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Monitoring of nucleophosmin oligomerization in live cells
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SYSNO ASEP 0490941 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Monitoring of nucleophosmin oligomerization in live cells Author(s) Holoubek, A. (CZ)
Herman, P. (CZ)
Sýkora, Jan (UFCH-W) RID
Brodská, B. (CZ)
Humpolíčková, Jana (UFCH-W) RID
Kráčmarová, M. (CZ)
Gášková, D. (CZ)
Hof, Martin (UFCH-W) RID, ORCID
Kuželová, K. (CZ)Article number 035016 Source Title Methods and Applications in Fluorescence. - : Institute of Physics Publishing - ISSN 2050-6120
Roč. 6, č. 3 (2018)Number of pages 13 s. Language eng - English Country GB - United Kingdom Keywords B23 ; FLIM-FRET ; nucleolus Subject RIV CF - Physical ; Theoretical Chemistry OECD category Physical chemistry R&D Projects GA16-06096S GA ČR - Czech Science Foundation (CSF) Institutional support UFCH-W - RVO:61388955 UT WOS 000436971700001 EID SCOPUS 85051424965 DOI 10.1088/2050-6120/aaccb9 Annotation Oligomerization plays a crucial role in the function of nucleophosmin (NPM), an abundant nucleolar phosphoprotein. Two dual-color methods based on modern fluorescence confocal microscopy are applied for trackingNPMaggregates in live cells: cross-correlation Number and Brightness analysis (ccN&B) combined with pulsed interleaved excitation (PIE) and fluorescence-lifetime imaging microscopy (FLIM) utilizing resonance energy transfer (FRET). HEK-293T cells were transfected with mixture of plasmids designed for tagging with fluorescent proteins so that the cells express mixed population ofNPMlabeled either with eGFP or mRFP1.Weobserve joint oligomers formed from the fluorescently labeled NPM. Having validated the in vivo methods, we study an effect of substitutions in cysteine 21 (Cys21) of theNPMN-terminus on the oligomerization to demonstrate applicability of the methods. Inhibitory effect of mutations of the Cys21 to nonpolar Ala or to aromatic Phe on the oligomerization was reported in literature using in vitro semi-native electrophoresis. However, we do not detect any break-up of the jointNPMoligomers due to the Cys21 mutations in live cells. In vivo microscopy observations are supported by an in vitro method, the GFP-Trap immunoprecipitation assay. Our results therefore show importance of utilizing several methods for detection of biologically relevant protein aggregates. In vivo monitoring of theNPMoligomerization, a potential cancer therapy target, by the presented methods offers a new way to monitor effects of drugs that are tested as NPMoligomerization inhibitors directly in live cells. Workplace J. Heyrovsky Institute of Physical Chemistry Contact Michaela Knapová, michaela.knapova@jh-inst.cas.cz, Tel.: 266 053 196 Year of Publishing 2019
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