Paxillin (PXN) is a focal adhesion protein that has been implicated in signal transduction from the extracellular matrix. Recently, it has been shown to shuttle between the cytoplasm and the nucleus. When inside the nucleus, paxillin promotes cell proliferation. Here, we introduce paxillin as a transcriptional regulator of IGF2 and H19 genes. It does not affect the allelic expression of the two genes; rather, it regulates long-range chromosomal interactions between the IGF2 or H19 promoter and a shared distal enhancer on an active allele. Specifically, paxillin stimulates the interaction between the enhancer and the IGF2 promoter, thus activating IGF2 gene transcription, whereas it restrains the interaction between the enhancer and the H19 promoter, downregulating the H19 gene. We found that paxillin interacts with cohesin and the mediator complex, which have been shown to mediate long-range chromosomal looping. We propose that these interactions occur at the IGF2 and H19 gene cluster and are involved in the formation of loops between the IGF2 and H19 promoters and the enhancer, and thus the expression of the corresponding genes. These observations contribute to a mechanistic explanation of the role of paxillin in proliferation and fetal development.

• MeSH
• buňky Hep G2 MeSH
• chromozomální proteiny, nehistonové genetika   MeSH
• extracelulární matrix genetika   MeSH
• fokální adheze genetika   MeSH
• genomový imprinting genetika   MeSH
• insulinu podobný růstový faktor II biosyntéza   genetika   MeSH
• lidé MeSH
• metylace DNA genetika   MeSH
• paxilin aplikace a dávkování   MeSH
• proliferace buněk účinky léků   genetika   MeSH
• promotorové oblasti (genetika) MeSH
• proteiny buněčného cyklu genetika   MeSH
• RNA dlouhá nekódující biosyntéza   genetika   MeSH
• signální transdukce účinky léků   MeSH
• vývoj plodu genetika   MeSH
• vývojová regulace genové exprese MeSH
• zesilovače transkripce 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

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

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

Inverse-electron-demand Diels-Alder (iEDDA) cycloaddition between 1,2,4,5-tetrazines and strained dienophiles belongs among the most popular bioconjugation reactions. In addition to its fast kinetics, this cycloaddition can be tailored to produce fluorescent products from non-fluorescent starting materials. Here we show that even the reaction intermediates formed in iEDDA cycloaddition can lead to the formation of new types of fluorophores. The influence of various substituents on their photophysical properties and the generality of the approach with use of various trans-cyclooctene derivatives were studied. Model bioimaging experiments demonstrate the application potential of fluorogenic iEDDA cycloaddition.

• MeSH
• cykloadiční reakce MeSH
• cyklooktany chemie   MeSH
• fluorescenční barviva chemická syntéza   chemie   MeSH
• fluorescenční mikroskopie metody   MeSH
• HeLa buňky MeSH
• heterocyklické sloučeniny bicyklické chemická syntéza   chemie   MeSH
• heterocyklické sloučeniny monocyklické chemie   MeSH
• konfokální mikroskopie metody   MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The development of highly active and selective enzyme inhibitors is one of the priorities of medicinal chemistry. Typically, various high-throughput screening methods are used to find lead compounds from a large pool of synthetic compounds, and these are further elaborated and structurally refined to achieve the desired properties. In an effort to streamline this complex and laborious process, new selection strategies based on different principles have recently emerged as an alternative. Herein, we compare three such selection strategies with the aim of identifying potent and selective inhibitors of human carbonic anhydrase II. All three approaches, in situ click chemistry, phage-display libraries and synthetic peptide libraries, led to the identification of more potent inhibitors when compared to the parent compounds. In addition, one of the inhibitor-peptide conjugates identified from the phage libraries showed greater than 100-fold selectivity for the enzyme isoform used for the compound selection. In an effort to rationalize the binding properties of the conjugates, we performed detailed crystallographic and NMR structural analysis, which revealed the structural basis of the compound affinity towards the enzyme and led to the identification of a novel exosite that could be utilized in the development of isoform specific inhibitors.

• Publikační typ
• časopisecké články MeSH

• Publikační typ
• abstrakt z konference 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