirmed by DNA-sequencing.  Finally, the method was adapted for simultaneous detection of individual mRNA molecules and endogenous protein modifications in single cells using padlock probes and in situ PLA. This assay will be useful for gene expression analysis and exploration of new drugs with vague effector sites.

To our knowledge, no other technique exists today that offers in situ transcript detection with single-nucleotide resolution in heterogeneous tissues. The method will especially be suitable for discrimination of highly similar transcripts, e.g. splice variants, SNPs and point mutations, within gene expression studies and for cancer diagnostics.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 59 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 656
Keyword
padlock probes, in situ, rolling circle amplification, mRNA, genotyping, mutation detection, cancer, tissue sections, diagnostics, single-molecule, single-cell, microscopy
National Category
Medical Genetics
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-149776 (URN)978-91-554-8034-9 (ISBN)
Public defence
2011-05-06, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2011-04-14 Created: 2011-03-23 Last updated: 2011-05-05Bibliographically approved
2. Visualizing Interacting Biomolecules In Situ
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. 55 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 674
Keyword
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: 2011-07-01Bibliographically approved

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Weibrecht, IreneGrundberg, IdaNilsson, MatsSöderberg, Ola

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