Reduction of a 4q35-encoded nuclear envelope protein in muscle differentiation

Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients with FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately re-activated it could affect muscle differentiation and contribute to disease pathology.     

Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) is a dynamic nuclear and sarcomeric protein

Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) is a candidate gene for mediating FSHD pathophysiology, however, very little is known about the endogenous FRG1 protein. This study uses immunocytochemistry (ICC) and histology to provide insight into FRG1's role in vertebrate muscle development and address its potential involvement in FSHD pathophysiology. In cell culture, primary myoblast/myotube cultures, and mouse and human muscle sections, FRG1 showed distinct nuclear and cytoplasmic localizations and nuclear shuttling assays indicated the subcellular pools of FRG1 are linked. During myoblast differentiation, FRG1's subcellular distribution changed dramatically with FRG1 eventually associating with the matured Z-discs. This Z-disc localization was confirmed using isolated mouse myofibers and found to be maintained in adult human skeletal muscle biopsies. Thus, FRG1 is not likely involved in the initial assembly and alignment of the Z-disc but may be involved in sarcomere maintenance or signaling. Further analysis of human tissue showed FRG1 is strongly expressed in arteries, veins, and capillaries, the other prominently affected tissue in FSHD. Overall, we show that in mammalian cells, FRG1 is a dynamic nuclear and cytoplasmic protein, however in muscle, FRG1 is also a developmentally regulated sarcomeric protein suggesting FRG1 may perform a muscle-specific function. Thus, FRG1 is the only FSHD candidate protein linked to the muscle contractile machinery and may address why the musculature and vasculature are specifically susceptible in FSHD.     

BFRF1 protein is involved in EBV-mediated autophagy manipulation

Viral egress and autophagy are two mechanisms that seem to be strictly connected in Herpesviruses’s biology. Several data suggest that the autophagic machinery facilitates the egress of viral capsids and thus the production of new infectious particles. In the Herpesvirus family, viral nuclear egress is controlled and organized by a well conserved group of proteins named Nuclear Egress Complex (NEC). In the case of EBV, NEC is composed by BFRF1 and BFLF2 proteins, although the alterations of the nuclear host cell architecture are mainly driven by BFRF1, a multifunctional viral protein anchored to the inner nuclear membrane of the host cell. BFRF1 shares a peculiar distribution with several nuclear components and with them it strictly interacts. In this study, we investigated the possible role of BFRF1 in manipulating autophagy, pathway that possibly originates from nucleus, regulating the interplay between autophagy and viral egress.     

Characterization of a novel Dp71 dystrophin-associated protein complex (DAPC) present in the nucleus of HeLa cells: members of the nuclear DAPC associate with the nuclear matrix

Dystrophin is an essential component in the assembly and maintenance of the dystrophin-associated protein complex (DAPC), which includes members of the dystroglycan, syntrophin, sarcoglycan and dystrobrevin protein families. Distinctive complexes have been described in the cell membrane of different tissues and cultured cells. In this work, we report the identification and characterization of a novel DAPC present in the nuclei of HeLa cells, which contains dystrophin Dp71 as a key component. Using confocal microscopy and cell fractionation analyses, we found the presence of Dp71, beta-sarcoglycan, beta-dystroglycan, alpha- and beta-syntrophin, alpha1- and beta-dystrobrevin and nNOS in the nuclei of HeLa cells. Furthermore, we demonstrated by co-immunoprecipitation experiments that most of these proteins form a complex in the nuclear compartment. Next, we analyze the possible association of the nuclear DAPC with the nuclear matrix. We found the presence of Dp71, beta-dystroglycan, nNOS, beta-sarcoglycan, alpha/beta syntrophin, alpha1-dystrobrevin and beta-dystrobrevin in the nuclear matrix protein fractions and in situ nuclear matrix preparations from HeLa cells. Moreover, we found that Dp71, beta-dystroglycan and beta-dystrobrevin co-immunoprecipitated with the nuclear matrix proteins lamin B1 and actin. The association of members of the nuclear DAPC with the nuclear matrix indicates that they may work as scaffolding proteins involved in nuclear architecture.     

Prelamin A-mediated recruitment of SUN1 to the nuclear envelope directs nuclear positioning in human muscle

Lamin A is a nuclear lamina constituent expressed in differentiated cells. Mutations in the LMNA gene cause several diseases, including muscular dystrophy and cardiomyopathy. Among the nuclear envelope partners of lamin A are Sad1 and UNC84 domain-containing protein 1 (SUN1) and Sad1 and UNC84 domain-containing protein 2 (SUN2), which mediate nucleo-cytoskeleton interactions critical to the anchorage of nuclei. In this study, we show that differentiating human myoblasts accumulate farnesylated prelamin A, which elicits upregulation and recruitment of SUN1 to the nuclear envelope and favors SUN2 enrichment at the nuclear poles. Indeed, impairment of prelamin A farnesylation alters SUN1 recruitment and SUN2 localization. Moreover, nuclear positioning in myotubes is severely affected in the absence of farnesylated prelamin A. Importantly, reduced prelamin A and SUN1 levels are observed in Emery-Dreifuss muscular dystrophy (EDMD) myoblasts, concomitant with altered myonuclear positioning. These results demonstrate that the interplay between SUN1 and farnesylated prelamin A contributes to nuclear positioning in human myofibers and may be implicated in pathogenetic mechanisms.     

Impaired nuclear functions lead to increased senescence and inefficient differentiation in human myoblasts with a dominant p.R545C mutation in the LMNA gene

We have studied myoblasts from a patient with a severe autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD) caused by an arginine 545 to cystein point mutation (p.R545C) in the carboxy-terminal domain of the lamin A/C gene. This mutation has pleiotropic cellular effects on these myoblasts as demonstrated by nuclear structural defects, exhibiting lobulations which increase with cell passages in culture. The organization of both lamin A/C and its inner nuclear membrane partner emerin are altered, eventually showing a honeycomb pattern upon immunofluorescence microscopy. In addition, the distribution of histone H3 trimethylated at lysine 27 and of phosphorylated RNA polymerase II, markers of inactive and active chromatin domains, respectively, are altered suggesting an impact on gene expression. Patient myoblasts also presented a high index of senescence in ex vivo culture. Moreover, our data show for the first time in an AD-EDMD context that the 20S core particle of the proteasome was inactivated. With cell passages, the 20S core protein progressively accumulated into discrete nuclear foci that largely colocalized with promyelocytic leukemia (PML) bodies while p21 accumulated throughout the nuclear compartment. Proteasome inactivation has been linked to normal cellular ageing. Our data indicate that it may also contribute to premature senescence in AD-EDMD patient myoblasts. Finally, when transferred to low-serum medium, patient myoblasts were deficient in ex vivo differentiation, as assessed by the absence of myotube formation and myogenin induction. Altogether, these data suggest that the LMNA mutation p.R545C impairs both proliferation and differentiation capacities of myoblasts as part of the pathogenesis of AD-EDMD.     

Nuclear envelope precursor vesicle targeting to chromatin is stimulated by protein phosphatase 1 in Xenopus egg extracts

The mechanism underlying targeting of the nuclear membrane to chromatin at the end of mitosis was studied using an in vitro cell-free system comprising Xenopus egg membrane and cytosol fractions, and sperm chromatin. The mitotic phase membrane, which was separated from a mitotic phase extract of Xenopus eggs and could not bind to chromatin, became able to bind to chromatin on pretreatment with a synthetic phase cytosol fraction of Xenopus eggs. When the cytosol fraction was depleted of protein phosphatase 1 (PP1) with anti-Xenopus PP1γ1 antibodies, this ability was lost. The addition of recombinant xPP1γ1 to the PP1-depleted cytosol fraction restored the ability. These and other results suggested that dephosphorylation of mitotic phosphorylation sites on membranes by PP1 in the synthetic phase cytosol fraction promoted targeting of the membranes to chromatin. On the other hand, a fragment containing the chromatin-binding domain of lamin B receptor (LBR) but not emerin inhibited targeting of membrane vesicles. It was also shown that PP1 dephosphorylates a phosphate group(s) responsible for regulation of the binding of LBR to chromatin. A possible mechanism involving PP1 and LBR for the regulation of nuclear membrane targeting to chromatin was discussed.     

Understanding the roles of nuclear A- and B-type lamins in brain development

The nuclear lamina is composed mainly of lamins A and C (A-type lamins) and lamins B1 and B2 (B-type lamins). Dogma has held that lamins B1 and B2 play unique and essential roles in the nucleus of every eukaryotic cell. Recent studies have raised doubts about that view but have uncovered crucial roles for lamins B1 and B2 in neuronal migration during the development of the brain. The relevance of lamins A and C in the brain remains unclear, but it is intriguing that prelamin A expression in the brain is low and is regulated by miR-9, a brain-specific microRNA.     

NCL1, a novel gene for a non-essential nuclear protein in Saccharomyces cerevisiae

The nucleolar protein Nop2p is an essential gene product that is required for pre-rRNA processing and ribosome biogenesis in Saccharomyces cerevisiae (Hong, B. et al., 1997, Mol. Cell. Biol., 17, 378–388). A search for proteins similar to Nop2p identified a novel yeast gene product that also shares significant homology with the human proliferation associated nucleolar protein p120. The gene encoding this 78 kDa protein was termed NCL1 (for nuclear protein 1; corresponding to YBL024w). Ncl1p and Nop2p contain an evolutionarily conserved motif that has been termed the ‘NOL1/NOP2/fmu family signature' (NOL1 encodes p120). Epitope tagged Ncl1p was found to be localized to the nucleus, including the nucleolus, and was concentrated at the nuclear periphery. NCL1 is not essential. Strains containing a disruption of NCL1, or strains overexpressing NCL1, grow essentially identically to wildtype NCL1 strains on a number of different media and at different temperatures. Disruption of NCL1 does not affect steady-state levels of large and small ribosome subunits, monoribosomes, and polyribosomes. However, disruption of NCL1 leads to increased sensitivity to the antibiotic paromomycin.     

Novel nuclear protein ALC-INTERACTING PROTEIN1 is expressed in vascular and mesocarp cells in Arabidopsis

Pod shattering is an agronomical trait that is a result of the coordinated action of cell differentiation and separation. In Arabidopsis, pod shattering is controlled by a complex genetic network in which ALCATRAZ (ALC), a member of the basic helix-loop-helix family, is critical for cell separation during fruit dehiscence. Herein, we report the identification of ALC-INTERACTiNG PROTEIN1 (ACI1) via the yeast two-hybrid screen. ACI1 encodes a nuclear protein with a lysine-rich domain and a C-terminal serine-rich domain. ACI1 is mainly expressed in the vascular system throughout the plant and mesocarp of the valve in siliques. Our data showed that ACI1 interacts strongly with the N-terminal portion of ALC in yeast cells and in plant cells in the nucleus as demonstrated by bimolecular fluorescence complementation assay. Both ACl1 and ALC share an overlapping expression pattern, suggesting that they likely function together in planta. However, no detectable phenotype was found in plants with reduced ACI1 expression by RNA interference technology, suggesting that ACI1 may be redundant. Taken together, these data indicate that ALC may interact with ACll and its homologs to control cell separation during fruit dehiscence in Arabidopsis.     

A novel hook-related protein family and the characterization of hook-related protein 1

The spatial organization of organelles within a cell is dependent on microtubules. Recently, members of the Hook family of proteins have been proposed to function in linking organelles to microtubules. We report the identification of a completely novel protein family, the Hook-related protein (HkRP) family, from which the Hook proteins have diverged. Bioinformatic analysis of the HkRP family revealed several conserved domains, including a unique C-terminal HkRP domain. The central region of each protein is comprised of an extensive coiled-coil domain, and the N-terminus contains a putative microtubule-binding domain. This domain has been shown to bind microtubules in the Hook protein and show that the HkRP1 protein is microtubule-associated. While endogenous HkRP1 has no distinct organelle association, expression of the C-terminal membrane-binding domain suggests a function of the HkRP1 in early endosome. Ultrastructural studies reveal that expression of the C-terminal HkRP1 domain causes an accumulation of internal membranes with an electron-dense coat. Co-localization studies show a concomitant redistribution of the early endosome marker sorting-nexin 1 but not the early endosome antigen-1 (EEA1). The steady-state distribution of the epidermal growth factor receptor is also specifically disrupted by expression of the C-terminal domain. We propose that HkRP1 is involved in the process of tubulation of sorting nexin-1 positive membranes from early endosome subdomains.     

Brl1p -- a novel nuclear envelope protein required for nuclear transport

In this article, we identify a cold-sensitive mutant of Xpo1p designated as xop1-2 (but will be referred to from here on as xpo1-ok) that is synthetically lethal with srm1-1, a Saccharomyces cerevisiae RCC1 homolog. xpo1-ok was a novel mutated allele with a single point mutation, T283P. Suppressors of xpo1-ok were isolated, and one of them was found to encode a novel nuclear envelope integral membrane protein designated as Brl1p (Brr6 like protein no. 1). Brl1p is homologous with Brr6p at the C-terminal domain, which is well conserved in the Brr6/Brl1 family. To characterize the function of Brl1p, a series of temperature-sensitive mutants of Brl1p were isolated. All of brl1 mutations were localized to the conserved C-terminal domain that is essential for a function of Brl1p. Some brl1 alleles showed defects in nuclear export of either mRNA or protein, and nuclear pore clustering, similar to brr6-1. The cellular localization of Brl1p is also similar to that of Brr6p. The genetic analysis suggested that Brl1p functionally interacts with Brr6p. An interaction of Brl1p with Brr6p was shown by the two-hybrid method. We hypothesize that Brl1p functions for nuclear export as a complex with Brr6p.     

A novel insertion of a rearranged L1 element in exon 44 of the dystrophin gene: further evidence for possible bias in retroposon integration

L1 elements are mammalian retrotransposons contributing to genome evolution and causing rare mutations in human. We describe a de novo insertion of an L1 element into the dystrophin gene resulting in skipping of exon 44 and causing Duchenne muscular dystrophy in a boy. The L1 element was rearranged due to the twin-priming mechanism, but contrary to all described L1 rearrangements the 5' region of the inverted L1 sequence ended within the poly(A) tail of the element. Furthermore, the target site for the insertion was located only 87 bp from the insertion site in another patient described previously. These findings can contribute to the understanding of the mechanisms of L1 element rearrangement, and may support the notion that some subregions of the human genome could be preferred targets for retroelements using the L1 enzymatic machinery.     

PAL31, a novel nuclear protein, expressed in the developing brain

We cloned a cDNA encoding a novel protein (PAL31) predominantly expressed in the fetal rat brain by differential display. PAL31 contains leucine-rich repeat domains, a highly acidic region and a putative nuclear localization signal. PAL31 has 50-70% similarity to SSP29, APRIL, LANP, PHAP I, and PP32. Expression of PAL31 mRNA in the brain was high during the fetal period and decreased after birth. Immunohistochemical studies showed that PAL31 is expressed in the entire embryonic brain, whereas in the adult brain its expression is restricted to the subventricular zone where there are neural progenitor cells. It was also revealed that PAL31 is colocalized with PCNA in the nucleus, indicating that the PAL31 expression is developmentally regulated. Considering the primary structure of PAL31 and its spatiotemporal expression pattern, PAL31 is a novel nuclear protein related to the development of the brain through the proliferation of neuronal cells.     

Construction of the nuclear matrix at the transition from maternal to zygotic control of development in the mouse: an immunocytochemical study.

The nuclear matrix is thought to be responsible for DNA organization, DNA replication, RNA synthesis, and RNA processing. We have looked for the presence of nuclear matrix antigens during early mouse embryogenesis. Antibodies to peripheral and interior antigens (P1, Pl1, Pl2, and lamin B) were used to immunolocalize nuclear matrix antigens in germinal vesicle oocytes, metaphase II oocytes, zygotes, two-cell-stage embryos, and eight-cell stage embryos. All antibodies reacted with the nuclei of germinal vesicle oocytes, and two- and eight-cell-stage embryos; however, only P1 and lamin B were present at the pronuclear stage. In eggs collected at the pronuclear stage and cultured to the late two-cell stage in the presence of alpha-amanitin, the matrix morphology was altered for Pl1 and Pl2. alpha-Amanitin had no affect on the distribution of P1 or lamin B antigens. If alpha-amanitin was added 2 hr after cleavage to the two-cell stage, the normal staining pattern of Pl2 was retained. These results suggest that the presence of specific components of an internal matrix is correlated with normal genomic activity.     

Identification of a novel protein, DMAP, which interacts with the myotonic dystrophy protein kinase and shows strong homology to D1 snRNP

The most common adult form of muscular dystrophy, myotonic dystrophy, is due to a triplet repeat (CTG) expansion in the 3 untranslated region of the myotonic dystrophy gene. Although this gene is known to encode a protein kinase, the mechanism by which a defect in this gene results in a disease state is not understood. To gain insight into this mechanism, the yeast two hybrid system was utilized to identify proteins which interact with myotonic dystrophy protein kinase. Eight positive clones were identified that interact specifically with the myotonic dystrophy protein kinase. One clone, which encodes a novel protein interacting with myotonic dystrophy protein kinase bothin vivo in yeast andin vitro, was characterized further. The gene encoding this protein may represent a member of a small gene family, and the protein (95 amino acids) exhibits a high degree of homology to an snRNP protein, D1. This novel protein may be a member of the signal transduction pathway which is responsible for the manifestation of this disease.     

LIM kinase 1, a key regulator of actin dynamics, is widely expressed in embryonic and adult tissues

The expression of endogenous LIM kinase 1 (LIMK1) protein was investigated in embryonic and adult mice using a rat monoclonal antibody (mAb), which recognizes specifically the PDZ domain of LIMK1 and not LIMK2. Immunoblotting analysis revealed widespread expression of LIMK1 existing as a 70-kDa protein in tissues and in cell lines, with a higher mass form (approximately 75 kDa) present in some tissues and cell lines. Smaller isoforms of approximately 50 kDa were also occasionally evident. Immunofluorescence analysis demonstrated LIMK1 subcellular localization at focal adhesions in fibroblasts as revealed by co-staining with actin, paxillin and vinculin in addition to perinuclear (Golgi) and occasional nuclear localization. Furthermore, an association between LIMK1 and paxillin but not vinculin was identified by co-immunoprecipitation analysis. LIMK1 is enriched in both axonal and dendritic growth cones of E18 rat hippocampal pyramidal neurons where it is found in punctae that extend far out into filopodia, as well as in a perinuclear region identified as Golgi. In situ, we identify LIMK1 protein expression in all embryonic and adult tissues examined, albeit at different levels and in different cell populations. The rat monoclonal LIMK1 antibody recognizes proteins of similar size in cell and tissue extracts from numerous species. Thus, LIMK1 is a widely expressed protein that exists as several isoforms.