Repetitive sequences are among the most unstable regions in the eukaryotic genome and defects in their maintenance correlate with premature aging and cancer development. Promyelocytic leukemia protein (PML) induces accumulation of proteins at distinct nuclear sites, thereby affecting a plethora of processes including DNA repair or maintenance of telomeres. Doxorubicin, the broadly used chemotherapeutic compound, induces formation of PML-nucleolar associations (PNAs). Nevertheless, molecular factors affecting formation of PNAs are still largely unknown. Here we show that PNAs can accumulate ribosomal DNA (rDNA) and, after restoration of RNA polymerase I activity, these structures transfer a fraction of rDNA outside the nucleolus. Mutagenesis of PML isoforms revealed that this process depends on the SUMO-interacting motif and adjacent serine-rich region, and is enhanced by exon8b present exclusively in PML IV isoform. Moreover, we demonstrate that PNAs formation is also regulated by p14ARF/p53 tumor suppressors and casein kinase 2. Our data elucidate how PML nucleolar compartment is assembled, bring the first evidence of PML interacting with rDNA, and show the PML-dependent translocation of rDNA away from the nucleolus.

• Klíčová slova
• Nucleolus, P14(ARF), PML, Phospho-SIM, rDNA,
• MeSH
• doxorubicin farmakologie   MeSH
• jaderné proteiny * metabolismus   MeSH
• nádorové supresorové proteiny genetika   metabolismus   MeSH
• peptidové nukleové kyseliny * MeSH
• protein - isoformy metabolismus   MeSH
• ribozomální DNA genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• doxorubicin MeSH
• jaderné proteiny * MeSH
• nádorové supresorové proteiny MeSH
• peptidové nukleové kyseliny * MeSH
• protein - isoformy MeSH
• ribozomální DNA MeSH

Subseafloor sediments present an untapped source of novel bacterial species with industrially important bioactivities. Subseafloor core samples collected during the Integrated Ocean Drilling Program Expeditions 315, 316, and 331 and stored in Kochi Core Center at -80 °C for 1 to 4 years were used for cultivation-based study of viable actinomycetes. In total, more than 100 actinomycete-like colonies were isolated from two deep-frozen subseafloor sediment samples. Isolated actinomycetes showed close similarity to known Actinotalea, Dietzia, Gordonia, Isoptericola, Microbacterium, Nocardia, Rhodococcus, Pseudonocardia, Streptomyces, and Tsukamurella species and were halotolerant. Bioactivity assays revealed that two of the isolates were producing potent antibacterial compound(s) and one isolate was having antifungal activity. Our study demonstrated that deep-frozen subseafloor core samples could be a potential source of viable actinomycetes, which may be used in drug discovery.

• MeSH
• Actinobacteria chemie   klasifikace   genetika   izolace a purifikace   MeSH
• biodiverzita * MeSH
• DNA bakterií genetika   MeSH
• geologické sedimenty chemie   mikrobiologie   MeSH
• nízká teplota MeSH
• RNA ribozomální 16S genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA bakterií MeSH
• RNA ribozomální 16S MeSH

Membrane microdomains play vital roles in the process of bacterial infection. The membrane microdomain-associated protein Flot1 acts in an endocytic pathway and is required for seedling development, however, whether Flot1 is a part of host defense mechanisms remains unknown. During an analysis of callose deposition, we found that Flot1 amiRNAi mutants exhibited defects in response to flg22. Using variable-angle total internal reflection fluorescence microscopy (VA-TIRFM), structured illumination microscopy (SIM) and fluorescence cross spectroscopy (FCS), we determined that the dynamic behavior of GFP-Flot1 in Arabidopsis thaliana cotyledon epidermal cells changed significantly in plants treated with the elicitor flg22. Moreover, we found that Flot1 was constitutively recycled via an endocytic pathway and that flg22 could promote endocytosis. Importantly, targeting of Flot1 to the late endosome/vacuole for degradation increased in response to flg22 treatment; immunoblot analysis showed that when triggered by flg22, GFP-Flot1 was gradually degraded in a time-dependent manner. Taken together, these findings support the hypothesis that the changing of dynamics and oligomeric states can promote the endocytosis and degradation of Flot1 under flg22 treatment in plant cells.

• Klíčová slova
• Dynamics, Endocytosis, Flot1, SIM, VA-TIRFM,
• MeSH
• Arabidopsis účinky léků   genetika   metabolismus   MeSH
• endocytóza účinky léků   genetika   fyziologie   MeSH
• flagelin farmakologie   MeSH
• fluorescenční mikroskopie MeSH
• geneticky modifikované rostliny genetika   metabolismus   MeSH
• kotyledon genetika   metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulace genové exprese u rostlin účinky léků   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• flagelin MeSH
• proteiny huseníčku MeSH

An important aspect of synaptic plasticity in the brain is axonal transport of essential components such as mitochondria from the soma to the synapse. For uninterrupted transport of cellular cargo down the axon, functional microtubules are required. Altered microtubule dynamics induced by changes in expression of microtubule-associated tau protein affects normal microtubule function and interferes with axonal transport. Here we investigate the effects of the nontaxoid-binding-site microtubule-stabilizing agents peloruside A (PelA) and laulimalide, compared with the taxoid-site-binding agents paclitaxel (Ptx) and ixabepilone, on axonal transport of mitochondria in 1-day-old rat pup cerebral cortical neuron cultures. The differences in effects of these two types of compound on mitochondrial trafficking were specifically compared under conditions of excess tau expression. PelA and laulimalide had no adverse effects on their own on mitochondrial transport compared with Ptx and ixabepilone, which inhibited mitochondrial run length at higher concentrations. PelA, like Ptx, was able to partially reverse the blocked mitochondrial transport seen in ECFP-htau40-overexpressing neurons, although at higher concentrations of microtubule-stabilizing agent, the PelA response was improved over the Ptx response. These results support a neuroprotective effect of microtubule stabilization in maintaining axonal transport in neurons overexpressing tau protein and may be beneficial in reducing the severity of neurodegenerative diseases such as Alzheimer's disease.

• Klíčová slova
• axonal transport, microtubule, microtubule-associated protein, neurodegeneration, tau proteins,
• MeSH
• axonální transport účinky léků   MeSH
• bicyklické sloučeniny heterocyklické farmakologie   MeSH
• epothilony farmakologie   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• laktony farmakologie   MeSH
• lidé MeSH
• makrolidy farmakologie   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• mikrotubuly účinky léků   MeSH
• mitochondrie fyziologie   MeSH
• modulátory tubulinu farmakologie   MeSH
• mozková kůra cytologie   MeSH
• neurony účinky léků   ultrastruktura   MeSH
• novorozená zvířata MeSH
• paclitaxel farmakologie   MeSH
• potkani Sprague-Dawley MeSH
• transfekce MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bicyklické sloučeniny heterocyklické MeSH
• enhanced cyan fluorescent protein MeSH Prohlížeč
• epothilony MeSH
• ixabepilone MeSH Prohlížeč
• laktony MeSH
• laulimalide MeSH Prohlížeč
• makrolidy MeSH
• membránové proteiny MeSH
• modulátory tubulinu MeSH
• paclitaxel MeSH
• peloruside A MeSH Prohlížeč
• tau 40 protein, human MeSH Prohlížeč
• zelené fluorescenční proteiny MeSH

The intercellular transport of auxin is driven by PIN-formed (PIN) auxin efflux carriers. PINs are localized at the plasma membrane (PM) and on constitutively recycling endomembrane vesicles. Therefore, PINs can mediate auxin transport either by direct translocation across the PM or by pumping auxin into secretory vesicles (SVs), leading to its secretory release upon fusion with the PM. Which of these two mechanisms dominates is a matter of debate. Here, we addressed the issue with a mathematical modeling approach. We demonstrate that the efficiency of secretory transport depends on SV size, half-life of PINs on the PM, pH, exocytosis frequency and PIN density. 3D structured illumination microscopy (SIM) was used to determine PIN density on the PM. Combining this data with published values of the other parameters, we show that the transport activity of PINs in SVs would have to be at least 1000× greater than on the PM in order to produce a comparable macroscopic auxin transport. If both transport mechanisms operated simultaneously and PINs were equally active on SVs and PM, the contribution of secretion to the total auxin flux would be negligible. In conclusion, while secretory vesicle-mediated transport of auxin is an intriguing and theoretically possible model, it is unlikely to be a major mechanism of auxin transport in planta.

• Klíčová slova
• 3D-SIM microscopy, PIN transporters, auxin, mathematical modeling, polar auxin transport, secretion,
• MeSH
• Arabidopsis metabolismus   MeSH
• biologické modely * MeSH
• biologický transport MeSH
• endocytóza MeSH
• kyseliny indoloctové metabolismus   MeSH
• permeabilita buněčné membrány MeSH
• proteiny huseníčku metabolismus   MeSH
• sekreční vezikuly metabolismus   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kyseliny indoloctové MeSH
• proteiny huseníčku MeSH
• zelené fluorescenční proteiny MeSH

In the biosynthesis of diverse natural bioactive products the adenylation domains (ADs) of nonribosomal peptide synthetases select specific precursors from the cellular pool and activate them for further incorporation into the scaffold of the final compound. Therefore, the drug discovery programs employing PCR-based screening studies of microbial collections or metagenomic libraries often use AD-coding genes as markers of relevant biosynthetic gene clusters. However, due to significant sequence diversity of ADs, the conventional approach using only one primer pair in a single screening experiment could be insufficient for maximal coverage of AD abundance. In this study, the widely used primer pair A3F/A7R was compared with the newly designed aa194F/aa413R one by 454 pyrosequencing of two sets of actinomycete strains from highly dissimilar environments: subseafloor sediments and forest soil. Individually, none of the primer pairs was able to cover the overall diversity of ADs. However, due to slightly shifted specificity of the primer pairs, the total number and diversity of identified ADs were noticeably extended when both primer pairs were used in a single assay. Additionally, the efficiency of AD detection by different primer combinations was confirmed on the model of Salinispora tropica genomic DNA of known sequence.

• Klíčová slova
• 454 Pyrosequencing, Actinomycetes, Adenylation domain, Nonribosomal peptide synthetase, The Integrated Ocean Drilling Program,
• MeSH
• Actinobacteria klasifikace   genetika   izolace a purifikace   MeSH
• DNA primery * MeSH
• interakční proteinové domény a motivy genetika   MeSH
• konsenzuální sekvence MeSH
• peptidsynthasy chemie   genetika   MeSH
• polymerázová řetězová reakce MeSH
• pozičně specifické skórovací matice MeSH
• půdní mikrobiologie MeSH
• rychlé screeningové testy * MeSH
• sekvence nukleotidů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA primery * MeSH
• non-ribosomal peptide synthase MeSH Prohlížeč
• peptidsynthasy MeSH

Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images.Abbreviations: 3D FISH: three-dimensional fluorescence in situ hybridization; 3D: three-dimensional; ASY1: ASYNAPTIC 1; CC: chromocenters; CO: Crossover; DAPI: 4',6-diamidino-2-phenylindole; DMC1: DNA MEIOTIC RECOMBINASE 1; DSB: Double-Strand Break; FISH: fluorescence in situ hybridization; GFP: GREEN FLUORESCENT PROTEIN; HEI10: HUMAN ENHANCER OF INVASION 10; NCO: Non-Crossover; NE: Nuclear Envelope; Oligo-FISH: oligonucleotide fluorescence in situ hybridization; RNPII: RNA Polymerase II; SC: Synaptonemal Complex; SIM: structured illumination microscopy; ZMM (ZIP: MSH4: MSH5 and MER3 proteins); ZYP1: ZIPPER-LIKE PROTEIN 1.

• Klíčová slova
• 3D organization, Nucleus, RNA Pol II, SIM, STED imaging, chromatin, chromosome, crossovers, image analysis, meiosis, metaphase, mitosis, nuclear bodies, nuclear speckles, oligo FISH, pachytene, quantification, segmentation, spatial distribution, transcription factories,
• MeSH
• buněčné jádro * MeSH
• chromatin * MeSH
• fluorescenční mikroskopie MeSH
• hybridizace in situ fluorescenční MeSH
• lidé MeSH
• průběh práce MeSH
• zelené fluorescenční proteiny MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin * MeSH
• zelené fluorescenční proteiny MeSH

BACKGROUND: The characterisation of dividing brain cells is fundamental for studies ranging from developmental and stem cell biology, to brain cancers. Whilst there is extensive anatomical data on these dividing cells, limited gene transcription data is available due to technical constraints. NEW METHOD: We focally isolated dividing cells whilst conserving RNA, from culture, primary neural tissue and xenografted glioma tumours, using a thymidine analogue that enables gene transcription analysis. RESULTS: 5-ethynyl-2-deoxyuridine labels the replicating DNA of dividing cells. Once labelled, cultured cells and tissues were dissociated, fluorescently tagged with a revised click chemistry technique and the dividing cells isolated using fluorescence-assisted cell sorting. RNA was extracted and analysed using real time PCR. Proliferation and maturation related gene expression in neurogenic tissues was demonstrated in acutely and 3 day old labelled cells, respectively. An elevated expression of marker and pathway genes was demonstrated in the dividing cells of xenografted brain tumours, with the non-dividing cells showing relatively low levels of expression. COMPARISON WITH EXISTING METHOD: BrdU "immune-labelling", the most frequently used protocol for detecting cell proliferation, causes complete denaturation of RNA, precluding gene transcription analysis. This EdU labelling technique, maintained cell integrity during dissociation, minimized copper exposure during labelling and used a cell isolation protocol that avoided cell lysis, thus conserving RNA. CONCLUSIONS: The technique conserves RNA, enabling the definition of cell proliferation-related changes in gene transcription of neural and pathological brain cells in cells harvested immediately after division, or following a period of maturation.

• Klíčová slova
• Cell division, Click chemistry, FACS, Gene transcription, Glioma, Stem cells,
• MeSH
• analýza jednotlivých buněk metody   MeSH
• čichová sliznice fyziologie   MeSH
• click chemie MeSH
• deoxyuridin analogy a deriváty   MeSH
• embryonální kmenové buňky fyziologie   MeSH
• gliom patofyziologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mozek * fyziologie   patofyziologie   MeSH
• myši inbrední C57BL MeSH
• myši inbrední NOD MeSH
• myši SCID MeSH
• nádory mozku * patofyziologie   MeSH
• nervové kmenové buňky fyziologie   MeSH
• neurogeneze * fyziologie   MeSH
• neurony * fyziologie   MeSH
• RNA metabolismus   MeSH
• stanovení celkové genové exprese metody   MeSH
• transplantace nádorů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5-ethynyl-2'-deoxyuridine MeSH Prohlížeč
• deoxyuridin MeSH
• RNA MeSH

The mobilization of nutrient reserves into the ovaries of Aedes aegypti mosquitoes after sugar-feeding plays a vital role in the female's reproductive maturation. In the present work, three-dimensional secondary ion mass spectrometry imaging (3D-SIMS) was used to generate ultrahigh spatial resolution (~1 μm) chemical maps and study the composition and spatial distribution of lipids at the single ovarian follicle level (~100 μm in size). 3D-Mass Spectrometry Imaging (3D-MSI) allowed the identification of cellular types in the follicle (oocyte, nurse and follicular cells) using endogenous markers, and revealed that most of the triacyglycerides (TGs) were compartmentalized in the oocyte region. By comparing follicles from water-fed and sugar-fed females (n=2), 3D-MSI-Time of Flight-SIMS showed that TGs were more abundant in ovarian follicles of sugar-fed females; despite relative sample reproducibility per feeding condition, more biological replicates will better support the trends observed. While the current 3D-MSI-TOF-SIMS does not permit MS/MS analysis of the lipid species, complementary LC-MS/MS analysis of the ovarian follicles aided tentative lipid assignments of the SIMS data. The combination of these MS approaches is giving us a first glimpse of the distribution of functionally relevant ovarian lipid molecules at the cellular level. These new tools can be used to investigate the roles of different lipids on follicle fitness and overall mosquito reproductive output.

• Klíčová slova
• SIMS, imaging, lipids, mass spectrometry, mosquitoes, ovary,
• Publikační typ
• časopisecké články MeSH

• MeSH
• cervikální hlen fyziologie   MeSH
• lidé MeSH
• motilita spermií * MeSH
• ženská infertilita etiologie   patofyziologie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH