Monitoring of nucleophosmin oligomerization in live cells
The result's identifiers
Result code in IS VaVaI
<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216208%3A11320%2F18%3A10385095" target="_blank" >RIV/00216208:11320/18:10385095 - isvavai.cz</a>
Alternative codes found
RIV/61388955:_____/18:00490941 RIV/61388963:_____/18:00491886 RIV/00023736:_____/18:00011996
Result on the web
<a href="https://doi.org/10.1088/2050-6120/aaccb9" target="_blank" >https://doi.org/10.1088/2050-6120/aaccb9</a>
DOI - Digital Object Identifier
<a href="http://dx.doi.org/10.1088/2050-6120/aaccb9" target="_blank" >10.1088/2050-6120/aaccb9</a>
Alternative languages
Result language
angličtina
Original language name
Monitoring of nucleophosmin oligomerization in live cells
Original language description
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 tracking NPM aggregates 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 of NPM labeled either with eGFP or mRFP1. We observe 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 the NPMN-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 joint NPM oligomers 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 the NPM oligomerization, a potential cancer therapy target, by the presented methods offers a new way to monitor effects of drugs that are tested as NPM oligomerization inhibitors directly in live cells.
Czech name
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Czech description
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Classification
Type
J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database
CEP classification
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OECD FORD branch
10610 - Biophysics
Result continuities
Project
Result was created during the realization of more than one project. More information in the Projects tab.
Continuities
P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace
Others
Publication year
2018
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů
Data specific for result type
Name of the periodical
Methods and Applications in Fluorescence
ISSN
2050-6120
e-ISSN
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Volume of the periodical
6
Issue of the periodical within the volume
3
Country of publishing house
GB - UNITED KINGDOM
Number of pages
13
Pages from-to
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UT code for WoS article
000436971700001
EID of the result in the Scopus database
2-s2.0-85051424965