The resolution achieved by conventional light microscopy is limited by light diffraction. This obstacle can be overcome either by optical super-resolution techniques or by the recently developed method to physically expand specimens, called expansion microscopy (ExM). The method utilizes polymer chemistry and the ability of a swellable polyelectrolyte hydrogel to absorb water, and thus to expand its size. The procedure was successfully applied to different species and tissue samples, mostly from the animal kingdom. Physically expanded nuclei and chromosomes in combination with specific protein labeling and super-resolution microscopy may provide new insight into the ultrastructure, dynamics, and function of plant chromatin. Here we provide a detailed protocol to expand isolated plant nuclei and visualize proteins by indirect immunolabeling. With the focus on chromatin structure, we expanded isolated barley nuclei from root tips and visualized the centromere-specific histone H3 variant CENH3. The achieved physical expansion of ~4.2 times allowed the detection of DAPI-labeled chromatin structures already by conventional wild-field (WF) microscopy with a maximal resolution of ~50-60nm. By applying structured illumination microscopy (SIM), doubling the WF resolution, chromatin structures at a resolution of ~25-35nm were observed. However, a certain distortion of the centromeric chromatin ultrastructure became obvious.

• Klíčová slova
• CENH3, Expansion microscopy, Isolated nuclei, Plant chromatin, Rabl configuration, Structured illumination microscopy,
• MeSH
• buněčné jádro * MeSH
• centromera * MeSH
• chromatin * MeSH
• histony genetika   MeSH
• mikroskopie MeSH
• rostliny * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin * MeSH
• histony MeSH

Exploring ultrafast magnetization control in 2D magnets via laser pulses is established, yet the interplay between spin dynamics and the lattice remains underexplored. Utilizing real-time time-dependent density functional theory (rt-TDDFT) coupled with Ehrenfest dynamics and nonadiabatic molecular dynamics (NAMD) simulations, we systematically investigate the laser-induced spin-nuclei dynamics with pre-excited A1g and E2g coherent phonons in the 2D ferromagnet Fe3GeTe2 (FGT) monolayer. Selective pre-excitation of coherent phonons under ultrafast laser irradiation significantly alters the local spin moment of FGT, consequently inducing additional spin loss attributed to the nuclear motion-induced asymmetric interatomic charge transfer. Excited spin-resolved charge undergoes a bidirectional spin-flip between spin-down and spin-up states, characterized by a subtle change in the spin moment within approximately 100 fs, followed by unidirectional spin-flip, which will further contribute to the spin moment loss of FGT within tens of picoseconds. Our results shed light on the coupling of coherent phonons with magnetization dynamics in 2D limit.

• Klíčová slova
• Fe3GeTe2, laser-induced coherent phonon, nuclei dynamics, real-time TDDFT, ultrafast spin dynamics,
• Publikační typ
• časopisecké články MeSH

The adult circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus is resilient to glucocorticoids (GCs). The fetal rodent SCN resembles that of the adult in its organization of GC-sensitive peripheral tissues. We tested the hypothesis that the fetal SCN clock is sensitive to changes in GC levels. Maternal GCs must pass through the placenta to reach the fetal SCN. We show that the maternal but not the fetal part of the placenta harbors the autonomous circadian clock, which is reset by dexamethasone (DEX) and rhythmically expresses Hsd11b2. The results suggest the presence of a mechanism for rhythmic GC passage through the placental barrier, which is adjusted according to actual GC levels. GC receptors are expressed rhythmically in the laser-dissected fetal SCN samples. We demonstrate that hypothalamic explants containing the SCN of the mPer2 Luc mouse prepared at embryonic day (E)15 spontaneously develop rhythmicity within several days of culture, with dynamics varying among fetuses from the same litter. Culturing these explants in media enriched with DEX accelerates the development. At E17, treatment of the explants with DEX induces phase advances and phase delays of the rhythms depending on the timing of treatments, and the shifts are completely blocked by the GC receptor antagonist, mifepristone. The DEX-induced phase-response curve differs from that induced by the vehicle. The fetal SCN is sensitive to GCs in vivo because DEX administration to pregnant rats acutely downregulates c-fos expression specifically in the laser-dissected fetal SCN. Our results provide evidence that the rodent fetal SCN clock may respond to changes in GC levels.

• Klíčová slova
• circadian clock, entrainment, glucocorticoids, mPer2 mouse, ontogenesis, suprachiasmatic nuclei,
• MeSH
• cirkadiánní hodiny účinky léků   genetika   fyziologie   MeSH
• cirkadiánní proteiny Period genetika   MeSH
• dexamethason farmakologie   MeSH
• glukokortikoidy farmakologie   fyziologie   MeSH
• hypothalamus fyziologie   MeSH
• krysa rodu Rattus MeSH
• myši MeSH
• nucleus suprachiasmaticus účinky léků   fyziologie   MeSH
• placenta fyziologie   MeSH
• plod fyziologie   MeSH
• těhotenství MeSH
• vývoj plodu * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cirkadiánní proteiny Period MeSH
• dexamethason MeSH
• glukokortikoidy MeSH
• Per2 protein, mouse MeSH Prohlížeč

OBJECTIVES: A novel evaluative approach was used to determine single unit activities of non-bursting intralaminar thalamic nuclei under neuropathic pain state following dorsal rhizotomy. SETTINGS AND DESIGN: Extensive dorsal rhizotomy at cervicothoracic level in rats was used as a model of central pain. After rhizotomy, rats were divided into two groups: rats without any signs of self-mutilation, and those presenting self-mutilation. Spontaneous single unit activities of neurons of intralaminar thalamic nuclei were recorded and interspike intervals (ISIs) of non-bursting cells were counted for both groups and compared with that of non-rhizotomized control rats. Chaodynamic methods were applied for the evaluation of the ISIs. RESULTS: In control rats Lyapunov exponents, Shannon entropy and mutual information average values were significantly higher than those of rhizotomized rats without any signs of self-mutilation. Paradoxically, in animals presenting self-mutilation following rhizotomy the evaluated parameters were similar to those of controls. Further, Lyapunov exponents were positive values in all animals indicating chaotic pattern of the neuronal firing. MAIN FINDINGS: 1. Neurons behave in chaotic way in all animals, 2. The most regular firing was found in non-mutilating rhizotomized animals, 3. Patterns of the firing in selfmutilating rats were similar to those in controls. CONCLUSIONS: It is concluded that pain feeling is not executed neither by changes of chaotic dynamics of non-bursting intralaminar thalamic neurons. On the other hand, the paradoxical firing of the neurons under pathological brain matrix might participate in modification pain feeling.

• MeSH
• akční potenciály MeSH
• entropie MeSH
• krysa rodu Rattus MeSH
• modely neurologické MeSH
• nelineární dynamika MeSH
• neuralgie patofyziologie   MeSH
• nuclei intralaminares thalami fyziologie   MeSH
• potkani Wistar MeSH
• rizotomie * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Despite much recent progress, our understanding of the principles of plant genome organization and its dynamics in three-dimensional space of interphase nuclei remains surprisingly limited. Notably, it is not clear how these processes could be affected by the size of a plant's nuclear genome. In this study, DNA replication timing and interphase chromosome positioning were analyzed in seven Poaceae species that differ in their genome size. To provide a comprehensive picture, a suite of advanced, complementary methods was used: labeling of newly replicated DNA by ethynyl-2'-deoxyuridine, isolation of nuclei at particular cell cycle phases by flow cytometric sorting, three-dimensional immunofluorescence in situ hybridization, and confocal microscopy. Our results revealed conserved dynamics of DNA replication in all species, and a similar replication timing order for telomeres and centromeres, as well as for euchromatin and heterochromatin regions, irrespective of genome size. Moreover, stable chromosome positioning was observed while transitioning through different stages of interphase. These findings expand upon earlier studies in suggesting that a more complex interplay exists between genome size, organization of repetitive DNA sequences along chromosomes, and higher order chromatin structure and its maintenance in interphase, albeit controlled by currently unknown factors.

• Klíčová slova
• Poaceae, DNA replication, EdU labeling, Rabl configuration, S phase, flow cytometry, three-dimensional fluorescence in situ hybridization (3D-FISH),
• MeSH
• buněčné jádro * genetika   MeSH
• centromera genetika   MeSH
• genom rostlinný MeSH
• interfáze MeSH
• replikace DNA MeSH
• umístění chromozomů * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The plant nucleus plays an irreplaceable role in cellular control and regulation by auxin (indole-3-acetic acid, IAA) mainly because canonical auxin signaling takes place here. Auxin can enter the nucleus from either the endoplasmic reticulum or cytosol. Therefore, new information about the auxin metabolome (auxinome) in the nucleus can illuminate our understanding of subcellular auxin homeostasis. Different methods of nuclear isolation from various plant tissues have been described previously, but information about auxin metabolite levels in nuclei is still fragmented and insufficient. Herein, we tested several published nucleus isolation protocols based on differential centrifugation or flow cytometry. The optimized sorting protocol leading to promising yield, intactness, and purity was then combined with an ultra-sensitive mass spectrometry analysis. Using this approach, we can present the first complex report on the auxinome of isolated nuclei from cell cultures of Arabidopsis and tobacco. Moreover, our results show dynamic changes in auxin homeostasis at the intranuclear level after treatment of protoplasts with free IAA, or indole as a precursor of auxin biosynthesis. Finally, we can conclude that the methodological procedure combining flow cytometry and mass spectrometry offers new horizons for the study of auxin homeostasis at the subcellular level.

• Klíčová slova
• auxin, auxin metabolism, flow cytometry, nucleus, subcellular fractionation,
• MeSH
• Arabidopsis účinky léků   metabolismus   ultrastruktura   MeSH
• buněčné jádro účinky léků   metabolismus   ultrastruktura   MeSH
• buněčné kultury MeSH
• centrifugace metody   MeSH
• frakcionace buněk přístrojové vybavení   metody   MeSH
• hmotnostní spektrometrie MeSH
• homeostáza fyziologie   MeSH
• indoly metabolismus   farmakologie   MeSH
• kyseliny indoloctové metabolismus   MeSH
• protoplasty chemie   MeSH
• průtoková cytometrie MeSH
• regulátory růstu rostlin metabolismus   MeSH
• rostlinné buňky účinky léků   metabolismus   ultrastruktura   MeSH
• tabák účinky léků   metabolismus   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• indole MeSH Prohlížeč
• indoly MeSH
• kyseliny indoloctové MeSH
• regulátory růstu rostlin MeSH

Mitosis and cytokinesis are fundamental processes through which somatic cells increase their numbers and allow plant growth and development. Here, we analyzed the organization and dynamics of mitotic chromosomes, nucleoli, and microtubules in living cells of barley root primary meristems using a series of newly developed stable fluorescent protein translational fusion lines and time-lapse confocal microscopy. The median duration of mitosis from prophase until the end of telophase was 65.2 and 78.2 min until the end of cytokinesis. We showed that barley chromosomes frequently start condensation before mitotic pre-prophase as defined by the organization of microtubules and maintain it even after entering into the new interphase. Furthermore, we found that the process of chromosome condensation does not finish at metaphase, but gradually continues until the end of mitosis. In summary, our study features resources for in vivo analysis of barley nuclei and chromosomes and their dynamics during mitotic cell cycle.

• Klíčová slova
• Hordeum vulgare, 3D nuclear organization, barley, chromatin, chromosomes, confocal microscopy, crops, live imaging, microtubules, mitosis,
• MeSH
• buněčné jádro MeSH
• chromozomy MeSH
• ječmen (rod) * genetika   MeSH
• mikrotubuly MeSH
• mitóza MeSH
• profáze MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

N6-methyladenosine (m6A) is the most abundant epitranscriptomic mark that regulates the fate of RNA molecules. Recent studies have revealed a bidirectional interaction between m6A modification and the circadian clock. However, the precise temporal dynamics of m6A global enrichment in the central circadian pacemaker have not been fully elucidated. Our study investigates the relationship between FTO demethylase and molecular clocks in primary cells of the suprachiasmatic nucleus (SCN). In addition, we examined the effects of lipopolysaccharide (LPS) on Fto expression and the role of FTO in LPS-induced reactive oxygen species (ROS) production in primary SCN cell culture. We observed circadian rhythmicity in the global m6A levels, which mirrored the rhythmic expression of the Fto demethylase. Silencing FTO using siRNA reduced the mesor of Per2 rhythmicity in SCN primary cells and extended the period of the PER2 rhythm in SCN primary cell cultures from PER2::LUC mice. When examining the immune response, we discovered that exposure to LPS upregulated global m6A levels while downregulating Fto expression in SCN primary cell cultures. Interestingly, we found a loss of circadian rhythmicity in Fto expression following LPS treatment, indicating that the decrease of FTO levels may contribute to m6A upregulation without directly regulating its circadian rhythm. To explore potential protective mechanisms against neurotoxic inflammation, we examined ROS production following LPS treatment in SCN primary cell cultures pretreated with FTO siRNA. We observed a time-dependent pattern of ROS induction, with significant peak at 32 h but not at 20 h after synchronization. Silencing the FTO demethylase abolished ROS induction following LPS exposure, supporting the hypothesis that FTO downregulation serves as a protective mechanism during LPS-induced neuroinflammation in SCN primary cell cultures.

• Klíčová slova
• Fto demethylase, circadian rhythms, lipopolysaccharide, m6A RNA methylation, rodents, suprachiasmatic nucleus,
• MeSH
• adenosin * analogy a deriváty   metabolismus   MeSH
• cirkadiánní hodiny * účinky léků   fyziologie   genetika   MeSH
• cirkadiánní proteiny Period metabolismus   genetika   MeSH
• cirkadiánní rytmus účinky léků   fyziologie   MeSH
• gen pro FTO * metabolismus   genetika   MeSH
• kultivované buňky MeSH
• lipopolysacharidy * farmakologie   MeSH
• methylace RNA MeSH
• metylace účinky léků   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• neurozánětlivé nemoci metabolismus   MeSH
• nucleus suprachiasmaticus * metabolismus   účinky léků   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• RNA genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosin * MeSH
• cirkadiánní proteiny Period MeSH
• FTO protein, mouse MeSH Prohlížeč
• gen pro FTO * MeSH
• lipopolysacharidy * MeSH
• N-methyladenosine MeSH Prohlížeč
• Per2 protein, mouse MeSH Prohlížeč
• reaktivní formy kyslíku MeSH
• RNA MeSH

We study classical trajectories corresponding to L=0 vibrations in the geometric collective model of nuclei with stable axially symmetric quadrupole deformations. It is shown that with increasing stability against the onset of triaxiality the dynamics passes between a fully regular and semiregular limiting regime. In the transitional region, an interplay of chaotic and regular motions results in complex oscillatory dependence of the regular phase space on the Hamiltonian parameter and energy.

• Publikační typ
• časopisecké články MeSH

DNA replication in cells takes place in domains scattered throughout the nucleoplasm. We have characterized the dynamics of DNA synthesis in synchronized mid-S-phase HeLa cells. Saponin-permeabilized cells were allowed to elongate nascent DNA chains in presence of biotin-dUTP for 5, 15, and 30 min (a pulse experiment), or for 5 min followed by an incubation with unlabeled precursors for 10 or 25 min (a pulse-and-chase experiment). The replication foci were then identified in ultrathin sections using immunogold labeling of the incorporated biotin. Total number of particles per nucleus, total scanned area of the nucleus, size, shape, and gold particle number of each labeled cluster, and the density of clusters per nucleus were evaluated. We have demonstrated that as replication proceeds, the labeled sites increase in size up to 240 nm (30 min incorporation) while maintaining a broadly round shape. In pulse-and-chase experiments the labeled DNA was shown to spread to occupy DNA foci of approximately 400 nm in diameter. These results demonstrate that DNA replication is compartmentalized within cell nuclei at the level of DNA foci and support the view that the synthetic centers are spatially constrained while the chromatin loops are dynamic during DNA synthesis.

• MeSH
• biotin chemie   MeSH
• buněčné jádro metabolismus   ultrastruktura   MeSH
• časové faktory MeSH
• chromatin chemie   MeSH
• DNA chemie   ultrastruktura   MeSH
• elektronová mikroskopie MeSH
• HeLa buňky MeSH
• kinetika MeSH
• lidé MeSH
• metoda Monte Carlo MeSH
• počítačové zpracování obrazu MeSH
• replikace DNA * MeSH
• saponiny farmakologie   MeSH
• vazebná místa MeSH
• zlato chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biotin MeSH
• chromatin MeSH
• DNA MeSH
• saponiny MeSH
• zlato MeSH