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High-throughput in situ mapping of phosphorylated protein complexes across the cell cycle and in response to drugs
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Science for Life Laboratory, SciLifeLab, Science for Life Laboratory, SciLifeLab. (Molekylära verktyg, Molecular tools)
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Science for Life Laboratory, SciLifeLab, Science for Life Laboratory, SciLifeLab. (Molekylära verktyg, Molecular tools)ORCID-id: 0000-0002-0762-9034
Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap. (Molekylära verktyg, Molecular tools)
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Science for Life Laboratory, SciLifeLab, Science for Life Laboratory, SciLifeLab. (Molekylära verktyg, Molecular tools)
Vise andre og tillknytning
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
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.

HSV kategori
Forskningsprogram
Biokemi; Molekylär cellbiologi; Molekylär bioteknik
Identifikatorer
URN: urn:nbn:se:uu:diva-374248OAI: oai:DiVA.org:uu-374248DiVA, id: diva2:1280486
Tilgjengelig fra: 2019-01-18 Laget: 2019-01-18 Sist oppdatert: 2019-01-21
Inngår i avhandling
1. Molecular Approaches to Explore Drug-Target Interactions
Åpne denne publikasjonen i ny fane eller vindu >>Molecular Approaches to Explore Drug-Target Interactions
2019 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2019. s. 46
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1533
Emneord
Drug discovery, target engagement, target engagement-mediated amplification, cellular thermal shift assay, proximity extension assay, in situ PLA, high-content imaging
HSV kategori
Forskningsprogram
Molekylär medicin
Identifikatorer
urn:nbn:se:uu:diva-374329 (URN)978-91-513-0560-8 (ISBN)
Disputas
2019-03-08, Svedbergsalen (B8), Biomedicinskt centrum, Husargatan 3, Uppsala, 13:15 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2019-02-11 Laget: 2019-01-21 Sist oppdatert: 2019-02-19

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