- Publikační typ
- abstrakt z konference MeSH
Mediator is a multiprotein complex that connects regulation mediated by transcription factors with RNA polymerase II transcriptional machinery and integrates signals from the cell regulatory cascades with gene expression. One of the Mediator subunits, Mediator complex subunit 28 (MED28), has a dual nuclear and cytoplasmic localization and function. In the nucleus, MED28 functions as part of Mediator and in the cytoplasm, it interacts with cytoskeletal proteins and is part of the regulatory cascades including that of Grb2. MED28 thus has the potential to bring cytoplasmic regulatory interactions towards the centre of gene expression regulation. In this study, we identified MDT-28, the nematode orthologue of MED28, as a likely target of lysine acetylation using bioinformatic prediction of posttranslational modifications. Lysine acetylation was experimentally confirmed using anti-acetyl lysine antibody on immunoprecipitated GFP::MDT-28 expressed in synchronized C. elegans. Valproic acid (VPA), a known inhibitor of lysine deacetylases, enhanced the lysine acetylation of GFP::MDT-28. At the subcellular level, VPA decreased the nuclear localization of GFP::MDT-28 detected by fluorescencelifetime imaging microscopy (FLIM). This indicates that the nuclear pool of MDT-28 is regulated by a mechanism sensitive to VPA and provides an indirect support for a variable relative proportion of MED28 orthologues with other Mediator subunits.
- MeSH
- acetylace MeSH
- buněčné jádro účinky léků metabolismus MeSH
- Caenorhabditis elegans účinky léků metabolismus MeSH
- denzitometrie MeSH
- jaderné proteiny chemie metabolismus MeSH
- kyselina valproová farmakologie MeSH
- larva účinky léků MeSH
- lidé MeSH
- lysin metabolismus MeSH
- mediátorový komplex chemie metabolismus MeSH
- proteiny Caenorhabditis elegans chemie metabolismus MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin * MeSH
- transport proteinů účinky léků MeSH
- výpočetní biologie MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
ALKB-8 is a 2-oxoglutarate-dependent dioxygenase homologous to bacterial AlkB, which oxidatively demethylates DNA substrates. The mammalian AlkB family contains AlkB homologues denominated ALKBH1 to 8 and FTO. The C. elegans genome includes five AlkB-related genes, homologues of ALKBH1, 4, 6, 7, and 8, but lacks homologues of ALKBH2, 3, and 5 and FTO. ALKBH8 orthologues differ from other AlkB family members by possessing an additional methyltransferase module and an RNA binding N-terminal module. The ALKBH8 methyltransferase domain generates the wobble nucleoside 5-methoxycarbonylmethyluridine from its precursor 5-carboxymethyluridine and its (R)- and (S)-5-methoxycarbonylhydroxymethyluridine hydroxylated forms in tRNA Arg/UCG and tRNA Gly/UCC. The ALKBH8/ALKB-8 methyltransferase domain is highly similar to yeast TRM9, which selectively modulates translation of mRNAs enriched with AGA and GAA codons under both normal and stress conditions. In this report, we studied the role of alkb-8 in C. elegans. We show that downregulation of alkb-8 increases detection of lysosome-related organelles visualized by Nile red in vivo. Reversely, forced expression of alkb-8 strongly decreases the detection of this compartment. In addition, overexpression of alkb-8 applied in a pulse during the L1 larval stage increases the C. elegans lifespan.
- MeSH
- Caenorhabditis elegans embryologie enzymologie genetika MeSH
- dioxygenasy metabolismus MeSH
- dlouhověkost MeSH
- down regulace genetika MeSH
- embryo nesavčí metabolismus MeSH
- geneticky modifikovaná zvířata MeSH
- kyseliny ketoglutarové metabolismus MeSH
- larva metabolismus MeSH
- lyzozomy metabolismus MeSH
- methyltransferasy metabolismus MeSH
- operon MeSH
- promotorové oblasti (genetika) MeSH
- proteiny Caenorhabditis elegans genetika metabolismus MeSH
- RNA interference MeSH
- S-adenosylmethionin metabolismus MeSH
- stárnutí metabolismus MeSH
- vývojová regulace genové exprese MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
In the present work we introduce a straightforward fluorescent assay that can be applied in studies of the transbilayer movement (flip-flop) of fluorescent lipid analogues across supported phospholipid bilayers (SPBs). The assay is based on the distance dependent fluorescence quenching by light absorbing surfaces. Applied to SPBs this effect leads to strong differences in fluorescence lifetimes when the dye moves from the outer lipid leaflet to the leaflet in contact with the support. Herein, we present the basic principles of this novel approach, and comment on its advantages over the commonly used methods for investigating flip-flop dynamics across lipid bilayers. We test the assay on the fluorescent lipid analog Atto633-DOPE and the 3-hydroxyflavone F2N12S probe in SPBs composed of DOPC/ DOPS lipids. Moreover, we compare and discuss the flip-flop rates of the probes with respect to their lateral diffusion coefficients.
- MeSH
- časové faktory MeSH
- chemické techniky analytické metody MeSH
- difuze MeSH
- fluorescence MeSH
- fluorescenční barviva chemie MeSH
- fosfatidylcholiny chemie MeSH
- fosfatidylethanolaminy chemie MeSH
- fosfatidylseriny chemie MeSH
- fosfolipidy chemie MeSH
- kinetika MeSH
- lipidové dvojvrstvy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
There is a wide range of techniques utilizing fluorescence of doxorubicin (Dox) commonly used for analysis of intracellular accumulation and destiny of various drug delivery systems containing this anthracycline antibiotic. Unfortunately, results of these studies can be significantly influenced by doxorubicin degradation product, 7,8-dehydro-9,10-desacetyldoxorubicinone (D*) forming spontaneously in aqueous environment, whose fluorescence strongly interfere with that of doxorubicin. Here, we define two microscopy techniques enabling to distinguish and separate Dox and D* emission based either on its spectral properties or on fluorescence lifetime analysis. To analyze influx and nuclear accumulation of Dox (free or polymer-bound) by flow cytometry, we propose using an indirect method based on its DNA intercalation competition with Hoechst 33342 rather than a direct measurement of doxorubicin fluorescence inside the cells.
- MeSH
- antibiotika antitumorózní metabolismus farmakologie MeSH
- buněčné jádro metabolismus MeSH
- buňky 3T3 MeSH
- DNA metabolismus MeSH
- doxorubicin analogy a deriváty metabolismus farmakologie MeSH
- fluorescenční spektrometrie MeSH
- hydrofobní a hydrofilní interakce MeSH
- lidé MeSH
- lymfom T-buněčný farmakoterapie metabolismus MeSH
- myši MeSH
- nádorové buňky kultivované MeSH
- polymery metabolismus MeSH
- průtoková cytometrie MeSH
- systémy cílené aplikace léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Expression of the nascent RNA transcript is regulated by its interaction with a number of proteins. The misregulation of such interactions can often result in impaired cellular functions that can lead to cancer and a number of diseases. Thus, our understanding of RNA-protein interactions within the cellular context is essential for the development of novel diagnostic and therapeutic tools. While there are many in vitro methods that analyze RNA-protein interactions in vivo approaches are scarce. Here we established a method based on fluorescence resonance energy transfer (FRET), which we term RNA-binding mediated FRET (RB-FRET), which determines RNA-protein interaction inside cells and tested it on hnRNP H protein binding to its cognate RNA. Using two different approaches, we provide evidence that RB-FRET is sensitive enough to detect specific RNA-protein interactions in the cell, providing a powerful tool to study spatial and temporal localization of specific RNA-protein complexes.
- MeSH
- genetické vektory genetika MeSH
- HeLa buňky MeSH
- lidé MeSH
- proteiny vázající RNA genetika metabolismus MeSH
- rezonanční přenos fluorescenční energie metody MeSH
- RNA analýza metabolismus MeSH
- sekvence nukleotidů MeSH
- substrátová specifita MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
One of the most actively developing areas in fluorescence microscopy is the achievement of spatial resolution below Abbe's diffraction limit, which restricts the resolution to several hundreds of nanometers. Most of the approaches in use at this time require a complex optical setup, a difficult mathematical treatment, or usage of dyes with special photophysical properties. In this work, we present a new, to our knowledge, approach in confocal microscopy that enhances the resolution moderately but is both technically and computationally simple. As it is based on the saturation of the transition from the ground state to the first excited state, it is universally applicable with respect to the dye used. The idea of the method presented is based on a principle similar to that underlying saturation excitation microscopy, but instead of applying harmonically modulated excitation light, the fluorophores are excited by picosecond laser pulses at different intensities, resulting in different levels of saturation. We show that the method can be easily combined with the concept of triplet relaxation, which by tuning the dark periods between pulses helps to suppress the formation of a photolabile triplet state and effectively reduces photobleaching. We demonstrate our approach imaging GFP-labeled protein patches within the plasma membrane of yeast cells.
- MeSH
- biofyzika metody MeSH
- buněčná membrána metabolismus MeSH
- design vybavení MeSH
- fluorescenční barviva MeSH
- konfokální mikroskopie metody přístrojové vybavení MeSH
- lasery MeSH
- membránové transportní proteiny metabolismus MeSH
- normální rozdělení MeSH
- optika a fotonika MeSH
- Saccharomyces cerevisiae - proteiny metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- světlo MeSH
- terciární struktura proteinů MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Publikační typ
- práce podpořená grantem MeSH
This work reveals the compaction mechanism of intermediate- sized DNA molecules. Using the FLCS it was shown that the compaction of 10 kbp circular plasmide is the all-or-none transition.
The compaction of DNA plays a role in the nuclei of several types of cells and becomes important in the non-viral gene therapy. Thus, it is in the scope of research interest. It was shown, that spermine-induced compaction of large DNA molecules occurs in a discrete "all-or-non" regime, where the coexistence of free and folded DNA molecules was observed. In the case of intermediate-sized DNA molecules (approximately 10 kbp), so far, it was stated that the mechanism of folding is continuous. Here, we show, that neither a standard benchmark technique-dynamic light scattering, nor a single molecule technique such as fluorescence correlation spectroscopy, can decide what kind of mechanism is undertaken in the compaction process. Besides, we introduce an application of a new approach-fluorescence lifetime correlation spectroscopy. The method takes an advantage of a subtle lifetime change of an intercalating dye PicoGreen during the titration with spermine and based on that, it reveals the discrete mechanism of the process. Furthermore, we show that it allows for observation of the equilibrium state transition dynamics.
The present study has two main objectives. The first is to characterize antimicrobial peptide (AMP) cryptdin-4 (Crp-4) interactions with biological membranes and to compare those interactions with those of magainin 2. The second is to combine the complementary experimental approaches of laser scanning microscopy (LSM), ellipsometry, and Z-scan fluorescence correlation spectroscopy (FCS) to acquire comprehensive information on mechanisms of AMP interactions with supported phospholipid bilayers (SPBs)-a popular model of biological membranes. LSM shows appearance of inhomogeneities in spatial distribution of lipids in the bilayer after treatment with Crp-4. Ellipsometric measurements show that binding of Crp-4 does not significantly change the lipid structure of the bilayer (increase in adsorbed mass without a change in thickness of adsorbed layer). Furthermore, Crp-4 slows the lateral diffusion of lipids within the membrane as shown by Z-scan FCS. All changes of the bilayer induced by Crp-4 can be partially reversed by flushing the sample with excess of buffer. Bilayer interactions of magainin 2 are significantly different, causing large loss of lipids and extensive damage to the bilayer. It seems likely that differences in peptide mode of action, readily distinguished using these combined experimental methods, are related to the distinctive beta-sheet and alpha-helical structures of the respective peptides.
