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High-throughput in situ mapping of phosphorylated protein complexes across the cell cycle and in response to drugs
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Science for Life Laboratory, SciLifeLab, Science for Life Laboratory, SciLifeLab. (Molekylära verktyg, Molecular tools)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Science for Life Laboratory, SciLifeLab, Science for Life Laboratory, SciLifeLab. (Molekylära verktyg, Molecular tools)ORCID iD: 0000-0002-0762-9034
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Molekylära verktyg, Molecular tools)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Science for Life Laboratory, SciLifeLab, Science for Life Laboratory, SciLifeLab. (Molekylära verktyg, Molecular tools)
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Interactions and posttranslational modifications (PTMs) of proteins orchestrate cellular responses to cytokines, drugs or other agents, but it has been difficult to monitor and characterize these dynamic events at high-throughput. Here, we have established a semi-automated system for large-scale in situ proximity ligation assays (isPLA). The protocol combines isPLA in microtiter wells with automated microscopy and computer-based image analysis whereby specific protein phosphorylations and interactions are digitally recorded in cells, along with measurements of morphological features. We demonstrate how this platform can improve analysis of cellular signaling by investigating TGF-b responsive Smad2 linker phosphorylations and complex formations over time and across millions of individual cells. We depict single cell responses in relation to e.g. local cell crowding and cell cycle progression via measurements of DNA content and nuclear size. Finally, we illustrate the application of the protocol for demonstrating drug effects by screening a library of phosphatase inhibitors. In summary, our approach expands the scope for image-based single cell analyses by combining observations of protein interactions and modifications with morphological details of individual cells at high throughput.

National Category
Natural Sciences
Research subject
Biochemistry; Molecular Cellbiology; Molecular Biotechnology
Identifiers
URN: urn:nbn:se:uu:diva-374248OAI: oai:DiVA.org:uu-374248DiVA, id: diva2:1280486
Available from: 2019-01-18 Created: 2019-01-18 Last updated: 2019-01-21
In thesis
1. Molecular Approaches to Explore Drug-Target Interactions
Open this publication in new window or tab >>Molecular Approaches to Explore Drug-Target Interactions
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Improved means to assess the clinical potential of drug candidates can critically influence development of new therapeutic entities, a central aim in medical life science. Drug discovery and development relies on construction and selection of small organic compounds or biological agents that bind targets of interest. This thesis includes new methodology to investigate target engagement - that is the tendency for these drugs and drug candidates to bind their intended target molecules versus any off-targets. This is a matter of great importance and current strong interest in the pharmaceutical industry as well as academically and an important aim for precision medicine. Paper I describes the target engagement-mediated amplification (TEMA) technique, an accurate, selective and physiological relevant techniques to monitor target binding by DNA-conjugated low molecular weight drug molecules. The DNA conjugated forms of the drugs are uniquely suited to accurately and sensitively reveal the binding characteristics of drugs directly in relevant tissues. Paper II describes the evaluation of cellular thermal shift assays (CETSA) by multiplex proximity extension assays (PEA), to sensitively measure binding of drugs to their proper targets and off-targets in minimal samples of cells and tissues, and for many targets and samples in parallel. The technique provides valuable advantages during drug development, and potentially also in clinical care. Paper III describes a high-throughput approach to use in situ proximity ligation assays to investigate protein interactions or modifications along with phenotypic responses to drugs or cytokines. The technique allows responses by large numbers of cells to be evaluated by automated microscopy and computer-based analysis. Our approach expands the scope for combined molecular and morphological profiling, offering an information-rich means to profile cellular responses to drugs and other agents at the single cell level.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 46
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1533
Keywords
Drug discovery, target engagement, target engagement-mediated amplification, cellular thermal shift assay, proximity extension assay, in situ PLA, high-content imaging
National Category
Medical and Health Sciences
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-374329 (URN)978-91-513-0560-8 (ISBN)
Public defence
2019-03-08, Svedbergsalen (B8), Biomedicinskt centrum, Husargatan 3, Uppsala, 13:15 (English)
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Supervisors
Available from: 2019-02-11 Created: 2019-01-21 Last updated: 2019-02-11

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Heldin, JohanGallini, RadiosaBjörkesten, JohanOelrich, JohanKamali-Moghaddam, MasoodLandegren, Ulf

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Lönn, PeterAl-Amin, Rasel A.Heldin, JohanGallini, RadiosaBjörkesten, JohanOelrich, JohanKamali-Moghaddam, MasoodLandegren, Ulf
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Department of Immunology, Genetics and PathologyScience for Life Laboratory, SciLifeLabDepartment of Pharmaceutical BiosciencesMolecular toolsDepartment of Medical Sciences
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