Regulation Of Nuclear Functions Research Articles

Intrafibrillar and perinuclear mitochondrial heterogeneity in adult cardiac myocytes

Mitochondria are involved in multiple cellular functions, in addition to their core role in energy metabolism. Mitochondria localized in different cellular locations may have different morphology, Ca2+ handling and biochemical properties and may interact differently with other intracellular structures, causing functional specificity. However, most prior studies have utilized isolated mitochondria, removed from their intracellular environment. Mitochondria in cardiac ventricular myocytes are highly organized, with a majority squeezed between the myofilaments in longitudinal chains (intrafibrillar mitochondria, IFM). There is another population of perinuclear mitochondria (PNM) around and between the two nuclei typical in myocytes. Here, we take advantage of live myocyte imaging to test for quantitative morphological and functional differences between IFM and PNM with respect to calcium fluxes, membrane potential, sensitivity to oxidative stress, shape and dynamics. Our findings show higher mitochondrial Ca2+ uptake and oxidative stress sensitivity for IFM vs. PNM, which may relate to higher local energy demand supporting the contractile machinery. In contrast to IFM which are remarkably static, PNM are relatively mobile, appear to participate readily in fission/fusion dynamics and appear to play a central role in mitochondrial genesis and turnover. We conclude that while IFM may be physiologically tuned to support local myofilament energy demands, PNM may be more critical in mitochondrial turnover and regulation of nuclear function and import/export. Thus, important functional differences are present in intrafibrillar vs. perinuclear mitochondrial subpopulations.

Nuclear phosphoproteomics analysis reveals that CDK1/2 are involved in EGF-regulated constitutive pre-mRNA splicing in MDA-MB-468 cells

The epidermal growth factor (EGF) receptor (EGFR) pathway is one of the most dysregulated and extensively investigated signaling pathways in human cancers and plays important roles in the regulation of nuclear functions through both cytoplasmic and nuclear EGFR pathways. However, the current understanding of the nuclear phosphorylation responses to activated EGFR pathways remains limited. In the present study, phosphoproteomics analysis revealed the increased phosphorylation of 90 nuclear proteins, primarily involved in RNA processing, pre-mRNA splicing and cell cycle regulation, upon EGF stimulation in MDA-MB-468 cells. Cellular splicing assays of the β-globin (HBB) minigene confirmed that EGF induced constitutive pre-mRNA splicing. Further analysis of phosphoproteomics data identified multiple CDK1/2 substrates in pre-mRNA splicing-related proteins, and both CDK1/2 inhibitors and CDK1/2 knockdowns reduced EGF-regulated pre-mRNA splicing. In conclusion, the results of the present study provide evidence that CDK1/2 participate in the regulation of constitutive pre-mRNA splicing by EGF stimulation in MDA-MB-468 cells. SignificanceIn this study, we successfully carried out a survey of nuclear phosphorylation changes in response to EGF stimulation. The results from the functional category analysis and pre-mRNA splicing assay strongly indicated that EGFR activation increased constitutive pre-mRNA splicing in MDA-MB-468 cells, revealing additional role of EGFR on regulation of mRNA maturation beyond alternative pre-mRNA splicing reported by previous studies. Furthermore, we found that CDK1/2 participated in constitutive pre-mRNA splicing regulation by EGF in MDA-MB-468 cells. Our study provides new knowledge for understanding the regulation of constitutive pre-mRNA splicing by EGF stimulation.

IQGAPs choreograph cellular signaling from the membrane to the nucleus.

Since its discovery in 1994, recognized cellular functions for the scaffold protein IQGAP1 have expanded immensely. Over 100 unique IQGAP1-interacting proteins have been identified, implicating IQGAP1 as a critical integrator of cellular signaling pathways. Initial research established functions for IQGAP1 in cell-cell adhesion, cell migration, and cell signaling. Recent studies have revealed additional IQGAP1 binding partners, expanding the biological roles of IQGAP1. These include crosstalk between signaling cascades, regulation of nuclear function, and Wnt pathway potentiation. Investigation of the IQGAP2 and IQGAP3 homologs demonstrates unique functions, some of which differ from those of IQGAP1. Summarized here are recent observations that enhance our understanding of IQGAP proteins in the integration of diverse signaling pathways.

Nuclear phosphoinositides and their impact on nuclear functions

Polyphosphoinositides (PPIn) are important lipid molecules whose levels are de-regulated in human diseases such as cancer, neurodegenerative disorders and metabolic syndromes. PPIn are synthesized and degraded by an array of kinases, phosphatases and lipases which are localized to various subcellular compartments and are subject to regulation in response to both extra- and intracellular cues. Changes in the activities of enzymes that metabolize PPIn lead to changes in the profiles of PPIn in various subcellular compartments. Understanding how subcellular PPIn are regulated and how they affect downstream signaling is critical to understanding their roles in human diseases. PPIn are present in the nucleus, and their levels are changed in response to various stimuli, suggesting that they may serve to regulate specific nuclear functions. However, the lack of nuclear downstream targets has hindered the definition of which pathways nuclear PPIn affect. Over recent years, targeted and global proteomic studies have identified a plethora of potential PPIn-interacting proteins involved in many aspects of transcription, chromatin remodelling and mRNA maturation, suggesting that PPIn signalling within the nucleus represents a largely unexplored novel layer of complexity in the regulation of nuclear functions.

Continuous Inhibition of Poly(ADP-ribose) Polymerase Does Not Reduce Reperfusion Injury in Isolated Rat Heart

Poly(ADP-ribose) polymerase (PARP), an enzyme that is important to the regulation of nuclear function, is activated by DNA strand breakage. In massive DNA damage, PARP is overactivated, exhausting nicotinamide adenine dinucleotide and leading to cell death. Recent studies have succeeded in reducing cellular damage in ischemia/reperfusion by inhibiting PARP. However, PARP plays an important part in the DNA repair system, and its inhibition may be hazardous in certain situations. We compared the short-time inhibition of PARP against continuous inhibition during ischemia/reperfusion using isolated rat hearts. The hearts were reperfused after 21 minutes of ischemia with a bolus injection of 3-aminobenzamide (3-AB) (10 mg/kg) followed by continuous 3-AB infusion (50 μM) for the whole reperfusion period or for the first 6 minutes or without 3-AB. At the end of reperfusion, contractile function, high-energy phosphate content, nicotinamide adenine dinucleotide content, and infarcted area were significantly preserved in the 3-AB 6-minute group. In the 3-AB continuous group, these advantages were not apparent. At the end of reperfusion, PARP cleavage had significantly proceeded in the 3-AB continuous group, indicating initiation of the apoptotic cascade. Thus, continuous PARP inhibition by 3-AB does not reduce reperfusion injury in the isolated rat heart, which may be because of acceleration of apoptosis.

Influence of the genomic sequence on the primary structure of chromatin

As an important actor in the regulation of nuclear functions, the nucleosomal organization of the 10 nm chromatin fiber is the subject of increasing interest. Recent high-resolution mapping of nucleosomes along various genomes ranging from yeast to human, have revealed a patchy nucleosome landscape with alternation of depleted, well positioned and fuzzy regions. For many years, the mechanisms that control nucleosome occupancy along eukaryotic chromosomes and their coupling to transcription and replication processes have been under intense experimental and theoretical investigation. A recurrent question is to what extent the genomic sequence dictates and/or constrains nucleosome positioning and dynamics? In that context we have recently developed a simple thermodynamical model that accounts for both sequence specificity of the histone octamer and for nucleosome–nucleosome interactions. As a main issue, our modelling mimics remarkably well in vitro data showing that the sequence signaling that prevails are high energy barriers that locally inhibit nucleosome formation and condition the collective positioning of neighboring nucleosomes according to thermal equilibrium statistical ordering. When comparing to in vivo data, our physical modelling performs as well as models based on statistical learning suggesting that in vivo bulk chromatin is to a large extent controlled by the underlying genomic sequence although it is also subject to finite-range remodelling action of external factors including transcription factors and ATP-dependent chromatin remodellers. On the highly studied S. cerevisiae organism, we discuss the implications of the highlighted ‘positioning via excluding’ mechanism on the structure and function of yeast genes. The generalization of our physical modelling to human is likely to provide new insight on the isochore structure of mammalian genomes in relation with their primary nucleosomal structure.

The role of nuclear pores in gene regulation, development and disease

Nuclear-pore complexes (NPCs) are large protein channels that span the nuclear envelope (NE), which is a double membrane that encloses the nuclear genome of eukaryotes. Each of the typically 2,000-4,000 pores in the NE of vertebrate cells is composed of multiple copies of 30 different proteins known as nucleoporins. The evolutionarily conserved NPC proteins have the well-characterized function of mediating the transport of molecules between the nucleoplasm and the cytoplasm. Mutations in nucleoporins are often linked to specific developmental defects and disease, and the resulting phenotypes are usually interpreted as the consequences of perturbed nuclear transport activity. However, recent evidence suggests that NPCs have additional functions in chromatin organization and gene regulation, some of which might be independent of nuclear transport. Here, we review the transport-dependent and transport-independent roles of NPCs in the regulation of nuclear function and gene expression.

The mobility of Bach2 nuclear foci is regulated by SUMO-1 modification

The small ubiquitin-like modifier-1 (SUMO-1) modulates the functions of nuclear proteins by changing their structure and/or subnuclear localization. Several nuclear proteins form dynamic higher order nuclear structures, termed non-chromatin nuclear domains, which are involved in the regulation of nuclear function. However, the role that SUMO modification of the component proteins plays in the regulation of the activity and function of nuclear domains is unclear. Here we demonstrate that nuclear domains formed by Bach2, a transcription repressor, show restricted movement and undergo fusion events upon oxidative stress. Mutation of the SUMO-acceptor lysines in Bach2 alters the behavior of these nuclear foci and results in a decreased frequency of fusion events. We propose that SUMO modification is an important regulatory system for the mobility of the nuclear domains formed by Bach2.

The Cell Adhesion Receptor Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 Regulates Nucleocytoplasmic Trafficking of DNA Polymerase δ-Interacting Protein 38

The homophilic cell-cell adhesion receptor CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1, CD66a) acts as a regulator of contact-dependent cell survival, differentiation, and growth. It is involved in the control of proliferation in hematopoietic and epithelial cells and can act as a tumor suppressor. In this study, we identify DNA polymerase delta-interacting protein 38 (PDIP38) as a novel binding partner for CEACAM1-L and CEACAM1-S. We show that PDIP38 can occur in the nucleus, in the cytoplasm and at the plasma membrane in NBT-II, IEC18, RBE, and HeLa cells and that the distribution in NBT-II cells is influenced by the confluency of the cells. We also demonstrate that the interaction of CEACAM1 and PDIP38 is of functional importance in NBT-II cells, which co-express the long and the short CEACAM1 isoform. In subconfluent, proliferating NBT-II cells, perturbation of CEACAM1 by antibody clustering induces increased binding to PDIP38 and results in rapid recruitment of PDIP38 to the plasma membrane. The same treatment of confluent, quiescent NBT-II cells leads to a different response, i.e. translocation of PDIP38 to the nucleus. Together, our data show that PDIP38 can shuttle between the cytoplasmic and the nuclear compartments and that its subcellular localization is regulated by CEACAM1, implicating that PDIP38 may constitute a novel downstream target of CEACAM1 signaling.

Hela l-CaD undergoes a DNA replication-associated switch in localization from the cytoplasm to the nuclei of endothelial cells/endothelial progenitor cells in human tumor vasculature

Caldesmon (CaD) is a major actin-binding protein distributed in a variety of cell types. So far no diversity in functions of the different isoforms were found in in vitro studies. The low molecular weight isoform (Hela l-CaD) was detected in the vasculature of a variety of tumor types in our previous study. Proliferation of endothelial cells/endothelial progenitor cells is a crucial event for formation of new blood vessels. Here we report the intranuclear translocation of Hela l-CaD in cell cycle activated ECs/EPCs in the vasculature of human tumors. The nuclear translocation coincides with phosphorylation of the molecule and the activation of intranuclear protein kinase p34cdc2. These findings point to a function of this molecule relating to DNA synthesis which is triggered by cell-cycle signalling pathways. The data challenge and update the generally accepted concept that CaD is a pure cytoplasmic protein in vitro study. It suggests that nuclear translocation of Hela l-CaD serves as an additional regulatory step in the control of mitotic initiation and triggers further investigations in the role of this protein in the regulation of nuclear functions.

Inositol polyphosphate kinases: regulators of nuclear function

Recent work has uncovered roles for inositide signalling pathways downstream of phospholipase C activation and inositol 1,4,5-trisphosphate in the regulation of nuclear processes including gene expression, mRNA export and DNA metabolism. The identification of several IPKs (inositol polyphosphate kinases) has renewed interest in the cellular roles of inositol tetra-, penta-, hexa- and pyro-phosphates. Discoveries of inositide receptors and novel mechanisms of inositide action have provided important insights into how such messengers couple to nuclear machinery. In this chapter, we discuss the IPK family members and the nuclear processes that their inositide products regulate.

Inositol polyphosphate kinases: regulators of nuclear function

Recent work has uncovered roles for inositide signalling pathways downstream of phospholipase C activation and inositol 1,4,5-trisphosphate in the regulation of nuclear processes including gene expression, mRNA export and DNA metabolism. The identification of several IPKs (inositol polyphosphate kinases) has renewed interest in the cellular roles of inositol tetra-, penta-, hexa- and pyro-phosphates. Discoveries of inositide receptors and novel mechanisms of inositide action have provided important insights into how such messengers couple to nuclear machinery. In this chapter, we discuss the IPK family members and the nuclear processes that their inositide products regulate.

Role of regucalcin in liver nuclear function: Binding of regucalcin to nuclear protein or DNA and modulation of tumor-related gene expression

Regucalcin is a regulatory protein in the intracellular signaling pathway which is related to regulation of nuclear function. In this study the binding of regucalcin to nuclear proteins or DNA in vitro was examined. The results of the Far-Western analysis showed the existence of protein components which bind to regucalcin in the nucleus isolated from rat liver. Whether regucalcin binds deoxyribonucleic acid (DNA) was analyzed using DNA cellulose in vitro. Regucalcin was incubated in reaction mixture containing DNA cellulose, and DNA-binding regucalcin was detected using Western blot analysis for regucalcin. The results showed that regucalcin binds DNA in vitro. Moreover, the expression of c-src, p53, and Rb mRNAs was examined in the cloned rat hepatoma H4-II-E cells cultured for 24 or 48 h in the presence of fetal bovine serum (10%), using reverse transcription-polymerase chain reaction (RT-PCR). The expression of oncogene c-src mRNA was significantly suppressed in the hepatoma cells (transfectants), overexpressing regucalcin. Meanwhile, the expression of the tumor suppressor gene p53 or Rb mRNA was significantly enhanced in transfectants. This study may support the view that regucalcin modulates the transcriptional process by binding to protein and DNA in the nucleus.

Regulation of nuclear functions – nucleocytoplasmic transport in context

A recent meeting of cell biologists in Cleveland, Ohio (April 22/23, 2004) highlighted the value of juxtaposing discussions of the functional organization of the nuclear periphery with themechanisms bywhich the nucleus receives signals, both from the cell surface and from defined sites in the cytoplasm. The event was jointly organized by theCell Biology Program of Case Western Reserve University School of Medicine and the Cell Biology Department and Lerner Research Institute of the Cleveland Clinic Foundation. In introductory remarks, Alan Tartakoff (Case Western Reserve University) highlighted the breadth of importance of dynamic nucleocytoplasmic communication. He described contemporary molecular understanding of TMsignaling∫ to the nucleus as one of the recent TMconquests∫ of cell biology, which characteristically insists on mechanistic questions which involve topologic or topographic issues. He also called attention to the realization that many examples of nuclear import and export do not directly involve the TMclassic∫ transport pathways which are driven by theRanGTPase andmembers of the family of importins/exportins (also known as TMkaryopherins∫) (Gˆrlich and Kutay, 1999; Xu and Massague, 2004). He further emphasized that net relocalization of proteins from the nucleus to the cytoplasm (and vice versa) can ± in many cases ± result from adjustment of the rate constants of ongoing shuttling, rather that being due to TMde novo∫ unidirectional transport (Fig. 1) (Tartakoff et al., 2000). As a prelude for subsequent lectures, he then enumerated additional circumstances which can cause individual transport cargoes to distribute asymmetrically across the nuclear envelope: unmasking of transport signals and protein-protein interaction domains (due to covalent changes or displacement of inhibitory peptide domains), tethering of cargo proteins to immobile structures (either covalently or non-covalently), etc.

Histone modification enzymes: novel targets for cancer drugs.

In eukaryotes, genomic DNA is packaged with histone proteins into the cell nucleus as chromatin, condensing the DNA > 10,000-fold. Chromatin is highly dynamic and exerts profound control on gene expression. Localised chromatin decondensation facilitates access of nuclear machinery. Chromatin displays epigenetic inheritance, in that changes in its structure can pass to the next generation independently of the DNA sequence itself. It is now clear that the post-translational modification of histones, for example, acetylation, methylation and phosphorylation, plays a crucial role in the regulation of nuclear function through the 'histone code'. There has been significant progress in identifying and understanding the enzymes that control these complex processes, in particular histone acetyltransferases and histone deacetylases. The exciting discovery that compounds inhibiting histone deacetylase activity also have antitumour properties has focused attention on their use as anticancer drugs. As a consequence, there is ongoing evaluation of several histone deacetylase inhibitor compounds in Phase I and II clinical trials with promising early results. It is likely that many of the enzymes involved in the control of histone modification will provide therapeutic opportunities for the treatment of cancer, including histone methyltransferases and Aurora kinases.

Barrier-to-autointegration factor plays crucial roles in cell cycle progression and nuclear organization in Drosophila.

Barrier-to-autointegration factor (BAF) is potentially a DNA-bridging protein, which directly associates with inner nuclear membrane proteins carrying LEM domains. These features point to a key role in regulation of nuclear function and organization, dependent on interactions between the nuclear envelope and chromatin. To understand the functions of BAF in vivo, Drosophila baf null mutants generated by P-element-mediated imprecise excision were analyzed. Homozygous null mutants showed a typical mitotic mutant phenotype: lethality at the larval-pupal transition with small brains and missing imaginal discs. Mitotic figures were decreased but a defined anaphase defect as reported for C. elegans RNAi experiments was not observed in these small brains, suggesting a different phase or phases of cell cycle arrest. Specific abnormalities in interphase nuclear structure were frequently found upon electron microscopic examination of baf null mutants, with partial clumping of chromatin and convolution of nuclear shape. At the light microscopic level, grossly aberrant nuclear lamina structure and B-type lamin distribution correlated well with the loss of detectable amounts of BAF protein from nuclei. Together, these data represent evidence of BAF's anticipated function in mediating interactions between the nuclear envelope and interphase chromosomes. We thus conclude that BAF plays essential roles in nuclear organization and that these BAF functions are required in both M phase and interphase of the cell cycle.

Nuclear lipid metabolism and signaling.

Evidence has been accumulating that nuclear lipid metabolism is involved in the regulation of nuclear functions. Here I describe an autonomous nuclear lipid signaling that has been found to be associated with the metabolism of such lipids as phosphoinositides, choline phospholipids, and the acylation and deacylation cycle. Some lipid signals from the plasma membrane ultimately reach the nucleus and regulate the nuclear function. In this case, however, generated lipids and their metabolites may not directly act on the nuclear factors involved in nuclear function. The unique and direct effects of nuclear lipids and their metabolites on nuclear factors are also discussed.

Alternative splicing, chromosome assignment and subcellular localization of the testicular haploid expressed gene (THEG)

We have previously isolated and characterized the mouse Testicular Haploid Expressed Gene (Theg) that is specifically expressed in haploid germ cells. We now describe the molecular cloning and characterization of the human homologue (THEG) of mouse Theg. Expression studies by using both dot blot and Northern blot techniques revealed that human THEG is expressed specifically in the testis. Additionally, we found two alternatively spliced transcripts (THEG major and THEG minor) for THEG by using reverse transcription-polymerase chain reaction on human testicular RNA. Sequence analysis of these PCR products demonstrated that the smaller transcript (THEG minor) lacks 72 bp which was also observed for the mouse Theg. We have isolated the cDNAs of human THEG major and THEG minor, containing the complete open reading frames, which encode putative nuclear proteins of 379 amino acids and 355 amino acids, respectively. Database searches identified two genomic clones on chromosome 19 harboring the human THEG gene, which is approximately 14 kb pairs in size, contains eight exons, and comparison of the two cDNA sequences with the genomic sequence indicated that the smaller transcript lacks exon 3. Furthermore, we assigned the human THEG gene (THEG) to human chromosome 19ptel→ p13 by fluorescence in situ hybridization. Moreover, we detected mouse THEG protein prominently in the nucleus of round spermatids by using an antibody against THEG on both testicular sections and cellular suspensions. Additionally, the subcellular localization of mouse THEG was confirmed by a green fluorescent protein (GFP) fusion protein of mouse THEG which was found mainly in the nucleus of transfected NIH3T3 cells. These data suggest that both human and mouse THEG are specifically expressed in the nucleus of haploid male germ cells and are involved in the regulation of nuclear functions.

Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions

Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions

Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions

Poly(ADP-ribosyl)ation is a post-translational modification of proteins. During this process, molecules of ADP-ribose are added successively on to acceptor proteins to form branched polymers. This modification is transient but very extensive in vivo, as polymer chains can reach more than 200 units on protein acceptors. The existence of the poly(ADP-ribose) polymer was first reported nearly 40 years ago. Since then, the importance of poly(ADP-ribose) synthesis has been established in many cellular processes. However, a clear and unified picture of the physiological role of poly(ADP-ribosyl)ation still remains to be established. The total dependence of poly(ADP-ribose) synthesis on DNA strand breaks strongly suggests that this post-translational modification is involved in the metabolism of nucleic acids. This view is also supported by the identification of direct protein-protein interactions involving poly(ADP-ribose) polymerase (113 kDa PARP), an enzyme catalysing the formation of poly(ADP-ribose), and key effectors of DNA repair, replication and transcription reactions. The presence of PARP in these multiprotein complexes, in addition to the actual poly(ADP-ribosyl)ation of some components of these complexes, clearly supports an important role for poly(ADP-ribosyl)ation reactions in DNA transactions. Accordingly, inhibition of poly(ADP-ribose) synthesis by any of several approaches and the analysis of PARP-deficient cells has revealed that the absence of poly(ADP-ribosyl)ation strongly affects DNA metabolism, most notably DNA repair. The recent identification of new poly(ADP-ribosyl)ating enzymes with distinct (non-standard) structures in eukaryotes and archaea has revealed a novel level of complexity in the regulation of poly(ADP-ribose) metabolism.