Centrosomes play a crucial role during immune cell interactions and initiation of the immune response. In proliferating cells, centrosome numbers are tightly controlled and generally limited to one in G1 and two prior to mitosis. Defects in regulating centrosome numbers have been associated with cell transformation and tumorigenesis. Here, we report the emergence of extra centrosomes in leukocytes during immune activation. Upon antigen encounter, dendritic cells pass through incomplete mitosis and arrest in the subsequent G1 phase leading to tetraploid cells with accumulated centrosomes. In addition, cell stimulation increases expression of polo-like kinase 2, resulting in diploid cells with two centrosomes in G1-arrested cells. During cell migration, centrosomes tightly cluster and act as functional microtubule-organizing centers allowing for increased persistent locomotion along gradients of chemotactic cues. Moreover, dendritic cells with extra centrosomes display enhanced secretion of inflammatory cytokines and optimized T cell responses. Together, these results demonstrate a previously unappreciated role of extra centrosomes for regular cell and tissue homeostasis.
- MeSH
- centrozom * metabolismus MeSH
- chemotaxe MeSH
- cytokiny metabolismus MeSH
- dendritické buňky * metabolismus MeSH
- kontrolní body buněčného cyklu MeSH
- lidé MeSH
- mitóza MeSH
- organizační centrum mikrotubulů MeSH
- pohyb buněk MeSH
- protein-serin-threoninkinasy metabolismus MeSH
- T-lymfocyty metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
The Drosophila salivary glands (SGs) were well known for the puffing patterns of their polytene chromosomes and so became a tissue of choice to study sequential gene activation by the steroid hormone ecdysone. One well-documented function of these glands is to produce a secretory glue, which is released during pupariation to fix the freshly formed puparia to the substrate. Over the past two decades SGs have been used to address specific aspects of developmentally-regulated programmed cell death (PCD) as it was thought that they are doomed for histolysis and after pupariation are just awaiting their fate. More recently, however, we have shown that for the first 3-4 h after pupariation SGs undergo tremendous endocytosis and vacuolation followed by vacuole neutralization and membrane consolidation. Furthermore, from 8 to 10 h after puparium formation (APF) SGs display massive apocrine secretion of a diverse set of cellular proteins. Here, we show that during the period from 11 to 12 h APF, the prepupal glands are very active in calcium oxalate (CaOx) extrusion that resembles renal or nephridial excretory activity. We provide genetic evidence that Prestin, a Drosophila homologue of the mammalian electrogenic anion exchange carrier SLC26A5, is responsible for the instantaneous production of CaOx by the late prepupal SGs. Its positive regulation by the protein kinases encoded by fray and wnk lead to increased production of CaOx. The formation of CaOx appears to be dependent on the cooperation between Prestin and the vATPase complex as treatment with bafilomycin A1 or concanamycin A abolishes the production of detectable CaOx. These data demonstrate that prepupal SGs remain fully viable, physiologically active and engaged in various cellular activities at least until early pupal period, that is, until moments prior to the execution of PCD.
- MeSH
- aktivní transport fyziologie MeSH
- Drosophila melanogaster MeSH
- protein-serin-threoninkinasy genetika metabolismus MeSH
- proteiny Drosophily genetika metabolismus MeSH
- proteiny přenášející anionty biosyntéza genetika metabolismus MeSH
- slinné žlázy sekrece MeSH
- šťavelan vápenatý metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Inosine-5'-monophosphate dehydrogenase catalyzes the critical step in the de novo synthesis of guanosine nucleotides: the oxidation of inosine monophosphate to xanthosine monophosphate. This reaction can be inhibited by specific inhibitors, such as ribavirin or mycophenolic acid, which are widely used in clinical treatment when required to inhibit the proliferation of viruses or cells. However, it was recently found that such an inhibition affects the cells, leading to a redistribution of IMPDH2 and the appearance of IMPDH2 inclusions in the cytoplasm. According to their shape, these inclusions have been termed "Rods and Rings" (R&R). In this work, we focused on the subcellular localization of IMPDH2 protein and the ultrastructure of R&R inclusions. Using microscopy and western blot analysis, we show the presence of nuclear IMPDH2 in human cells. We also show that the nuclear pool has an ability to form Rod structures after inhibition by ribavirin. Concerning the ultrastructure, we observed that R&R inclusions in cellulo correspond to the accumulation of fibrous material that is not surrounded by a biological membrane. The individual fibers are composed of regularly repeating subunits with a length of approximately 11 nm. Together, our findings describe the localization of IMPDH2 inside the nucleus of human cells as well as the ultrastructure of R&R inclusions.
- MeSH
- aktivní transport - buněčné jádro účinky léků MeSH
- buněčné jádro enzymologie metabolismus ultrastruktura MeSH
- cytoplazma enzymologie ultrastruktura MeSH
- HeLa buňky MeSH
- IMP-dehydrogenasa antagonisté a inhibitory chemie metabolismus ultrastruktura MeSH
- inhibitory enzymů farmakologie MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The spatial organization of the cell nucleus into separated domains with a specific macromolecular composition seems to be the fundamental principle that regulates its functioning. Because of the importance of regulation at the nuclear level, the cell nucleus and its domains have been intensively studied. This review is focused on the nuclear domain termed the Polycomb (PcG) body. We summarize and discuss data reported in the literature on different components of the PcG body that could form its structural basis. First, we describe the protein nature of the PcG body and the gene silencing factory model. Second, we review the target genes of Polycomb-mediated silencing and discuss their essentiality for the structural nature of the PcG body. In this respect, two different schematic models are presented. Third, we mention new data on the importance of RNAs, insulator elements and insulator proteins for the structure of PcG bodies. With this review, we hope to illustrate the importance of understanding the nature of the PcG subcompartment. The structural basis of a subcompartment directly reflects its status in the cell nucleus and the mechanism of its function.
The limited specimen tilting range that is typically available in electron tomography gives rise to a region in the Fourier space of the reconstructed object where experimental data are unavailable - the missing wedge. Since this region is sharply delimited from the area of available data, the reconstructed signal is typically hampered by convolution with its impulse response, which gives rise to the well-known missing wedge artefacts in 3D reconstructions. Despite the recent progress in the field of reconstruction and regularization techniques, the missing wedge artefacts remain untreated in most current reconstruction workflows in structural biology. Therefore we have designed a simple Fourier angular filter that effectively suppresses the ray artefacts in the single-axis tilting projection acquisition scheme, making single-axis tomographic reconstructions easier to interpret in particular at low signal-to-noise ratio in acquired projections. The proposed filter can be easily incorporated into current electron tomographic reconstruction schemes.
- MeSH
- artefakty MeSH
- Fourierova analýza MeSH
- krysa rodu rattus MeSH
- líska ultrastruktura MeSH
- mozeček ultrastruktura MeSH
- počítačové zpracování obrazu * MeSH
- poměr signál - šum MeSH
- pyl ultrastruktura MeSH
- tomografie elektronová metody MeSH
- Trypanosoma brucei brucei ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
- Klíčová slova
- Inosin 5-mono-fosfát dehydrogenáza, inkluze, ultrastruktura,
- MeSH
- elektronová mikroskopie využití MeSH
- inhibitory enzymů * izolace a purifikace terapeutické užití MeSH
- konformace proteinů * účinky léků MeSH
- lidé MeSH
- nukleotidy biosyntéza MeSH
- prospektivní studie MeSH
- zmrazování MeSH
- Check Tag
- lidé MeSH
BACKGROUND INFORMATION: A Polycomb (PcG) body is an orphan nuclear subcompartment characterised by accumulations of Polycomb repressive complex 1 (PRC1) proteins. However, seemingly contradictory reports have appeared that describe the PcG bodies either as protein-based bodies in the interchromatin compartment or chromatin domains. In this respect, molecular crowding is an important factor for the assembly and stability of nuclear subcompartments. In order to settle this contradiction, crowding experiments, that represent a convenient model distinguishing between interchromatin and chromatin compartments, were carried out. RESULTS: In sucrose-hypertonically induced crowding, we observed in U-2 OS cells that PcG bodies disappeared, but persisted as nuclear domains characterised by accumulations of DNA. This phenomenon was also observed in cells hypertonically treated with sorbitol and NaCl. Importantly, the observed changes were quickly reversible after re-incubation of cells in normal medium. We found that the PcG foci disappearance and the dissociation of PRC1 proteins (BMI1 and RING1a proteins) from chromatin were associated with their hyper-phosphorylation. In addition, under hyper- and hypotonic conditions, the behaviour of the PcG bodies differed from that of the typical nucleoplasmic body. CONCLUSION: PRC1 proteins accumulations do not represent a genuine nuclear subcompartment. The PcG body is a chromosomal domain, rather than a nucleoplasmic body.
- MeSH
- anthrachinony metabolismus MeSH
- barvení a značení MeSH
- chromatin metabolismus MeSH
- fluorescence MeSH
- fosforylace účinky léků MeSH
- genetická transkripce účinky léků MeSH
- hypertonické roztoky farmakologie MeSH
- lidé MeSH
- makromolekulární látky metabolismus MeSH
- nádorové buněčné linie MeSH
- polycomb proteiny metabolismus MeSH
- PRC1 metabolismus MeSH
- RNA genetika metabolismus MeSH
- sacharosa farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Polycomb group (PcG) proteins of the Polycomb repressive complex 1 (PRC1) are found to be diffusely distributed in nuclei of cells from various species. However they can also be localized in intensely fluorescent foci, whether imaged using GFP fusions to proteins of PRC1 complex, or by conventional immunofluorescence microscopy. Such foci are termed PcG bodies, and are believed to be situated in the nuclear intechromatin compartment. However, an ultrastructural description of the PcG body has not been reported to date. To establish the ultrastructure of PcG bodies in human U-2 OS cells stably expressing recombinant polycomb BMI1-GFP protein, we used correlative light-electron microscopy (CLEM) implemented with high-pressure freezing, cryosubstitution and on-section labeling of BMI1 protein with immunogold. This approach allowed us to clearly identify fluorescent PcG bodies, not as distinct nuclear bodies, but as nuclear domains enriched in separated heterochromatin fascicles. Importantly, high-pressure freezing and cryosubstitution allowed for a high and clear-cut immunogold BMI1 labeling of heterochromatin structures throughout the nucleus. The density of immunogold labeled BMI1 in the heterochromatin fascicles corresponding to fluorescent "PcG bodies" did not differ from the density of labeling of heterochromatin fascicles outside of the "PcG bodies". Accordingly, an appearance of the fluorescent "PcG bodies" seems to reflect a local accumulation of the labeled heterochromatin structures in the investigated cells. The results of this study should allow expansion of the knowledge about the biological relevance of the "PcG bodies" in human cells.
- MeSH
- elektronová mikroskopie MeSH
- heterochromatin metabolismus MeSH
- imunohistochemie MeSH
- kryoprezervace MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- represorové proteiny chemie metabolismus MeSH
- světlo MeSH
- tlak MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The essential role of MCM 2–7 proteins in the initiation of DNA replication in all eukaryotes is well known. Their role in replication elongation is supported by numerous studies, but there is still a knowledge gap in this respect. Even though biochemical studies have established an association of MCM proteins with replication forks, previous immunofluorescence studies in mammalian cells have suggested that MCM 2–7 proteins are displaced after replication initiation from sites of DNA replication. Therefore, we used a robust statistical method to more precisely analyse immunofluorescence localization of MCM 2 proteins with respect to the DNA replication foci. We show that despite the predominantly different localization of MCM 2 and replication signals, there is still a small but significant fraction of MCM 2 proteins that co-localize with DNA replication foci during most of S phase. The fluorescence localization of the MCM 2 proteins and DNA replication may thus reflect an active function of MCM 2 proteins associated with the replication foci and partially explain one facet of the “MCM paradox”.
- MeSH
- buněčné jádro metabolismus MeSH
- chromatin metabolismus MeSH
- financování organizované MeSH
- fluorescenční protilátková technika MeSH
- HeLa buňky MeSH
- jaderné proteiny metabolismus MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- neparametrická statistika MeSH
- počítačové zpracování obrazu metody MeSH
- proteiny buněčného cyklu metabolismus MeSH
- replikační počátek MeSH
- S fáze MeSH
- Check Tag
- lidé MeSH
