Juno and CD9 protein, expressed in oolemma, are known to be essential for sperm-oocyte binding and fusion. Although evidence exists that these two proteins cooperate, their interaction has not yet been demonstrated. Here in, we present Juno and CD9 mutual localization over the surface of mouse metaphase II oocytes captured using the 3D STED super-resolution technique. The precise localization of examined proteins was identified in different compartments of oolemma such as the microvillar membrane, planar membrane between individual microvilli, and the membrane of microvilli-free region. Observed variance in localization of Juno and CD9 was confirmed by analysis of transmission and scanning electron microscopy images, which showed a significant difference in the presence of proteins between selected membrane compartments. Colocalization analysis of super-resolution images based on Pearson's correlation coefficient supported evidence of Juno and CD9 mutual position in the oolemma, which was identified by proximity ligation assay. Importantly, the interaction between Juno and CD9 was detected by co-immunoprecipitation and mass spectrometry in HEK293T/17 transfected cell line. For better understanding of experimental data, mouse Juno and CD9 3D structure were prepared by comparative homology modelling and several protein-protein flexible sidechain dockings were performed using the ClusPro server. The dynamic state of the proteins was studied in real-time at atomic level by molecular dynamics (MD) simulation. Docking and MD simulation predicted Juno-CD9 interactions and stability also suggesting an interactive mechanism. Using the multiscale approach, we detected close proximity of Juno and CD9 within microvillar oolemma however, not in the planar membrane or microvilli-free region. Our findings show yet unidentified Juno and CD9 interaction within the mouse oolemma protein network prior to sperm attachment. These results suggest that a Juno and CD9 interactive network could assist in primary Juno binding to sperm Izumo1 as a prerequisite to subsequent gamete membrane fusion.
- Publikační typ
- časopisecké články MeSH
Cholesterol is not only a major component of the cell membrane, but also plays an important role in a wide range of biological processes and pathologies. It is therefore crucial to develop appropriate tools for visualizing intracellular cholesterol transport. Here, we describe new cationic analogues of BODIPY-Cholesterol (TopFluor-Cholesterol, TF-Chol), which combine a positive charge on the sterol side chain and a BODIPY group connected via a C-4 linker. In contrast to TF-Chol, the new analogues TF-1 and TF-3 possessing acetyl groups on the A ring (C-3 position on steroid) internalized much faster and displayed slightly different levels of intracellular localization. Their applicability for cholesterol monitoring was indicated by the fact that they strongly label compartments with accumulated cholesterol in cells carrying a mutation of the Niemann-Pick disease-associated cholesterol transporter, NPC1.
- MeSH
- biologický transport MeSH
- buněčné linie MeSH
- cholesterol analogy a deriváty analýza chemická syntéza metabolismus MeSH
- lidé MeSH
- optické zobrazování MeSH
- sloučeniny boru analýza chemická syntéza chemie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The monitoring of intracellular cholesterol homeostasis and trafficking is of great importance because their imbalance leads to many pathologies. Reliable tools for cholesterol detection are in demand. This study presents the design and synthesis of fluorescent probes for cholesterol recognition and demonstrates their selectivity by a variety of methods. The construction of dedicated library of 14 probes was based on heterocyclic (pyridine)-sterol derivatives with various attached fluorophores. The most promising probe, a P1-BODIPY conjugate FP-5, was analysed in detail and showed an intensive labelling of cellular membranes followed by intracellular redistribution into various cholesterol rich organelles and vesicles. FP-5 displayed a stronger signal, with faster kinetics, than the commercial TF-Chol probe. In addition, cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, exhibited strong and fast FP-5 signal in the endo/lysosomal compartment, co-localizing with filipin staining of cholesterol. Hence, FP-5 has high potential as a new probe for monitoring cholesterol trafficking and its disorders.
- MeSH
- biologický transport MeSH
- buněčná membrána chemie metabolismus MeSH
- buněčné linie MeSH
- cholesterol analýza metabolismus MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční mikroskopie metody MeSH
- lidé MeSH
- lyzozomální nemoci z ukládání diagnóza metabolismus MeSH
- pyridiny chemie MeSH
- sloučeniny boru chemie MeSH
- steroly chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: The search for new anticancer compounds is a crucial element of natural products research. PURPOSE: In this study the effects of naturally occurring homochelidonine in comparison to chelidonine on cell cycle progression and cell death in leukemic T-cells with different p53 status are described. METHODS: The mechanism of cytotoxic, antiproliferative, apoptosis-inducing effects and the effect on expressions of cell cycle regulatory proteins was investigated using XTT assay, Trypan blue exclusion assay, flow cytometry, Western blot analysis, xCELLigence, epi-fluorescence and 3D super resolution microscopy. A549 cells were used for xCELLigence, clonogenic assay and for monitoring microtubule stability. RESULTS: We found that homochelidonine and chelidonine displayed significant cytotoxicity in examined blood cancer cells with the exception of HEL 92.1.7 and U-937 exposed to homochelidonine. Unexpectedly, homochelidonine and chelidonine-induced cytotoxicity was more pronounced in Jurkat cells contrary to MOLT-4 cells. Homochelidonine showed an antiproliferative effect on A549 cells but it was less effective compared to chelidonine. Biphasic dose-depended G1 and G2/M cell cycle arrest along with the population of sub-G1 was found after treatment with homochelidonine in MOLT-4 cells. In variance thereto, an increase in G2/M cells was detected after treatment with homochelidonine in Jurkat cells. Treatment with chelidonine induced cell cycle arrest in the G2/M cell cycle in both MOLT-4 and Jurkat cells. MOLT-4 and Jurkat cells treated with homochelidonine and chelidonine showed features of apoptosis such as phosphatidylserine exposure, a loss of mitochondrial membrane potential and an increase in the caspases -3/7, -8 and -9. Western blots indicate that homochelidonine and chelidonine exposure activates Chk1 and Chk2. Studies conducted with fluorescence microscopy demonstrated that chelidonine and homochelidonine inhibit tubulin polymerization in A549 cells. CONCLUSION: Collectively, the data indicate that chelidonine and homochelidonine are potent inducers of cell death in cancer cell lines, highlighting their potential relevance in leukemic cells.
- MeSH
- apoptóza účinky léků MeSH
- benzofenantridiny farmakologie MeSH
- berberinové alkaloidy farmakologie MeSH
- Chelidonium chemie MeSH
- Jurkat buňky MeSH
- kaspasy metabolismus MeSH
- kontrolní body buněčného cyklu účinky léků MeSH
- lidé MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- nádorové buněčné linie účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Skupinu chorob vyznačujících se vysokou hladinou imunoglobulinu G4 (IgG4) v krevním séru označujeme jako IgG4 asociovaná onemocnění. Nejčastěji se vyskytující nemocí z této skupiny je autoimunitní forma pankreatitidy. Onemocnění je charakterizované v úvodu nepříliš intenzivními subjektivními potížemi, spíše pouze břišním dyskomfortem, ale prvním příznakem může být bezbolestný obstrukční ikterus. Zobrazovacími metodami lze zobrazit difuzně nebo segmentárně zvětšený pankreas, což může činit obtíže v odlišení od pankreatického karcinomu. Pro autoimunitní formu pankreatitidy, především pro její 1. typ, je charakteristickým nález zvýšené hodnoty IgG4 krevního séra, kdy 97% specificita a 95% senzitivita predikují diagnózu autoimunitní formy pankreatitidy. V literatuře se také objevila kazuistická sdělení uvádějící nález vysoké hladiny IgG4 u osob s pankreatickým karcinomem. Proto jsme v prospektivní studii vyšetřili soubor osob s histologicky verifikovaným pankreatickým karcinomem a stanovili u nich IgG4 v krevním séru. Materiál a metodika: V průběhu 52 měsíců byla v krevním séru vyšetřena hladina IgG4 metodou nefelometrickou u 81 osob s histologicky verifikovaným pankreatickým karcinomem. Podle literárních standardů je mezní hodnotou hladina IgG4 vyšší než 135 mg/dl. Výsledky: Ve sledovaném souboru byla hladina IgG4 vyšší než 135 mg/dl prokázána u osmi osob (9,8 %). Průměrná hodnota IgG4 byla 173 mg/dl, tj. o 12,9 % vyšší oproti hraniční hodnotě. U osob, které měly prokázánu vyšší hladinu IgG4, bylo pět osob, kde lze předpokládat vznik karcinomu vterénu chronické pankreatitidy, protože diagnóza chronické pankreatitidy byla stanovena více než pět let před stanovením diagnózy pankreatického karcinomu. Ani jedna z osmi osob s vyšší hladinou IgG4 neměla jakékoli diagnostické znaky autoimunitní formy pankreatitidy. Závěr: Vyšší hladina IgG4 může provázet i nález pankreatického karcinomu, avšak hodnota IgG4 je zvýšena ne více než o 20 % nad tzv. limitní hodnotu IgG4 v séru. Stanovení IgG4 není markerem vhodným pro diferenciální diagnostiku mezi autoimunitní formou pankreatitidy a pankreatickým karcinomem, jak bylo původně předpokládáno, a lze souhlasit s názory, že alespoň dvojnásobné zvýšení IgG4 krevního séra je významným diagnostickým znakem pro autoimunitní formu pankreatitidy, resp. pro celou skupinu chorob označených IgG4 asociované nemoci.
The group of illnesses marked by high level of immunoglobulin G4 (IgG4) in blood serum are designated as IgG4-associated diseases. The most common disease in this group is an autoimmune form of pancreatitis. In its initial stage, this is characterised by mild, subjective complaints, which more resemble stomach discomfort. The first symptom, however, may be a painless, obstructive icterus. Imaging methods can be used to view the diffuse or segmental enlargement of the pancreas, which can cause difficulties in distinguishing it from a pancreatic carcinoma. A characteristic feature of the autoimmune form of pancreatitis, and in particular first type, is an increased level of IgG4 of the blood serum, where a 97% specificity and 95% sensitivity predict a diagnosis of the autoimmune form of pancreatitis. Case studies have also appeared in specialist literature showing a high level of IgG4 in patients with a pancreatic carcinoma. In our prospective study, we therefore examined a set of patients with histologically verified pancreatic carcinomas and determined the level of IgG4 in their blood serum. Materials and methods: Over the course of a 52-month period, the level of IgG4 in blood serum was analysed using the nephelometric method in 81 patients with histologically verified pancreatic carcinomas. According to standards set in literature, the borderline IgG4 value is higher than 135 mg/dl. Results: In the observed group an IgG4 level exceeding 135 mg/dl was demonstrated in 8 patients (9.8). The average IgG4 level was 173 mg/dl, i.e. 12.9% higher against the borderline value. Amongst the patients showing a greater IgG4 value were five in whom the creation of a carcinoma during the course of chronic pancreatitis could be anticipated, as the diagnosis of chronic pancreatitis was determined more than 5 years before the determination of the diagnosis of the pancreatic carcinoma. None of the 8 patients with a higher IgG4 value showed any diagnostic signs of the autoimmune form of pancreatitis. Conclusion: A higher level of IgG4 can accompany the finding of a pancreatic carcinoma; however, the IgG4 value is increased by no more than 20% above the so-called serum IgG4 limit value. Determination of IgG4 is not a suitable marker for differential diagnostics between the autoimmune form of pancreatitis and a pancreatic carcinoma, as was originally assumed, and it is possible to agree with opinions that an at least twofold increase in IgG4 of the blood serum is a significant diagnostic sign for the autoimmune form of pancreatitis and the entire group of diseases designated as IgG4-associated diseases.
- MeSH
- autoimunitní nemoci diagnóza etiologie MeSH
- autoimunitní pankreatitida MeSH
- biopsie metody využití MeSH
- diagnostické zobrazování metody využití MeSH
- diferenciální diagnóza MeSH
- imunoglobulin G imunologie izolace a purifikace krev MeSH
- klinický obraz nemoci MeSH
- lidé MeSH
- nádory slinivky břišní diagnóza etiologie imunologie MeSH
- pankreatitida diagnóza etiologie imunologie MeSH
- prospektivní studie MeSH
- senzitivita a specificita MeSH
- sérologické testy metody využití MeSH
- sérum imunologie MeSH
- statistika jako téma MeSH
- výsledky a postupy - zhodnocení (zdravotní péče) MeSH
- Check Tag
- lidé MeSH
Processing bodies (P-bodies) are dynamic cytoplasmic structures involved in mRNA degradation, but the mechanism that governs their formation is poorly understood. In this paper, we address a role of Like-Sm (LSm) proteins in formation of P-bodies and provide evidence that depletion of nuclear LSm8 increases the number of P-bodies, while LSm8 overexpression leads to P-body loss. We show that LSm8 knockdown causes relocalization of LSm4 and LSm6 proteins to the cytoplasm and suggest that LSm8 controls nuclear accumulation of all LSm2-7 proteins. We propose a model in which redistribution of LSm2-7 to the cytoplasm creates new binding sites for other P-body components and nucleates new, microscopically visible structures. The model is supported by prolonged residence of two P-body proteins, DDX6 and Ago2, in P-bodies after LSm8 depletion, which indicates stronger interactions between these proteins and P-bodies. Finally, an increased number of P-bodies has negligible effects on microRNA-mediated translation repression and nonsense mediated decay, further supporting the view that the function of proteins localized in P-bodies is independent of visible P-bodies.
- MeSH
- autoantigeny metabolismus MeSH
- buněčné jádro metabolismus MeSH
- cytoplazmatická granula metabolismus MeSH
- DEAD-box RNA-helikasy metabolismus MeSH
- fluorescenční mikroskopie MeSH
- lidé MeSH
- malý jaderný ribonukleoprotein U4-U6 metabolismus fyziologie MeSH
- N-terminální acetyltransferáza C metabolismus fyziologie MeSH
- posttranskripční úpravy RNA * MeSH
- proteiny vázající RNA metabolismus MeSH
- protoonkogenní proteiny metabolismus MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- ribonukleoproteiny malé jaderné metabolismus MeSH
- transport proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The U4/U6·U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) is an essential pre-mRNA splicing factor, which is assembled in a stepwise manner before each round of splicing. It was previously shown that the tri-snRNP is formed in Cajal bodies (CBs), but little is known about the dynamics of this process. Here we created a mathematical model of tri-snRNP assembly in CBs and used it to fit kinetics of individual snRNPs monitored by fluorescence recovery after photobleaching. A global fitting of all kinetic data determined key reaction constants of tri-snRNP assembly. Our model predicts that the rates of di-snRNP and tri-snRNP assemblies are similar and that ∼230 tri-snRNPs are assembled in one CB per minute. Our analysis further indicates that tri-snRNP assembly is approximately 10-fold faster in CBs than in the surrounding nucleoplasm, which is fully consistent with the importance of CBs for snRNP formation in rapidly developing biological systems. Finally, the model predicted binding between SART3 and a CB component. We tested this prediction by Förster resonance energy transfer and revealed an interaction between SART3 and coilin in CBs.
- MeSH
- antigeny nádorové genetika metabolismus MeSH
- buněčné jádro genetika metabolismus MeSH
- Cajalova tělíska genetika metabolismus MeSH
- HeLa buňky MeSH
- jaderné proteiny metabolismus MeSH
- kinetika MeSH
- lidé MeSH
- malý jaderný ribonukleoprotein U4-U6 genetika metabolismus MeSH
- malý jaderný ribonukleoprotein U5 genetika metabolismus MeSH
- molekulární modely MeSH
- nádorové buněčné linie MeSH
- prekurzory RNA genetika metabolismus MeSH
- proteiny vázající RNA genetika metabolismus MeSH
- ribonukleoproteiny malé jaderné genetika metabolismus MeSH
- RNA-helikasy genetika metabolismus MeSH
- sestřih RNA genetika MeSH
- spliceozomy genetika metabolismus MeSH
- vazba proteinů genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Cajal body (CB) is a nuclear structure closely associated with import and biogenesis of small nuclear ribonucleoprotein particles (snRNPs). Here, we tested whether CBs also contain mature snRNPs and whether CB integrity depends on the ongoing snRNP splicing cycle. Sm proteins tagged with photoactivatable and color-maturing variants of fluorescent proteins were used to monitor snRNP behavior in living cells over time; mature snRNPs accumulated in CBs, traveled from one CB to another, and they were not preferentially replaced by newly imported snRNPs. To test whether CB integrity depends on the snRNP splicing cycle, two human orthologues of yeast proteins involved in distinct steps in spliceosome disassembly after splicing, hPrp22 and hNtr1, were depleted by small interfering RNA treatment. Surprisingly, depletion of either protein led to the accumulation of U4/U6 snRNPs in CBs, suggesting that reassembly of the U4/U6.U5 tri-snRNP was delayed. Accordingly, a relative decrease in U5 snRNPs compared with U4/U6 snRNPs was observed in CBs, as well as in nuclear extracts of treated cells. Together, the data show that particular phases of the spliceosome cycle are compartmentalized in living cells, with reassembly of the tri-snRNP occurring in CBs.
- MeSH
- biologické markery metabolismus MeSH
- Cajalova tělíska metabolismus MeSH
- financování organizované MeSH
- HeLa buňky MeSH
- lidé MeSH
- malá interferující RNA metabolismus MeSH
- malý jaderný ribonukleoprotein U4-U6 metabolismus MeSH
- malý jaderný ribonukleoprotein U5 metabolismus MeSH
- protein přežití motorických neuronů 1 metabolismus MeSH
- ribonukleoproteiny malé jaderné metabolismus MeSH
- spliceozomy metabolismus MeSH
- transportní proteiny metabolismus MeSH
- Check Tag
- lidé MeSH
