9 results on '"Michaela Blažíková"'
Search Results
2. The Wavelet-Based Denoising Of Images in Fiji, With Example Applications in Structured Illumination Microscopy
- Author
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Martin Čapek, Michaela Blažíková, Ivan Novotný, Helena Chmelová, David Svoboda, Barbora Radochová, Jiří Janáček, and Ondrej Horváth
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discrete wavelet transform ,fiji plugin ,image filtration ,structured illumination microscopy ,Medicine (General) ,R5-920 ,Mathematics ,QA1-939 - Abstract
Filtration of super-resolved microscopic images brings often troubles with removing undesired image parts like, e.g., noise, inhomogenous background and reconstruction artifacts. Standard filtration techniques, e.g., convolution- or Fourier transform-based methods are not always appropriate, since they may lower image resolution that was acquired by hi-tech and expensive microscopy systems. Thus, in this article it is proposed to filter such images using discrete wavelet transform (DWT). Newly developed Wavelet_Denoise plugin for free available Fiji software package demonstrates important possibilities of applying DWT to images: Decomposition of a filtered picture using various wavelet filters and levels of details with showing decomposed images and visualization of effects of back transformation of the picture with chosen level of suppression or denoising of wavelet coefficients. The Fiji framework allows, for example, using a plethora of various microscopic image formats for data opening, users can easily install the plugin through a menu command and the plugin supports processing 3D images in Z-stacks. The application of the plugin for removal of reconstruction artifacts and undesirable background in images acquired by super-resolved structured illumination microscopy is demonstrated as well.
- Published
- 2021
- Full Text
- View/download PDF
3. The F-Actin-Binding MPRIP Forms Phase-Separated Condensates and Associates with PI(4,5)P2 and Active RNA Polymerase II in the Cell Nucleus
- Author
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Can Balaban, Martin Sztacho, Michaela Blažíková, and Pavel Hozák
- Subjects
MPRIP ,phase separation ,PIP2 ,actin ,nucleus ,Cytology ,QH573-671 - Abstract
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.
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- 2021
- Full Text
- View/download PDF
4. Structural and Functional Modulation of Perineuronal Nets: In Search of Important Players with Highlight on Tenascins
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Ana Jakovljević, Pavle R. Andjus, Andrej Korenić, Michaela Blažíková, Yannis F. Missirlis, Milena Tucić, and Vera Stamenković
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QH301-705.5 ,extracellular matrix ,Nerve Tissue Proteins ,Review ,Biology ,Mechanotransduction, Cellular ,03 medical and health sciences ,0302 clinical medicine ,super-resolution microscopy ,Neuroplasticity ,Image Processing, Computer-Assisted ,Animals ,Premovement neuronal activity ,Biology (General) ,Mechanotransduction ,mechanotransduction ,030304 developmental biology ,Neurons ,Extracellular Matrix Proteins ,perineuronal nets ,0303 health sciences ,Neuronal Plasticity ,synaptic plasticity ,Mechanism (biology) ,Perineuronal net ,Neurogenesis ,General Medicine ,neurogenesis ,Synaptic plasticity ,tenascin-C ,Neuroscience ,030217 neurology & neurosurgery ,Function (biology) - Abstract
The extracellular matrix (ECM) of the brain plays a crucial role in providing optimal conditions for neuronal function. Interactions between neurons and a specialized form of ECM, perineuronal nets (PNN), are considered a key mechanism for the regulation of brain plasticity. Such an assembly of interconnected structural and regulatory molecules has a prominent role in the control of synaptic plasticity. In this review, we discuss novel ways of studying the interplay between PNN and its regulatory components, particularly tenascins, in the processes of synaptic plasticity, mechanotransduction, and neurogenesis. Since enhanced neuronal activity promotes PNN degradation, it is possible to study PNN remodeling as a dynamical change in the expression and organization of its constituents that is reflected in its ultrastructure. The discovery of these subtle modifications is enabled by the development of super-resolution microscopy and advanced methods of image analysis.
- Published
- 2021
5. The F-Actin-Binding MPRIP Forms Phase-Separated Condensates and Associates with PI(4,5)P2 and Active RNA Polymerase II in the Cell Nucleus
- Author
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Michaela Blažíková, Can Balaban, Pavel Hozák, and Martin Sztacho
- Subjects
Phosphatidylinositol 4,5-Diphosphate ,Green Fluorescent Proteins ,Phosphatase ,RNA polymerase II ,macromolecular substances ,Article ,Glycols ,Myosin Type I ,chemistry.chemical_compound ,PIP2 ,Protein Domains ,Transcription (biology) ,Cell Line, Tumor ,Myosin ,medicine ,Humans ,Phosphatidylinositol ,MPRIP ,lcsh:QH301-705.5 ,Actin ,Adaptor Proteins, Signal Transducing ,Cell Nucleus ,biology ,nucleus ,General Medicine ,Actins ,Cell nucleus ,medicine.anatomical_structure ,lcsh:Biology (General) ,chemistry ,biology.protein ,Biophysics ,RNA Polymerase II ,phase separation ,actin ,Nucleus ,Protein Binding ,Subcellular Fractions - Abstract
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.
- Published
- 2021
6. Juno and CD9 protein network organization in oolemma of mouse oocyte
- Author
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Michaela Frolikova, Vishma Pratap Sur, Ivan Novotny, Michaela Blazikova, Jana Vondrakova, Ondrej Simonik, Lukas Ded, Eliska Valaskova, Lenka Koptasikova, Ales Benda, Pavla Postlerova, Ondrej Horvath, and Katerina Komrskova
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oocyte ,Juno ,CD9 ,oolemma compartments ,protein interaction ,STED ,Biology (General) ,QH301-705.5 - Abstract
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.
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- 2023
- Full Text
- View/download PDF
7. In vivo kinetics of U4/U6·U5 tri-snRNP formation in Cajal bodies
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Petr Herman, Ivan Novotný, Jan Malinsky, David Staneˇk, and Michaela Blažíková
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Models, Molecular ,Ribonucleoprotein, U4-U6 Small Nuclear ,RNA Splicing ,Coiled Bodies ,Biology ,environment and public health ,digestive system ,Antigens, Neoplasm ,Cell Line, Tumor ,RNA Precursors ,Humans ,snRNP ,Molecular Biology ,Ribonucleoprotein, U5 Small Nuclear ,Ribonucleoprotein ,Cell Nucleus ,Nucleoplasm ,digestive, oral, and skin physiology ,Nuclear Functions ,Ribonucleoprotein particle ,Nuclear Proteins ,RNA-Binding Proteins ,Fluorescence recovery after photobleaching ,Articles ,Cell Biology ,Ribonucleoproteins, Small Nuclear ,Molecular biology ,digestive system diseases ,Kinetics ,Förster resonance energy transfer ,nervous system ,Cajal body ,Spliceosomes ,Biophysics ,Coilin ,RNA Helicases ,HeLa Cells ,Protein Binding - Abstract
A combination of mathematical modeling and live-cell measurements was applied to determine the dynamics of small nuclear ribonucleoprotein (snRNP) formation in Cajal bodies of living cells. Our results indicate that a substantial fraction of tri-snRNPs is formed in Cajal bodies in cells with many Cajal bodies per nucleus., 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.
- Published
- 2011
8. Enhanced Membrane Fluidization and Cholesterol Displacement by 1-Heptanol Inhibit Mast Cell Effector Functions
- Author
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Viktor Bugajev, Lubica Draberova, Pavol Utekal, Michaela Blazikova, Magda Tumova, and Petr Draber
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alkanol ,membrane fluidizer ,heat shock response ,STIM1-ORAI1 coupling ,store-operated calcium entry ,FcεRI signaling ,Cytology ,QH573-671 - Abstract
Signal transduction by the high-affinity IgE receptor (FcεRI) depends on membrane lipid and protein compartmentalization. Recently published data show that cells treated with 1-heptanol, a cell membrane fluidizer, exhibit changes in membrane properties. However, the functional consequences of 1-heptanol-induced changes on mast cell signaling are unknown. This study shows that short-term exposure to 1-heptanol reduces membrane thermal stability and dysregulates mast cell signaling at multiple levels. Cells treated with 1-heptanol exhibited increased lateral mobility and decreased internalization of the FcεRI. However, this did not affect the initial phosphorylation of the FcεRI-β chain and components of the SYK/LAT1/PLCγ1 signaling pathway after antigen activation. In contrast, 1-heptanol inhibited SAPK/JNK phosphorylation and effector functions such as calcium response, degranulation, and cytokine production. Membrane hyperfluidization induced a heat shock-like response via increased expression of the heat shock protein 70, increased lateral diffusion of ORAI1-mCherry, and unsatisfactory performance of STIM1-ORAI1 coupling, as determined by flow-FRET. Furthermore, 1-heptanol inhibited the antigen-induced production of reactive oxygen species and potentiated stress-induced plasma membrane permeability by interfering with heat shock protein 70 activity. The combined data suggest that 1-heptanol-mediated membrane fluidization does not interfere with the earliest biochemical steps of FcεRI signaling, such as phosphorylation of the FcεRI-β chain and components of the SYK/LAT/PLCγ1 signaling pathway, instead inhibiting the FcεRI internalization and mast cell effector functions, including degranulation and cytokine production.
- Published
- 2023
- Full Text
- View/download PDF
9. Spliceosomal Small Nuclear Ribonucleoprotein Particles Repeatedly Cycle through Cajal Bodies
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David Staněk, Ivan Novotný, Karla M. Neugebauer, Martina Huranova, Aparna K. Sapra, Xin Wen, Michaela Blažíková, and Jarmila Přidalová-Hnilicová
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Small interfering RNA ,Spliceosome ,Ribonucleoprotein, U4-U6 Small Nuclear ,Coiled Bodies ,Biology ,environment and public health ,Humans ,snRNP ,RNA, Small Interfering ,Molecular Biology ,Ribonucleoprotein, U5 Small Nuclear ,Yeast Proteins ,RNA-Binding Proteins ,Cell Biology ,Articles ,Ribonucleoproteins, Small Nuclear ,Molecular biology ,Survival of Motor Neuron 1 Protein ,Cell biology ,Cajal body ,RNA splicing ,Spliceosomes ,Carrier Proteins ,Small nuclear ribonucleoprotein ,Biogenesis ,Biomarkers ,HeLa Cells - Abstract
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.
- Published
- 2008
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