Tick-borne encephalitis virus (TBEV) causes serious, potentially fatal neurological infections that affect humans in endemic regions of Europe and Asia. Neurons are the primary target for TBEV infection in the central nervous system. However, knowledge about this viral infection and virus-induced neuronal injury is fragmental. Here, we directly examined the pathology that occurs after TBEV infection in human primary neurons. We exploited the advantages of advanced high-pressure freezing and freeze-substitution techniques to achieve optimal preservation of infected cell architecture. Electron tomographic (ET) reconstructions elucidated high-resolution 3D images of the proliferating endoplasmic reticulum, and individual tubule-like structures of different diameters in the endoplasmic reticulum cisternae of single cells. ET revealed direct connections between the tubule-like structures and viral particles in the endoplasmic reticulum. Furthermore, ET showed connections between cellular microtubules and vacuoles that harbored the TBEV virions in neuronal extensions. This study was the first to characterize the 3D topographical organization of membranous whorls and autophagic vacuoles in TBEV-infected human neurons. The functional importance of autophagy during TBEV replication was studied in human neuroblastoma cells; stimulation of autophagy resulted in significantly increased dose-dependent TBEV production, whereas the inhibition of autophagy showed a profound, dose-dependent decrease of the yield of infectious virus.
- MeSH
- autofagie účinky léků genetika MeSH
- benzylaminy farmakologie MeSH
- chinazoliny farmakologie MeSH
- endoplazmatické retikulum účinky léků ultrastruktura virologie MeSH
- lidé MeSH
- mikrotubuly účinky léků ultrastruktura virologie MeSH
- nádorové buněčné linie MeSH
- neurony účinky léků ultrastruktura virologie MeSH
- nokodazol farmakologie MeSH
- primární buněčná kultura MeSH
- replikace viru účinky léků MeSH
- sirolimus farmakologie MeSH
- tomografie elektronová MeSH
- virion účinky léků růst a vývoj ultrastruktura MeSH
- viry klíšťové encefalitidy účinky léků růst a vývoj ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Automated imaging screens are performed mostly on fixed and stained samples to simplify the workflow and increase throughput. Some processes, such as the movement of cells and organelles or measuring membrane integrity and potential, can be measured only in living cells. Developing such assays to screen large compound or RNAi collections is challenging in many respects. Here, we develop a live-cell high-content assay for tracking endocytic organelles in medium throughput. We evaluate the added value of measuring kinetic parameters compared with measuring static parameters solely. We screened 2000 compounds in U-2 OS cells expressing Lamp1-GFP to label late endosomes. All hits have phenotypes in both static and kinetic parameters. However, we show that the kinetic parameters enable better discrimination of the mechanisms of action. Most of the compounds cause a decrease of motility of endosomes, but we identify several compounds that increase endosomal motility. In summary, we show that kinetic data help to better discriminate phenotypes and thereby obtain more subtle phenotypic clustering.
- MeSH
- analýza buněčné migrace metody MeSH
- analýza hlavních komponent MeSH
- automatizace MeSH
- endozomy metabolismus MeSH
- fenotyp MeSH
- fluorescenční mikroskopie MeSH
- lidé MeSH
- multivariační analýza MeSH
- nádorové buněčné linie MeSH
- nokodazol chemie MeSH
- počítačové zpracování obrazu MeSH
- pohyb MeSH
- reprodukovatelnost výsledků MeSH
- RNA interference MeSH
- techniky kombinatorické chemie MeSH
- zelené fluorescenční proteiny chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as 'funicular cabin' for the cell nucleus, and actin cables as intracellular 'funicular' suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.
- MeSH
- aktiny analýza MeSH
- bicyklické sloučeniny heterocyklické farmakologie MeSH
- buněčné jádro ultrastruktura MeSH
- Cryptococcus neoformans účinky léků fyziologie ultrastruktura MeSH
- dimethylsulfoxid farmakologie MeSH
- elektronová mikroskopie MeSH
- faloidin analogy a deriváty MeSH
- fluorescenční mikroskopie MeSH
- mikrofilamenta ultrastruktura MeSH
- mikrotubuly účinky léků MeSH
- modulátory tubulinu farmakologie MeSH
- mořské toxiny farmakologie MeSH
- nokodazol farmakologie MeSH
- rhodaminy MeSH
- scavengery volných radikálů farmakologie MeSH
- thiazolidiny farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Chk1 is phosphorylated at Ser317 and Ser345 by ATR in response to stalled replication and genotoxic stresses. This Chk1 activation is thought to play critical roles in the prevention of premature mitosis. However, the behavior of Chk1 in mitosis remains largely unknown. Here we reported that Chk1 was phosphorylated in mitosis. The reduction of this phosphorylation was observed at the metaphase-anaphase transition. Two-dimensional phosphopeptide mapping revealed that Chk1 phosphorylation sites in vivo were completely overlapped with the in vitro sites by cyclin-dependent protein kinase (Cdk) 1 or by p38 MAP kinase. Ser286 and Ser301 were identified as novel phosphorylation sites on Chk1. Treatment with Cdk inhibitor butyrolactone I induced the reduction of Chk1-S301 phosphorylation, although treatment with p38-specific inhibitor SB203580 or siRNA did not. In addition, ionizing radiation (IR) or ultraviolet (UV) light did not induce Chk1 phosphorylation at Ser317 and Ser345 in nocodazole-arrested mitotic cells. These observations imply the regulation of mitotic Chk1 function through Chk1 phosphorylation at novel sites by Cdk1.
- MeSH
- biologické modely MeSH
- CHO buňky MeSH
- fosforylace MeSH
- HeLa buňky MeSH
- imunoblotting MeSH
- ionizující záření MeSH
- křečci praví MeSH
- lidé MeSH
- mitóza * fyziologie účinky léků MeSH
- molekulární sekvence - údaje MeSH
- nokodazol farmakologie metabolismus MeSH
- proteinkinasa CDC2 * metabolismus MeSH
- proteinkinasy genetika chemie metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- serin genetika metabolismus MeSH
- transfekce MeSH
- ultrafialové záření MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- lidé MeSH
- zvířata MeSH
Microtubule disruptors, widely known as antimitotics, have broad applications in human medicine, especially as anti-neoplastic agents. They are subject to biotransformation within human body frequently involving cytochromes P450. Therefore antimitotics are potential culprits of drug-drug interactions on the level of activity as well as expression of cytochromes P450. This review discusses the effects of four well-known natural antimitotics: colchicine, taxol (paclitaxel), vincristine, and vinblastine, and a synthetic microtubule disruptor nocodazole on transcriptional activity of glucocorticoid and aryl hydrocarbon receptors. It appears that microtubules disarray restricts the signaling by these two nuclear receptors regardless of cell cycle phase. Consequently, intact microtubules play an important role in the regulation of expression of cytochromes P450, which are under direct or indirect control of the two nuclear receptors.
- MeSH
- antimitotika farmakologie MeSH
- biotransformace MeSH
- kolchicin farmakologie MeSH
- lékové interakce MeSH
- lidé MeSH
- mikrotubuly účinky léků MeSH
- nokodazol farmakologie MeSH
- paclitaxel farmakologie MeSH
- receptory aromatických uhlovodíků metabolismus MeSH
- receptory glukokortikoidů metabolismus MeSH
- signální transdukce MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- vinblastin farmakologie MeSH
- vinkristin farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
In this study we examined whether microtubules and heat shock protein 90 (Hsp90) are involved in phorbol myristate acetate (PMA) and N-formyl-Met-Leu-Phe (fMLP)-induced oxidative burst in DMSO-differentiated HL-60 cells. Our results showed that microtubule interfering agents, paclitaxel (1-5 microM), colchicine (1-100 microM), nocodazole (1-20 microM), and vincristine (1-50 microM), did not affect either PMA or fMLP-induced oxidative burst. In contrast, radicicol, an inhibitor of Hsp90, inhibited fMLP-induced oxidative burst in time and concentration-dependent manner where IC50 value for 30 min pre-incubation was 16.5 +/- 3.5 microM radicicol. We conclude that both PMA and fMLP-induced oxidative burst in DMSO-differentiated HL-60 cells is microtubule-independent while the latter requires Hsp90 activity.
- MeSH
- buněčná diferenciace MeSH
- dimethylsulfoxid farmakologie MeSH
- HL-60 buňky MeSH
- kolchicin farmakologie MeSH
- lidé MeSH
- mikrotubuly účinky léků fyziologie MeSH
- N-formylmethionin-leucyl-fenylalanin farmakologie MeSH
- neutrofily metabolismus MeSH
- nokodazol farmakologie MeSH
- paclitaxel farmakologie MeSH
- proteiny tepelného šoku HSP90 metabolismus MeSH
- respirační vzplanutí účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH