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      The Subcellular Localization and Oligomerization Preferences of NME1/NME2 upon Radiation-Induced DNA Damage

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          Abstract

          Nucleoside diphosphate kinases (NDPK/NME/Nm23) are enzymes composed of subunits NME1/NDPK A and NME2/NDPK B, responsible for the maintenance of the cellular (d)NTP pool and involved in other cellular processes, such as metastasis suppression and DNA damage repair. Although eukaryotic NDPKs are active only as hexamers, it is unclear whether other NME functions require the hexameric form, and how the isoenzyme composition varies in different cellular compartments. To examine the effect of DNA damage on intracellular localization of NME1 and NME2 and the composition of NME oligomers in the nucleus and the cytoplasm, we used live-cell imaging and the FRET/FLIM technique. We showed that exogenous NME1 and NME2 proteins co-localize in the cytoplasm of non-irradiated cells, and move simultaneously to the nucleus after gamma irradiation. The FRET/FLIM experiments imply that, after DNA damage, there is a slight shift in the homomer/heteromer balance between the nucleus and the cytoplasm. Collectively, our results indicate that, after irradiation, NME1 and NME2 engage in mutual functions in the nucleus, possibly performing specific functions in their homomeric states. Finally, we demonstrated that fluorophores fused to the N-termini of NME polypeptides produce the largest FRET effect and thus recommend this orientation for use in similar studies.

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          Creating new fluorescent probes for cell biology.

          Fluorescent probes are one of the cornerstones of real-time imaging of live cells and a powerful tool for cell biologists. They provide high sensitivity and great versatility while minimally perturbing the cell under investigation. Genetically-encoded reporter constructs that are derived from fluorescent proteins are leading a revolution in the real-time visualization and tracking of various cellular events. Recent advances include the continued development of 'passive' markers for the measurement of biomolecule expression and localization in live cells, and 'active' indicators for monitoring more complex cellular processes such as small-molecule-messenger dynamics, enzyme activation and protein-protein interactions.
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            Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter

            Constitutive promoters are used routinely to drive ectopic gene expression. Here, we carried out a systematic comparison of eight commonly used constitutive promoters (SV40, CMV, UBC, EF1A, PGK and CAGG for mammalian systems, and COPIA and ACT5C for Drosophila systems). We also included in the comparison the TRE promoter, which can be activated by the rtTA transcriptional activator in a doxycycline-inducible manner. To make our findings representative, we conducted the comparison in a variety of cell types derived from several species. We found that these promoters vary considerably from one another in their strength. Most promoters have fairly consistent strengths across different cell types, but the CMV promoter can vary considerably from cell type to cell type. At maximal induction, the TRE promoter is comparable to a strong constitutive promoter. These results should facilitate more rational choices of promoters in ectopic gene expression studies.
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              A Guide to Fluorescent Protein FRET Pairs

              Förster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies.
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                Author and article information

                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                29 March 2020
                April 2020
                : 21
                : 7
                : 2363
                Affiliations
                [1 ]Division of Molecular Medicine, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb, Croatia; Martina.Radic@ 123456irb.hr (M.R.); slade@ 123456irb.hr (N.S.)
                [2 ]Division of Molecular Biology, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb, Croatia; Marko.Sostar@ 123456irb.hr (M.Š.); iweber@ 123456irb.hr (I.W.); cetkovic@ 123456irb.hr (H.Ć.)
                Author notes
                [* ]Correspondence: mherak@ 123456irb.hr ; Tel.: +385-1-456-0996
                Author information
                https://orcid.org/0000-0003-4296-3166
                Article
                ijms-21-02363
                10.3390/ijms21072363
                7177722
                32235358
                ffe50f5b-fefe-4ad3-a174-0a1b50e792d3
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 23 December 2019
                : 26 March 2020
                Categories
                Article

                Molecular biology
                nme,ndpk,nm23,nucleoside diphosphate kinase,fret,flim,live-cell imaging
                Molecular biology
                nme, ndpk, nm23, nucleoside diphosphate kinase, fret, flim, live-cell imaging

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