Lamins, the nuclear intermediate filaments, are important regulators of nuclear structural integrity as well as nuclear functional processes such as DNA transcription, replication and repair, and epigenetic regulations. A portion of phosphorylated lamin A/C localizes to the nuclear interior in interphase, forming a lamin A/C pool with specific properties and distinct functions. Nucleoplasmic lamin A/C molecular functions are mainly dependent on its binding partners; therefore, revealing new interactions could give us new clues on the lamin A/C mechanism of action. In the present study, we show that lamin A/C interacts with nuclear phosphoinositides (PIPs), and with nuclear myosin I (NM1). Both NM1 and nuclear PIPs have been previously reported as important regulators of gene expression and DNA damage/repair. Furthermore, phosphorylated lamin A/C forms a complex with NM1 in a phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent manner in the nuclear interior. Taken together, our study reveals a previously unidentified interaction between phosphorylated lamin A/C, NM1, and PI(4,5)P2 and suggests new possible ways of nucleoplasmic lamin A/C regulation, function, and importance for the formation of functional nuclear microdomains.

• Keywords
• NM1, PI(4,5)P2, cell nucleus, lamin A/C, nuclear lamina, nuclear myosin 1, nucleoplasm, phosphoinositides, phosphorylation,
• MeSH
• Cell Nucleus * metabolism   MeSH
• Interphase MeSH
• Intermediate Filaments metabolism   MeSH
• Lamin Type A * metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Lamin Type A * MeSH

Simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. While fluorescent microscopy allows for simultaneous detection of multiple antigens, the sensitive electron microscopy immunodetection is limited to only two antigens. In order to overcome this limitation, we prepared a set of novel, shape-coded metal nanoparticles readily discernible in transmission electron microscopy which can be conjugated to antibodies or other bioreactive molecules. With the use of novel nanoparticles, various combinations with commercial gold nanoparticles can be made to obtain a set for simultaneous labeling. For the first time in ultrastructural histochemistry, up to five molecular targets can be identified simultaneously. We demonstrate the usefulness of the method by mapping of the localization of nuclear lipid phosphatidylinositol-4,5-bisphosphate together with four other molecules crucial for genome function, which proves its suitability for a wide range of biomedical applications.

• MeSH
• Actins metabolism   MeSH
• Staining and Labeling methods   MeSH
• Cell Nucleus MeSH
• Microscopy, Electron MeSH
• Phosphatidylinositol 4,5-Diphosphate metabolism   MeSH
• HeLa Cells MeSH
• Immunohistochemistry methods   MeSH
• Nuclear Proteins metabolism   MeSH
• Metal Nanoparticles chemistry   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Nucleophosmin MeSH
• Cell Cycle Proteins MeSH
• Antibodies immunology   MeSH
• Ribonucleoproteins, Small Nuclear metabolism   MeSH
• Carrier Proteins metabolism   MeSH
• Gold chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Actins MeSH
• Phosphatidylinositol 4,5-Diphosphate MeSH
• Nuclear Proteins MeSH
• Nucleophosmin MeSH
• Cell Cycle Proteins MeSH
• Antibodies MeSH
• Ribonucleoproteins, Small Nuclear MeSH
• SMC2 protein, human MeSH Browser
• Carrier Proteins MeSH
• Gold 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
• Cell Nucleus metabolism   MeSH
• DNA Primers genetics   MeSH
• Phenotype * MeSH
• Genotype MeSH
• Immunoprecipitation MeSH
• Myosin Type I genetics   metabolism   MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Plasmids genetics   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Protein Isoforms genetics   metabolism   MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Primers MeSH
• Myo1c protein, mouse MeSH Browser
• Myosin Type I MeSH
• Protein Isoforms MeSH

Lamins are the best characterized cytoskeletal components of the cell nucleus that help to maintain the nuclear shape and participate in diverse nuclear processes including replication or transcription. Nuclear actin is now widely accepted to be another cytoskeletal protein present in the nucleus that fulfills important functions in the gene expression. Some viruses replicating in the nucleus evolved the ability to interact with and probably utilize nuclear actin for their replication, e.g., for the assembly and transport of capsids or mRNA export. On the other hand, lamins play a role in the propagation of other viruses since nuclear lamina may represent a barrier for virions entering or escaping the nucleus. This review will summarize the current knowledge about the roles of nuclear actin and lamins in viral infections.

• Keywords
• viruses, cytoskeleton, lamin, nuclear actin, nuclear lamina, nucleus,
• MeSH
• Actins metabolism   MeSH
• Baculoviridae metabolism   pathogenicity   MeSH
• Cell Nucleus metabolism   virology   MeSH
• Cytoskeleton MeSH
• Herpesviridae metabolism   pathogenicity   MeSH
• Herpesviridae Infections metabolism   pathology   virology   MeSH
• Lamins metabolism   MeSH
• Humans MeSH
• Virus Replication * MeSH
• Retroviridae metabolism   pathogenicity   MeSH
• Retroviridae Infections metabolism   pathology   virology   MeSH
• Virus Assembly * MeSH
• Virus Diseases metabolism   virology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Actins MeSH
• Lamins 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
• Adenosine Diphosphate metabolism   MeSH
• Actins metabolism   MeSH
• Active Transport, Cell Nucleus MeSH
• Cell Nucleus metabolism   MeSH
• Cell Line MeSH
• Nuclear Localization Signals MeSH
• Calmodulin metabolism   MeSH
• Karyopherins metabolism   MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Myosin Type I chemistry   metabolism   MeSH
• Myosins chemistry   metabolism   MeSH
• Mice MeSH
• Amino Acid Sequence MeSH
• Protein Structure, Tertiary MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Diphosphate MeSH
• Actins MeSH
• Nuclear Localization Signals MeSH
• Calmodulin MeSH
• Karyopherins MeSH
• Myo1c protein, mouse MeSH Browser
• Myosin Type I MeSH
• Myosins 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
• Actins metabolism   MeSH
• Cell Nucleolus metabolism   MeSH
• Transcription, Genetic physiology   MeSH
• HeLa Cells MeSH
• Immunohistochemistry MeSH
• Humans MeSH
• Myosin Type I metabolism   MeSH
• DNA, Ribosomal metabolism   MeSH
• RNA, Ribosomal metabolism   MeSH
• RNA Polymerase I metabolism   MeSH
• Pol1 Transcription Initiation Complex Proteins metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Actins MeSH
• Myosin Type I MeSH
• DNA, Ribosomal MeSH
• RNA, Ribosomal MeSH
• RNA Polymerase I MeSH
• transcription factor UBF MeSH Browser
• Pol1 Transcription Initiation Complex Proteins MeSH

Actin is a well-known protein that has shown a myriad of activities in the cytoplasm. However, recent findings of actin involvement in nuclear processes are overwhelming. Actin complexes in the nucleus range from very dynamic chromatin-remodeling complexes to structural elements of the matrix with single partners known as actin-binding proteins (ABPs). This review summarizes the recent findings of actin-containing complexes in the nucleus. Particular attention is given to key processes like chromatin remodeling, transcription, DNA replication, nucleocytoplasmic transport and to actin roles in nuclear architecture. Understanding the mechanisms involving ABPs will definitely lead us to the principles of the regulation of gene expression performed via concerting nuclear and cytoplasmic processes.

• MeSH
• Actins chemistry   metabolism   MeSH
• Models, Biological MeSH
• Cell Nucleus chemistry   metabolism   MeSH
• Humans MeSH
• Microfilament Proteins chemistry   metabolism   MeSH
• DNA Repair MeSH
• DNA Replication MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Actins MeSH
• Microfilament Proteins 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
• Cell Nucleus metabolism   MeSH
• Cell Line MeSH
• Gene Expression MeSH
• Phylogeny MeSH
• Transcription, Genetic MeSH
• Conserved Sequence MeSH
• Humans MeSH
• Myosin Type I chemistry   genetics   isolation & purification   metabolism   MeSH
• Mice MeSH
• Vertebrates genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Nucleic Acid MeSH
• Serum chemistry   MeSH
• Nucleic Acid Amplification Techniques MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Myosin Type I MeSH