This paper describes a novel type of nuclear structure - nuclear lipid islets (NLIs). They are of 40-100 nm with a lipidic interior, and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] molecules comprise a significant part of their surface. Most of NLIs have RNA at the periphery. Consistent with that, RNA is required for their integrity. The NLI periphery is associated with Pol II transcription machinery, including the largest Pol II subunit, transcription factors and NM1 (also known as NMI). The PtdIns(4,5)P2-NM1 interaction is important for Pol II transcription, since NM1 knockdown reduces the Pol II transcription level, and the overexpression of wild-type NM1 [but not NM1 mutated in the PtdIns(4,5)P2-binding site] rescues the transcription. Importantly, Pol II transcription is dependent on NLI integrity, because an enzymatic reduction of the PtdIns(4,5)P2 level results in a decrease of the Pol II transcription level. Furthermore, about half of nascent transcripts localise to NLIs, and transcriptionally active transgene loci preferentially colocalise with NLIs. We hypothesize that NLIs serve as a structural platform that facilitates the formation of Pol II transcription factories, thus participating in the formation of nuclear architecture competent for transcription.
Phosphoinositides (PIs) are glycerol-based phospholipids containing hydrophilic inositol ring. The inositol ring is mono-, bis-, or tris-phosphorylated yielding seven PIs members. Ample evidence shows that PIs localize both to the cytoplasm and to the nucleus. However, tools for direct visualization of nuclear PIs are limited and many studies thus employ indirect approaches, such as staining of their metabolic enzymes. Since localization and mobility of PIs differ from their metabolic enzymes, these approaches may result in incomplete data. In this paper, we tested commercially available PIs antibodies by light microscopy on fixed cells, tested their specificity using protein-lipid overlay assay and blocking assay, and compared their staining patterns. Additionally, we prepared recombinant PIs-binding domains and tested them on both fixed and live cells by light microscopy. The results provide a useful overview of usability of the tools tested and stress that the selection of adequate tools is critical. Knowing the localization of individual PIs in various functional compartments should enable us to better understand the roles of PIs in the cell nucleus.
- MeSH
- buněčné jadérko chemie metabolismus MeSH
- fluorescenční mikroskopie MeSH
- fluorescenční protilátková technika nepřímá MeSH
- fosfatidylinositoly analýza imunologie metabolismus MeSH
- konfokální mikroskopie MeSH
- kultivované buňky MeSH
- lidé MeSH
- protilátky imunologie MeSH
- reakce antigenu s protilátkou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
RNA polymerase I (Pol I) transcription is essential for the cell cycle, growth and protein synthesis in eukaryotes. In the present study, we found that phosphatidylinositol 4,5-bisphosphate (PIP2) is a part of the protein complex on the active ribosomal promoter during transcription. PIP2 makes a complex with Pol I and the Pol I transcription factor UBF in the nucleolus. PIP2 depletion reduces Pol I transcription, which can be rescued by the addition of exogenous PIP2. In addition, PIP2 also binds directly to the pre-rRNA processing factor fibrillarin (Fib), and co-localizes with nascent transcripts in the nucleolus. PIP2 binding to UBF and Fib modulates their binding to DNA and RNA, respectively. In conclusion, PIP2 interacts with a subset of Pol I transcription machinery, and promotes Pol I transcription.
- MeSH
- buněčné jadérko genetika metabolismus MeSH
- chromozomální proteiny, nehistonové genetika metabolismus MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- fosfatidylinositol-4,5-difosfát genetika metabolismus MeSH
- genetická transkripce genetika MeSH
- HeLa buňky MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- prekurzory RNA genetika metabolismus MeSH
- promotorové oblasti (genetika) genetika MeSH
- RNA-polymerasa I genetika metabolismus MeSH
- transkripční iniciační komplex Pol1 - proteiny genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
To maintain growth and division, cells require a large-scale production of rRNAs which occurs in the nucleolus. Recently, we have shown the interaction of nucleolar phosphatidylinositol 4,5-bisphosphate (PIP2) with proteins involved in rRNA transcription and processing, namely RNA polymerase I (Pol I), UBF, and fibrillarin. Here we extend the study by investigating transcription-related localization of PIP2 in regards to transcription and processing complexes of Pol I. To achieve this, we used either physiological inhibition of transcription during mitosis or inhibition by treatment the cells with actinomycin D (AMD) or 5,6-dichloro-1β-d-ribofuranosyl-benzimidazole (DRB). We show that PIP2 is associated with Pol I subunits and UBF in a transcription-independent manner. On the other hand, PIP2/fibrillarin colocalization is dependent on the production of rRNA. These results indicate that PIP2 is required not only during rRNA production and biogenesis, as we have shown before, but also plays a structural role as an anchor for the Pol I pre-initiation complex during the cell cycle. We suggest that throughout mitosis, PIP2 together with UBF is involved in forming and maintaining the core platform of the rDNA helix structure. Thus we introduce PIP2 as a novel component of the NOR complex, which is further engaged in the renewed rRNA synthesis upon exit from mitosis.
- MeSH
- buněčné jadérko metabolismus MeSH
- buněčný cyklus MeSH
- chromozomální proteiny, nehistonové metabolismus MeSH
- genetická transkripce MeSH
- HeLa buňky MeSH
- lidé MeSH
- mitóza MeSH
- nádorové buněčné linie MeSH
- organizátor jadérka metabolismus MeSH
- rekombinantní proteiny metabolismus MeSH
- ribozomální DNA MeSH
- RNA ribozomální MeSH
- RNA-polymerasa I metabolismus MeSH
- transkripční iniciační komplex Pol1 - proteiny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the "cytoplasmic" myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. CONCLUSIONS/SIGNIFICANCE: We have shown that the novel specific NLS brings to the cell nucleus not only the "nuclear" isoform of myosin I (NM1 protein) but also its "cytoplasmic" isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus.
- MeSH
- adenosindifosfát metabolismus MeSH
- aktiny metabolismus MeSH
- aktivní transport - buněčné jádro MeSH
- buněčné jádro metabolismus MeSH
- buněčné linie MeSH
- jaderné lokalizační signály MeSH
- kalmodulin metabolismus MeSH
- karyoferiny metabolismus MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- myosin typu I chemie metabolismus MeSH
- myosiny chemie metabolismus MeSH
- myši MeSH
- sekvence aminokyselin MeSH
- terciární struktura proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Rosiglitazone, peroxisome proliferator-activated receptor-gamma agonist, is an insulin sensitizing agent in peripheral tissues. This study investigated islet hormones and hormone-like peptides expression patterns in rosiglitazone treated streptozotocin (STZ)-diabetic rats by using immunohistochemistry and in situ hybridization methods. Animals were divided into four groups. I. Group: Intact control rats. II. Group: Rosiglitazone-treated controls. III. Group: STZ-diabetic rats. IV. Group: Rosiglitazone-treated diabetic animals. Rosiglitazone was given for 7 days at a dose of 20 mg/kg body weight. In the STZ-diabetic group, there were significant differences in islet hormones and hormone like peptides cell numbers compared to rosiglitazone control group and intact control group. There were significant differences in cocaine- and amphetamine-regulated transcript (CART) and pancreatic polypeptide (PP) cell numbers between rosiglitazone control group and rosiglitazone + STZ-diabetic group. We detected a significant decrease in glucagon mRNA signals in rosiglitazone-treated control group compared to intact controls. We found a statistically significant difference in islet amyloid polypeptide (IAPP) mRNA signals between the STZ-diabetic group and the rosiglitazone + STZ-diabetic group. Besides, we also demonstrated co-localization of peptides by using double and triple histochemistry. In conclusion, our results show that short-term rosiglitazone treatment had a preservative effect to some extent on the expression of islet hormones and hormone-like peptides to maintain the islet function.
- MeSH
- experimentální diabetes mellitus MeSH
- hormony metabolismus MeSH
- hybridizace in situ MeSH
- hypoglykemika farmakologie MeSH
- imunohistochemie MeSH
- krysa rodu rattus MeSH
- Langerhansovy ostrůvky cytologie metabolismus účinky léků MeSH
- peptidy metabolismus MeSH
- potkani Wistar MeSH
- PPAR gama agonisté MeSH
- thiazolidindiony farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
