BACKGROUND: EGFP is a fluorescent tag extensively used in biological and biomedical research. Over the years many researches have gathered collections of cell lines bearing specific EGFP-tagged proteins. Despite its popularity some photochemical properties of EGFP remain undocumented and unused. We report on so far unexplored lifetime photoconversion of EGFP usable in FLIM. METHODS: Fluorescence lifetime imaging and spectral FLIM has been used for characterization of the EGFP photoconversion and protein tracking. RESULT: Our data suggest that EGFP can be permanently photoconverted to a short-fluorescence-lifetime form (PC-EGFP) by intense blue irradiation. PC-EGFP cannot be reverted back by 405 nm light and exhibits the same spectral emission properties with blue-shifted absorption compared to the unconverted EGFP. Fluorescence of PC-EGFP is pH-independent and the photoconversion efficiency decreases with the solvent viscosity. Utilization of the EGFP photoconversion was demonstrated by tracking of a nucleophosmin mutant in live HEK-293 T cells during its cytoplasm-nuclear relocalization induced by Leptomycin B. CONCLUSIONS: Besides potential FLIM artifacts caused by an unintended EGFP photoconversion, the controlled photoconversion turns EGFP to an excellent tool for kinetic FLIM applications. Since the photoconversion occurs in the lifetime domain, PC-EGFP can be easily distinguished from the unconverted tag by time-resolved detection while all other spectral channels stay free for multicolor labeling. GENERAL SIGNIFICANCE: The reported lifetime photoconversion lines up EGFP with other photoconvertible fluorescent proteins with special advantage for fluorescence lifetime imaging where lifetime-photoconvertible labels are scarce.

• MeSH
• buněčná adheze MeSH
• fluorescence MeSH
• fluorescenční mikroskopie metody   MeSH
• fotochemie metody   MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• jaderné proteiny chemie   MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• mutace MeSH
• nenasycené mastné kyseliny chemie   MeSH
• rozpouštědla chemie   MeSH
• viskozita MeSH
• zelené fluorescenční proteiny chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Microtubules of all eukaryotic cells are formed by α- and β-tubulin heterodimers. In addition to the well known cytoplasmic tubulins, a subpopulation of tubulin can occur in the nucleus. So far, the potential function of nuclear tubulin has remained elusive. In this work, we show that α- and β-tubulins of various organisms contain multiple conserved nuclear export sequences, which are potential targets of the Exportin 1/CRM1 pathway. We demonstrate exemplarily that these NES motifs are sufficient to mediate export of GFP as model cargo and that this export can be inhibited by leptomycin B, an inhibitor of the Exportin 1/CRM1 pathway. Likewise, leptomycin B causes accumulation of GFP-tagged tubulin in interphase nuclei, in both plant and animal model cells. Our analysis of nuclear tubulin content supports the hypothesis that an important function of nuclear tubulin export is the exclusion of tubulin from interphase nuclei, after being trapped by nuclear envelope reassembly during telophase.

• MeSH
• aktivní transport - buněčné jádro fyziologie   MeSH
• buněčné jádro metabolismus   MeSH
• buněčné linie MeSH
• cytoplazma metabolismus   MeSH
• eukaryotické buňky metabolismus   MeSH
• karyoferiny metabolismus   MeSH
• lidé MeSH
• mikrotubuly metabolismus   MeSH
• receptory cytoplazmatické a nukleární metabolismus   MeSH
• tabák metabolismus   MeSH
• transport proteinů fyziologie   MeSH
• tubulin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Drugs targeting MDM2's hydrophobic pocket activate p53. However, these agents act allosterically and have agonist effects on MDM2's protein interaction landscape. Dominant p53-independent MDM2-drug responsive-binding proteins have not been stratified. We used as a variable the differential expression of MDM2 protein as a function of cell density to identify Nutlin-3 responsive MDM2-binding proteins that are perturbed independent of cell density using SWATH-MS. Dihydrolipoamide dehydrogenase, the E3 subunit of the mitochondrial pyruvate dehydrogenase complex, was one of two Nutlin-3 perturbed proteins identified fours hour posttreatment at two cell densities. Immunoblotting confirmed that dihydrolipoamide dehydrogenase was induced by Nutlin-3. Depletion of MDM2 using siRNA also elevated dihydrolipoamide dehydrogenase in Nutlin-3 treated cells. Mitotracker confirmed that Nutlin-3 inhibits mitochondrial activity. Enrichment of mitochondria using TOM22+ immunobeads and TMT labeling defined key changes in the mitochondrial proteome after Nutlin-3 treatment. Proximity ligation identified rearrangements of cellular protein-protein complexes in situ. In response to Nutlin-3, a reduction of dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase protein complexes highlighted a disruption of the pyruvate dehydrogenase complex. This coincides with an increase in MDM2/dihydrolipoamide dehydrogenase complexes in the nucleus that was further enhanced by the nuclear export inhibitor Leptomycin B. The data suggest one therapeutic impact of MDM2 drugs might be on the early perturbation of specific protein-protein interactions within the mitochondria. This methodology forms a blueprint for biomarker discovery that can identify rearrangements of MDM2 protein-protein complexes in drug-treated cells.

• MeSH
• dihydrolipoamiddehydrogenasa metabolismus   MeSH
• HCT116 buňky MeSH
• imidazoly farmakologie   MeSH
• lidé MeSH
• mapy interakcí proteinů účinky léků   MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• piperaziny farmakologie   MeSH
• protoonkogenní proteiny c-mdm2 metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

γ-Tubulin is assumed to be a typical cytosolic protein necessary for nucleation of microtubules from microtubule organizing centers. Using immunolocalization and cell fractionation techniques in combination with siRNAi and expression of FLAG-tagged constructs, we have obtained evidence that γ-tubulin is also present in nucleoli of mammalian interphase cells of diverse cellular origins. Immunoelectron microscopy has revealed γ-tubulin localization outside fibrillar centers where transcription of ribosomal DNA takes place. γ-Tubulin was associated with nucleolar remnants after nuclear envelope breakdown and could be translocated to nucleoli during mitosis. Pretreatment of cells with leptomycin B did not affect the distribution of nuclear γ-tubulin, making it unlikely that rapid active transport via nuclear pores participates in the transport of γ-tubulin into the nucleus. This finding was confirmed by heterokaryon assay and time-lapse imaging of photoconvertible protein Dendra2 tagged to γ-tubulin. Immunoprecipitation from nuclear extracts combined with mass spectrometry revealed an association of γ-tubulin with tumor suppressor protein C53 located at multiple subcellular compartments including nucleoli. The notion of an interaction between γ-tubulin and C53 was corroborated by pull-down and co-immunoprecipitation experiments. Overexpression of γ-tubulin antagonized the inhibitory effect of C53 on DNA damage G(2) /M checkpoint activation. The combined results indicate that aside from its known role in microtubule nucleation, γ-tubulin may also have nuclear-specific function(s).

• MeSH
• astrocyty metabolismus   MeSH
• buněčné jadérko metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• časosběrné zobrazování MeSH
• fluorescenční protilátková technika MeSH
• glioblastom metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• imunoelektronová mikroskopie MeSH
• imunoprecipitace MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• mikrotubuly metabolismus   MeSH
• mitóza fyziologie   MeSH
• nádorové buněčné linie MeSH
• nádory mozku metabolismus   MeSH
• proteiny nervové tkáně metabolismus   MeSH
• transport proteinů fyziologie   MeSH
• tubulin metabolismus   MeSH
• tumor supresorové geny MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH