We firstly identified 48 kDa molecular form of the unconventional myosin 1c (p48/Myo1C), and isolated it from blood serum of multiple sclerosis patients. The amount of p48/Myo1C in human blood serum correlated with some autoimmune, hemato-oncological and neurodegenerative diseases and thus may serve as a potential molecular biomarker. The biological functions of this protein in human blood remain unknown. Previously, we used the monodisperse magnetic poly (glycidyl methacrylate)(mag-PGMA-NH2 ) microspheres with immobilized 48/Myo1C and western-blot analysis, which allowed us to identify IgM and IgG immunoglobulins presenting an affinity to this protein. Here, we used mass spectrometry followed by the western blotting in order to identify other blood serum proteins with affinity to 48/Myo1C. The obtained data demonstrate that 48/Myo1C binds to component 3 of the complement and the antithrombin-III proteins. A combination of magnetic microparticle-based affinity chromatography with MALDI-TOF mass spectrometry and an in silico analysis provided an opportunity to identify the partners of interaction of 48/Myo1C with other proteins, in particular those participating in complement and coagulation cascades.
- MeSH
- chromatografie afinitní metody MeSH
- krevní proteiny analýza chemie metabolismus MeSH
- lidé MeSH
- magnety MeSH
- mikrosféry MeSH
- molekulární modely MeSH
- myosin typu I chemie metabolismus MeSH
- prognóza MeSH
- roztroušená skleróza krev MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- vazba proteinů MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Here, we provide evidence for the presence of Myosin phosphatase rho-interacting protein (MPRIP), an F-actin-binding protein, in the cell nucleus. The MPRIP protein binds to Phosphatidylinositol 4,5-bisphosphate (PIP2) and localizes to the nuclear speckles and nuclear lipid islets which are known to be involved in transcription. We identified MPRIP as a component of RNA Polymerase II/Nuclear Myosin 1 complex and showed that MPRIP forms phase-separated condensates which are able to bind nuclear F-actin fibers. Notably, the fibrous MPRIP preserves its liquid-like properties and reforms the spherical shaped condensates when F-actin is disassembled. Moreover, we show that the phase separation of MPRIP is driven by its long intrinsically disordered region at the C-terminus. We propose that the PIP2/MPRIP association might contribute to the regulation of RNAPII transcription via phase separation and nuclear actin polymerization.
- MeSH
- adaptorové proteiny signální transdukční chemie metabolismus MeSH
- aktiny metabolismus MeSH
- buněčné jádro účinky léků metabolismus MeSH
- fosfatidylinositol-4,5-difosfát metabolismus MeSH
- glykoly farmakologie MeSH
- lidé MeSH
- myosin typu I metabolismus MeSH
- nádorové buněčné linie MeSH
- proteinové domény MeSH
- RNA-polymerasa II metabolismus MeSH
- subcelulární frakce metabolismus MeSH
- vazba proteinů účinky léků MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Nuclear myosin 1 (NM1) has been implicated in key nuclear functions. Together with actin, it has been shown to initiate and regulate transcription, it is part of the chromatin remodeling complex B-WICH, and is responsible for rearrangements of chromosomal territories in response to external stimuli. Here we show that deletion of NM1 in mouse embryonic fibroblasts leads to chromatin and transcription dysregulation affecting the expression of DNA damage and cell cycle genes. NM1 KO cells exhibit increased DNA damage and changes in cell cycle progression, proliferation, and apoptosis, compatible with a phenotype resulting from impaired p53 signaling. We show that upon DNA damage, NM1 forms a complex with p53 and activates the expression of checkpoint regulator p21 (Cdkn1A) by PCAF and Set1 recruitment to its promoter for histone H3 acetylation and methylation. We propose a role for NM1 in the transcriptional response to DNA damage response and maintenance of genome stability.
- MeSH
- apoptóza MeSH
- buněčné jádro účinky léků genetika metabolismus patologie MeSH
- buněčné linie MeSH
- buněčný cyklus MeSH
- epigeneze genetická MeSH
- etoposid toxicita MeSH
- genetická transkripce * MeSH
- histonlysin-N-methyltransferasa genetika metabolismus MeSH
- inhibitor p21 cyklin-dependentní kinasy genetika metabolismus MeSH
- myosin typu I genetika metabolismus MeSH
- myši MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- poškození DNA * MeSH
- proliferace buněk MeSH
- restrukturace chromatinu * MeSH
- transkripční faktory p300-CBP genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension.
- MeSH
- buněčná membrána metabolismus MeSH
- buněčné jádro metabolismus MeSH
- exocytóza fyziologie MeSH
- fibroblasty cytologie metabolismus MeSH
- HeLa buňky MeSH
- kultivované buňky MeSH
- kůže cytologie metabolismus MeSH
- lidé MeSH
- mikrofilamenta metabolismus MeSH
- myosin typu I metabolismus MeSH
- myši knockoutované MeSH
- myši MeSH
- pohyb buněk MeSH
- tvar buňky MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. METHODOLOGY/PRINCIPAL FINDINGS: In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. CONCLUSION/SIGNIFICANCE: We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes.
- MeSH
- buněčné jádro metabolismus MeSH
- DNA primery genetika MeSH
- fenotyp * MeSH
- genotyp MeSH
- imunoprecipitace MeSH
- myosin typu I genetika metabolismus MeSH
- myši knockoutované MeSH
- myši MeSH
- plazmidy genetika MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- protein - isoformy genetika metabolismus MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata 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
Nuclear actin and nuclear myosin I (NMI) are important players in transcription of ribosomal genes. Transcription of rDNA takes place in highly organized intranuclear compartment, the nucleolus. In this study, we characterized the localization of these two proteins within the nucleolus of HeLa cells with high structural resolution by means of electron microscopy and gold-immunolabeling. We demonstrate that both actin and NMI are localized in specific compartments within the nucleolus, and the distribution of NMI is transcription-dependent. Moreover, a pool of NMI is present in the foci containing nascent rRNA transcripts. Actin, in turn, is present both in transcriptionally active and inactive regions of the nucleolus and colocalizes with RNA polymerase I and UBF. Our data support the involvement of actin and NMI in rDNA transcription and point out to other functions of these proteins in the nucleolus, such as rRNA maturation and maintenance of nucleolar architecture.
- MeSH
- aktiny metabolismus MeSH
- buněčné jadérko metabolismus MeSH
- genetická transkripce fyziologie MeSH
- HeLa buňky MeSH
- imunohistochemie MeSH
- lidé MeSH
- myosin typu I metabolismus MeSH
- ribozomální DNA metabolismus MeSH
- RNA ribozomální metabolismus 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
Nuclear myosin I (NMI) is a single-headed member of myosin superfamily localized in the cell nucleus which participates along with nuclear actin in transcription and chromatin remodeling. We demonstrate that NMI is present in cell nuclei of all mouse tissues examined except for cells in terminal stages of spermiogenesis. Quantitative PCR and western blots demonstrate that the expression of NMI in tissues varies with the highest levels in the lungs. The expression of NMI is lower in serum-starved cells and it increases after serum stimulation. The lifespan of NMI is longer than 16 h as determined by cycloheximide translation block. A homologous protein is expressed in human, chicken, Xenopus, and zebrafish as shown by RACE analysis. The analysis of genomic sequences indicates that almost identical homologous NMI genes are expressed in mammals, and similar NMI genes in vertebrates.
- MeSH
- buněčné jádro metabolismus MeSH
- buněčné linie MeSH
- exprese genu MeSH
- financování organizované MeSH
- fylogeneze MeSH
- genetická transkripce MeSH
- konzervovaná sekvence MeSH
- lidé MeSH
- myosin typu I genetika chemie izolace a purifikace metabolismus MeSH
- myši MeSH
- obratlovci genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie nukleových kyselin MeSH
- sérum chemie MeSH
- techniky amplifikace nukleových kyselin MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
As previous studies suggested, nuclear myosin I (NMI) and actin have important roles in DNA transcription. In this study, we characterized the dynamics of these two proteins during transcriptional activation in phytohemagglutinin (PHA) stimulated human lymphocytes. The stimulation led to strong up-regulation of NMI both on the mRNA and protein level, while actin was relatively stably expressed. The intranuclear distribution of actin and NMI was evaluated using immunogold labeling. In nucleoli of resting cells, actin was localized predominantly to fibrillar centers (FCs), while NMI was located mainly to the dense fibrillar component (DFC). Upon stimulation, FCs remained the main site of actin localization, however, an accumulation of both actin and NMI in the DFC and in the granular component was observed. In the nucleoplasm of resting lymphocytes, both actin and NMI were localized mostly in condensed chromatin. Following stimulation, the majority of both proteins shifted towards the decondensed chromatin. In transcriptionally active cells, both actin and NMI colocalized with nucleoplasmic transcription sites. These results demonstrate that actin and NMI are compartmentalized in the nuclei where they can dynamically translocate depending on transcriptional activity of the cells.
- MeSH
- aktiny metabolismus MeSH
- buněčné jadérko metabolismus účinky léků ultrastruktura MeSH
- buněčné jádro metabolismus účinky léků ultrastruktura MeSH
- financování organizované MeSH
- fytohemaglutininy farmakologie MeSH
- genetická transkripce imunologie MeSH
- lidé MeSH
- lymfocyty mikrobiologie účinky záření virologie MeSH
- messenger RNA metabolismus MeSH
- myosin typu I metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- transmisní elektronová mikroskopie MeSH
- Check Tag
- lidé MeSH
