Nuclear Rim Research Articles

Positioning Heterochromatin at the Nuclear Periphery Suppresses Histone Turnover to Promote Epigenetic Inheritance

In eukaryotes, heterochromatin is generally located at the nuclear periphery. This study investigates the biological significance of perinuclear positioning for heterochromatin maintenance and gene silencing. We identify the nuclear rim protein Amo1NUPL2 as a factor required for the propagation of heterochromatin at endogenous and ectopic sites in the fission yeast genome. Amo1 associates with the Rix1PELP1-containing RNA processing complex RIXC and with the histone chaperone complex FACT. RIXC, which binds to heterochromatin protein Swi6HP1 across silenced chromosomal domains and to surrounding boundary elements, connects heterochromatin with Amo1 at the nuclear periphery. In turn, the Amo1-enriched subdomain is critical for Swi6 association with FACT that precludes histone turnover to promote gene silencing and preserve epigenetic stability ofheterochromatin. In addition to uncovering conserved factors required for perinuclear positioning of heterochromatin, these analyses elucidate a mechanism by which a peripheral subdomain enforces stable gene repression and maintains heterochromatin in a heritable manner.

Nesprin-1-alpha2 associates with kinesin at myotube outer nuclear membranes, but is restricted to neuromuscular junction nuclei in adult muscle

Nesprins, nuclear envelope spectrin-repeat proteins encoded by the SYNE1 and SYNE2 genes, are involved in localization of nuclei. The short isoform, nesprin-1-alpha2, is required for relocation of the microtubule organizer function from centromeres to the nuclear rim during myogenesis. Using specific antibodies, we now show that both nesprin-1-alpha2 and nesprin-1-giant co-localize with kinesin at the junctions of concatenated nuclei and at the outer poles of nuclear chains in human skeletal myotubes. In adult muscle, nesprin-1-alpha2 was found, together with kinesin, only on nuclei associated with neuromuscular junctions, whereas all adult cardiomyocyte nuclei expressed nesprin-1-alpha2. In a proteomics study, kinesin heavy and light chains were the only significant proteins in myotube extracts pulled down by nesprin-1-alpha2, but not by a mutant lacking the highly-conserved STAR domain (18 amino-acids, including the LEWD motif). The results support a function for nesprin-1-alpha2 in the specific localization of skeletal muscle nuclei mediated by kinesins and suggest that its primary role is at the outer nuclear membrane.

Nucleoporin Nup58 localizes to centrosomes and mid-bodies during mitosis

BackgroundNuclear pore complexes (NPCs) act as nano-turnstiles within nuclear membranes between the cytoplasm and nucleus of mammalian cells. NPC proteins, called nucleoporins (Nups), mediate trafficking of proteins and RNA into and out of the nucleus, and are involved in a variety of mitotic processes. We previously reported that Nup62 localizes to the centrosome and mitotic spindle during mitosis, and plays a role in centrosome homeostasis. However, whether Nup58, a Nup62 subcomplex protein, also localizes to spindle poles is unknown.ResultHerein, we show that Nup58 localizes to the nuclear rim during interphase, and to mitotic spindles, centrosomes, and midbodies during mitosis. Our confocal microscopy, live-cell imaging, and stimulated emission depletion nanoscopy results also demonstrated that Nup58 localized to the centrosomes during metaphase and relocalized to midbodies during abscission. Depletion of Nup58 resulted in centrosomal abnormalities and delayed abscission.ConclusionNup58 localized at the centrosomes and mitotic spindle during metaphase and relocalized at midbodies during abscission. This study highlights the important role of Nup58 in mitosis.

Identification and characterization of WD40 superfamily genes in peach

The WD40 transcription factor family is a superfamily found in all eukaryotes that plays important roles in regulating growth and development. To our knowledge, to date, WD40 superfamily genes have been identified and characterized in several plant species, but little information is available on the WD40 superfamily genes in peach. In this study, we identified 220 members of the WD40 superfamily in the peach genome, and these members were further classified into five subfamilies based on phylogenetic comparison with those in Arabidopsis. The members within each subfamily had conserved motifs and gene structures. The WD40 genes were unevenly distributed on chromosomes 1 to 8 of the peach genome. Additionally, 58 pairs of paralog WD40 members were found on eight chromosomes in peach, and 242 pairs of orthologous WD40 genes in peach and Arabidopsis were matched. The 54 selected putative WD40 genes in peach had diverse expression patterns in red-fleshed and white-fleshed peach fruits at five developmental stages. Prupe.6G211800.1 was located only on the cytomembrane, while Prupe.1G428200.1 and Prupe.I003200.1 were located on both the cytomembrane and in the nucleus; Prupe.1G558700.1 was densely localized around the nuclear rim but relatively faintly localized in the nucleoplasm; Prupe.5G116300.1 was located in the nucleus and cytomembrane with strong signals but showed weak signals in the cytoplasm; and Prupe.8G212400.1 and Prupe.1G053600.1 were located mainly in the nuclear envelope and cytomembrane but relatively faintly in the nucleoplasm. This study provides a foundation for the further functional verification of WD40 genes in peach.

A rapid technique for en masse soft cataract phacoemulsification.

Purpose: To describe a new, en masse, stepwise technique for purely soft cataracts.Methods: RAPID, a soft cataract phacoemulsification technique, is an acronym-based procedure where R is rotation of nucleus, A is alignment of phacoemulsification tip sideways, P is placement of tip adjacent to the nuclear rim, I is impaling of tip into nuclear rim and D is devouring wherein nucleus is aspirated/emulsified. RAPID technique was performed in 54 eyes of 54 patients (31 males and 23 females; mean age 46.35±3.95 years). The soft nucleus was emulsified, after performing hydrodissection, in a stepwise manner in the safe zone away from posterior capsule and corneal endothelium. Centurion/Infiniti Phacoemulsification system (Alcon Laboratories, Inc.) was used with vacuum parameters at 475 mm Hg and an aspiration flow rate of 0–45 mL/min in linear mode. The primary outcome measures were cumulative dissipated energy (CDE), ultrasound time (UST), amount of fluid used, surgical complications and mean endothelial cell loss.Results: Phacoemulsification with IOL implantation was performed successfully in all patients without any intraoperative complications. CDE was 1.03±0.61. Total UST for nuclear emulsification was 3.84±3.27 seconds and fluid used was 10±2.35 milliliters. Postoperative follow-up examinations were done on 1, 4, 14, 30 and 90 days. Mean percentage of endothelial cell loss was 7.05±2.65% (mean endothelial cell counts were 2383.75±105.21 cells/mm2 preoperatively and 2215.78±114.9 cells/mm2 3 months postoperatively).Conclusion: RAPID is an en masse non-fragmentation technique for purely soft cataracts. This technique requires neither any specialized instrumentations nor the use of high vacuum with complimenting surge preventing software. Simple stepwise multi-planer approach of RAPID technique allows easy and fast emulsification of soft cataracts with simultaneous safeguarding of posterior capsule and corneal endothelium.

Evidence for a role of spindle matrix formation in cell cycle progression by antibody perturbation.

In Drosophila it has recently been demonstrated that a spindle matrix in the form of a membrane-less macromolecular assembly embeds the microtubule-based spindle apparatus. In addition, two of its constituents, Megator and Chromator, were shown to function as spatial regulators of spindle checkpoint proteins. However, whether the spindle matrix plays a wider functional role in spatially regulating cell cycle progression factors was unknown. Here using a live imaging approach we provide evidence that a number of key cell cycle proteins such as Cyclin B, Polo, and Ran co-localize with the spindle matrix during mitosis. Furthermore, prevention of spindle matrix formation by injection of a function blocking antibody against the spindle matrix protein Chromator results in cell cycle arrest prior to nuclear envelope breakdown. In such embryos the spatial dynamics of Polo and Cyclin B enrichment at the nuclear rim and kinetochores is abrogated and Polo is not imported into the nucleus. This is in contrast to colchicine-arrested embryos where the wild-type dynamics of these proteins are maintained. Taken together these results suggest that spindle matrix formation may be a general requirement for the localization and proper dynamics of cell cycle factors promoting signaling events leading to cell cycle progression.

Pseudo Rabies Virus Protein UL34 Interacted with UL31 to Disrupt the Human Lamin A

Pseudo rabies virus (PRV) egresses from the nucleus by budding from the inner nuclear membrane (INM). The nuclear lamina forms a rigid meshwork of intermediate filaments underlying the INM. It remains unknown whether PRV infection induces the disruption of lamina. In this paper, it can be observed that nuclear Lamin A became fractured during PRV infection. UL34 was localized at the nuclear rim, but UL31 was accumulated in the nucleus as distinct patches. Interestingly, a part of UL31 was localized at the INM in the presence of UL34. Immunoprecipitation (IP) assay confirmed that PRV UL31 and UL34 interacted in the transfected cells. Importantly, the co-expression of UL31 and UL34 directly disrupted Lamin A, resembling that observed during PRV infection. In conclusion, PRV infection induces the disruption of Lamin A, and UL34 and UL31 play a critical role in the disruption of Lamin A.

Linkage between lipid droplet formation and nuclear deformation in HeLa human cervical cancer cells

Because lipid droplets (LDs) and the nucleus are cellular organelles that regulate seemingly very different biochemical processes, very little attention has been focused on their possible interplay. Here, we report a correlation between nuclear morphology and cytoplasmic LD formation in HeLa human cervical cells. When the cells were treated with oleic acid (OA), LDs were formed in the cytoplasm, but not in the nucleoplasm. Interestingly, cells harboring OA-induced cytoplasmic LDs showed deformity of the nucleus, particularly at the nuclear rim. Conversely, when alteration from a single spherical nuclear shape to a multinucleated form was enforced by coadministration of paclitaxel and reversine, a significant amount of LDs was detected in the cytoplasm of the multinucleated cells. These two distinct pharmacological culture conditions not only allow analysis of the previously underappreciated organelle relationship, but also provide insights into the mutual affectability of LD formation and nuclear deformation.

The role of vimentin in the tumor marker Nup88-dependent multinucleated phenotype

BackgroundNucleoporin Nup88, a component of nuclear pore complexes, is known to be overexpressed in several types of tumor tissue. The overexpression of Nup88 has been reported to promote the early step of tumorigenesis by inducing multinuclei in both HeLa cells and a mouse model. However, the molecular basis of how Nup88 leads to a multinucleated phenotype remains unclear because of a lack of information concerning its binding partners. In this study, we characterize a novel interaction between Nup88 and vimentin. We also examine the involvement of vimentin in the Nup88-dependent multinucleated phenotype.MethodsCells overexpressing tagged versions of Nup88, vimentin and their truncations were used in this study. Coprecipitation and GST-pulldown assays were carried out to analyze protein-protein interactions. Vimentin knockdown by siRNA was performed to examine the functional role of the Nup88-vimentin interaction in cells. The phosphorylation status of vimentin was analyzed by immunoblotting using an antibody specific for its phosphorylation site.ResultsVimentin was identified as a Nup88 interacting partner, although it did not bind to other nucleoporins, such as Nup50, Nup214, and Nup358, in HeLa cell lysates. The N-terminal 541 amino acid residues of Nup88 was found to be responsible for its interaction with vimentin. Recombinant GST-tagged Nup88 bound to recombinant vimentin in a GST-pulldown assay. Although overexpression of Nup88 in HeLa cells was observed mainly at the nuclear rim and in the cytoplasm, colocalization with vimentin was only partially detected at or around the nuclear rim. Disruption of the Nup88-vimentin interaction by vimentin specific siRNA transfection suppressed the Nup88-dependent multinucleated phenotype. An excess amount of Nup88 in cell lysates inhibited the dephosphorylation of a serine residue (Ser83) within the vimentin N-terminal region even in the absence and presence of an exogenous phosphatase. The N-terminal 96 amino acid residues of vimentin interacted with both full-length and the N-terminal 541 residues of Nup88.ConclusionsNup88 can affect the phosphorylation status of vimentin, which may contribute to the Nup88-dependent multinucleated phenotype through changing the organization of vimentin.

Stoichiometry and compositional plasticity of the yeast nuclear pore complex revealed by quantitative fluorescence microscopy

The nuclear pore complex (NPC) is an eightfold symmetrical channel providing selective transport of biomolecules across the nuclear envelope. Each NPC consists of ∼30 different nuclear pore proteins (Nups) all present in multiple copies per NPC. Significant progress has recently been made in the characterization of the vertebrate NPC structure. However, because of the estimated size differences between the vertebrate and yeast NPC, it has been unclear whether the NPC architecture is conserved between species. Here, we have developed a quantitative image analysis pipeline, termed nuclear rim intensity measurement (NuRIM), to precisely determine copy numbers for almost all Nups within native NPCs of budding yeast cells. Our analysis demonstrates that the majority of yeast Nups are present at most in 16 copies per NPC. This reveals a dramatic difference to the stoichiometry determined for the human NPC, suggesting that despite a high degree of individual Nup conservation, the yeast and human NPC architecture is significantly different. Furthermore, using NuRIM, we examined the effects of mutations on NPC stoichiometry. We demonstrate for two paralog pairs of key scaffold Nups, Nup170/Nup157 and Nup192/Nup188, that their altered expression leads to significant changes in the NPC stoichiometry inducing either voids in the NPC structure or substitution of one paralog by the other. Thus, our results not only provide accurate stoichiometry information for the intact yeast NPC but also reveal an intriguing compositional plasticity of the NPC architecture, which may explain how differences in NPC composition could arise in the course of evolution.

Transient and Partial Nuclear Lamina Disruption Promotes Chromosome Movement in Early Meiotic Prophase

SummaryMeiotic chromosome movement is important for the pairwise alignment of homologous chromosomes, which is required for correct chromosome segregation. Movement is driven by cytoplasmic forces, transmitted to chromosome ends by nuclear membrane-spanning proteins. In animal cells, lamins form a prominent scaffold at the nuclear periphery, yet the role lamins play in meiotic chromosome movement is unclear. We show that chromosome movement correlates with reduced lamin association with the nuclear rim, which requires lamin phosphorylation at sites analogous to those that open lamina network crosslinks in mitosis. Failure to remodel the lamina results in delayed meiotic entry, altered chromatin organization, unpaired or interlocked chromosomes, and slowed chromosome movement. The remodeling kinases are delivered to lamins via chromosome ends coupled to the nuclear envelope, potentially enabling crosstalk between the lamina and chromosomal events. Thus, opening the lamina network plays a role in modulating contacts between chromosomes and the nuclear periphery during meiosis.

A Role for Myosin Va in Human Cytomegalovirus Nuclear Egress.

Herpesviruses replicate and package their genomes into capsids in replication compartments within the nuclear interior. Capsids then move to the inner nuclear membrane for envelopment and release into the cytoplasm in a process called nuclear egress. We previously found that nuclear F-actin is induced upon infection with the betaherpesvirus human cytomegalovirus (HCMV) and is important for nuclear egress and capsid localization away from replication compartment-like inclusions toward the nuclear rim. Despite these and related findings, it has not been shown that any specific motor protein is involved in herpesvirus nuclear egress. In this study, we have investigated whether the host motor protein, myosin Va, could be fulfilling this role. Using immunofluorescence microscopy and coimmunoprecipitation, we observed associations between a nuclear population of myosin Va and the viral major capsid protein, with both concentrating at the periphery of replication compartments. Immunoelectron microscopy showed that nearly 40% of assembled nuclear capsids associate with myosin Va. We also found that myosin Va and major capsid protein colocalize with nuclear F-actin. Importantly, antagonism of myosin Va with RNA interference or a dominant negative mutant revealed that myosin Va is important for the efficient production of infectious virus, capsid accumulation in the cytoplasm, and capsid localization away from replication compartment-like inclusions toward the nuclear rim. Our results lead us to suggest a working model whereby human cytomegalovirus capsids associate with myosin Va for movement from replication compartments to the nuclear periphery during nuclear egress.IMPORTANCE Little is known regarding how newly assembled and packaged herpesvirus capsids move from the nuclear interior to the periphery during nuclear egress. While it has been proposed that an actomyosin-based mechanism facilitates intranuclear movement of alphaherpesvirus capsids, a functional role for any specific myosin in nuclear egress has not been reported. Furthermore, the notion that an actomyosin-based mechanism facilitates intranuclear capsid movement is controversial. Here we show that human cytomegalovirus capsids associate with nuclear myosin Va and F-actin and that antagonism of myosin Va impairs capsid localization toward the nuclear rim and nuclear egress. Together with our previous results showing that nuclear F-actin is induced upon HCMV infection and is also important for these processes, our results lend support to the hypothesis that nascent human cytomegalovirus capsids migrate to the nuclear periphery via actomyosin-based movement. These results shed light on a poorly understood viral process and the cellular machinery involved.

Differential staining of peripheral nuclear chromatin with Acridine orange implies an A-form epichromatin conformation of the DNA

ABSTRACTThe chromatin observed by conventional electron microscopy under the nuclear envelope constitutes a single layer of dense 30–35 nm granules, while ∼30 nm fibrils laterally attached to them, form large patches of lamin-associated domains (LADs). This particular surface “epichromatin” can be discerned by specific (H2A+H2B+DNA) conformational antibody at the inner nuclear envelope and around mitotic chromosomes. In order to differentiate the DNA conformation of the peripheral chromatin we applied an Acridine orange (AO) DNA structural test involving RNAse treatment and the addition of AO after acid pre-treatment. MCF-7 cells treated in this way revealed yellow/red patches of LADs attached to a thin green nuclear rim and with mitotic chromosomes outlined in green, topologically corresponding to epichromatin epitope staining by immunofluorescence. Differentially from LADs, the epichromatin was unable to provide metachromatic staining by AO, unless thermally denatured at 94oC. DNA enrichment in GC stretches has been recently reported for immunoprecipitated ∼ 1Kb epichromatin domains. Together these data suggest that certain epichromatin segments assume the relatively hydrophobic DNA A-conformation at the nuclear envelope and surface of mitotic chromosomes, preventing AO side dimerisation. We hypothesize that epichromatin domains form nucleosome superbeads. Hydrophobic interactions stack these superbeads and align them at the nuclear envelope, while repulsing the hydrophilic LADs. The hydrophobicity of epichromatin explains its location at the surface of mitotic chromosomes and its function in mediating chromosome attachment to the restituting nuclear envelope during telophase.

Autographa californica Multiple Nucleopolyhedrovirus ac75 Is Required for the Nuclear Egress of Nucleocapsids and Intranuclear Microvesicle Formation.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf75 (ac75) is a highly conserved gene of unknown function. In this study, we constructed an ac75 knockout AcMNPV bacmid and investigated the role of ac75 in the baculovirus life cycle. The expression and distribution of the Ac75 protein were characterized, and its interaction with another viral protein was analyzed to further understand its function. Our data indicated that ac75 was required for the nuclear egress of nucleocapsids, intranuclear microvesicle formation, and subsequent budded virion (BV) formation, as well as occlusion-derived virion (ODV) envelopment and embedding of ODVs into polyhedra. Western blot analyses showed that two forms, of 18 and 15 kDa, of FLAG-tagged Ac75 protein were detected. Ac75 was associated with both nucleocapsid and envelope fractions of BVs but with only the nucleocapsid fraction of ODVs; the 18-kDa form was associated with only BVs, whereas the 15-kDa form was associated with both types of virion. Ac75 was localized predominantly in the intranuclear ring zone during infection and exhibited a nuclear rim distribution during the early phase of infection. A phase separation assay suggested that Ac75 was not an integral membrane protein. A coimmunoprecipitation assay revealed an interaction between Ac75 and the integral membrane protein Ac76, and bimolecular fluorescence complementation assays identified the sites of the interaction within the cytoplasm and at the nuclear membrane and ring zone in AcMNPV-infected cells. Our results have identified ac75 as a second gene that is required for both the nuclear egress of nucleocapsids and the formation of intranuclear microvesicles.IMPORTANCE During the baculovirus life cycle, the morphogenesis of both budded virions (BVs) and occlusion-derived virions (ODVs) is proposed to involve a budding process at the nuclear membrane, which occurs while nucleocapsids egress from the nucleus or when intranuclear microvesicles are produced. However, the exact mechanism of virion morphogenesis remains unknown. In this study, we identified ac75 as a second gene, in addition to ac93, that is essential for the nuclear egress of nucleocapsids, intranuclear microvesicle formation, and subsequent BV formation, as well as ODV envelopment and embedding of ODVs into polyhedra. Ac75 is not an integral membrane protein. However, it interacts with an integral membrane protein (Ac76) and is associated with the nuclear membrane. These data enhance our understanding of the commonalities between nuclear egress of nucleocapsids and intranuclear microvesicle formation and may help to reveal insights into the mechanism of baculovirus virion morphogenesis.

Baculovirus infection induces disruption of the nuclear lamina

Baculovirus nucleocapsids egress from the nucleus primarily via budding at the nuclear membrane. The nuclear lamina underlying the nuclear membrane represents a substantial barrier to nuclear egress. Whether the nuclear lamina undergoes disruption during baculovirus infection remains unknown. In this report, we generated a clonal cell line, Sf9-L, that stably expresses GFP-tagged Drosophila lamin B. GFP autofluorescence colocalized with immunofluorescent anti-lamin B at the nuclear rim of Sf9-L cells, indicating GFP-lamin B was incorporated into the nuclear lamina. Meanwhile, virus was able to replicate normally in Sf9-L cells. Next, we investigated alterations to the nuclear lamina during baculovirus infection in Sf9-L cells. A portion of GFP-lamin B localized diffusely at the nuclear rim, and some GFP-lamin B was redistributed within the nucleus during the late phase of infection, suggesting the nuclear lamina was partially disrupted. Immunoelectron microscopy revealed associations between GFP-lamin B and the edges of the electron-dense stromal mattes of the virogenic stroma, intranuclear microvesicles, and ODV envelopes and nucleocapsids within the nucleus, indicating the release of some GFP-lamin B from the nuclear lamina. Additionally, GFP-lamin B phosphorylation increased upon infection. Based on these data, baculovirus infection induced lamin B phosphorylation and disruption of the nuclear lamina.

EBV early lytic protein BFRF1 alters emerin distribution and post-translational modification

The nuclear envelope (NE), a structural element of fundamental importance for the cell, is the first barrier that meets a virus in the early stages of viral maturation. Therefore, in order to allow the passage of nucleocapsids, viruses are known to modulate the architecture of the nuclear membrane to permit a proficient viral infection. Epstein-Barr Virus (EBV), a pathogen from Herpesvirus family, possesses two well conserved proteins, BFRF1 and BFLF2, which together form the heterodimeric nuclear egress complex (NEC) that is involved in the early steps of nuclear egress. Here we show that EBV replication causes the delocalization of emerin, a cellular LEM-domain protein normally distributed on the nuclear rim. We also demonstrate that the early lytic protein BFRF1 is responsible for emerin delocalization. Expression of BFRF1 alone or in combination with BFLF2 induces emerin hyperphosphorylation. Altogether, these results suggest a novel mechanism by which EBV exploits the cellular machinery for nucleocapsid egress.

A Role for Nuclear F-Actin Induction in Human Cytomegalovirus Nuclear Egress.

ABSTRACTHerpesviruses, which include important pathogens, remodel the host cell nucleus to facilitate infection. This remodeling includes the formation of structures called replication compartments (RCs) in which herpesviruses replicate their DNA. During infection with the betaherpesvirus, human cytomegalovirus (HCMV), viral DNA synthesis occurs at the periphery of RCs within the nuclear interior, after which assembled capsids must reach the inner nuclear membrane (INM) for translocation to the cytoplasm (nuclear egress). The processes that facilitate movement of HCMV capsids to the INM during nuclear egress are unknown. Although an actin-based mechanism of alphaherpesvirus capsid trafficking to the INM has been proposed, it is controversial. Here, using a fluorescently-tagged, nucleus-localized actin-binding peptide, we show that HCMV, but not herpes simplex virus 1, strongly induced nuclear actin filaments (F-actin) in human fibroblasts. Based on studies using UV inactivation and inhibitors, this induction depended on viral gene expression. Interestingly, by 24 h postinfection, nuclear F-actin formed thicker structures that appeared by super-resolution microscopy to be bundles of filaments. Later in infection, nuclear F-actin primarily localized along the RC periphery and between the RC periphery and the nuclear rim. Importantly, a drug that depolymerized nuclear F-actin caused defects in production of infectious virus, capsid accumulation in the cytoplasm, and capsid localization near the nuclear rim, without decreasing capsid accumulation in the nucleus. Thus, our results suggest that for at least one herpesvirus, nuclear F-actin promotes capsid movement to the nuclear periphery and nuclear egress. We discuss our results in terms of competing models for these processes.

The absence of p53 during Human Cytomegalovirus infection leads to decreased UL53 expression, disrupting UL50 localization to the inner nuclear membrane, and thereby inhibiting capsid nuclear egress

Our electron microscopy study (Kuan et al., 2016) found HCMV nuclear capsid egress was significantly reduced in p53 knockout cells (p53KOs), correlating with inhibited formation of infoldings of the inner nuclear membrane (IINMs). Molecular examination of these phenomena has found p53KOs expressed UL97 and phosphorylated lamins, however the lamina failed to remodel. The nuclear egress complex (NEC) protein UL50 was expressed in almost all cells. UL50 re-localized to the inner nuclear membrane (INM) in ~90% of wt cells, but only ~35% of p53KOs. UL53 expression was significantly reduced in p53KOs, and cells lacking UL50 nuclear staining, expressed no UL53. Re-introduction of p53 into p53KOs largely recovered UL53 positivity and UL50 nuclear re-localization. Nuclear rim located UL50/53 puncta, which co-localized with the major capsid protein, were largely absent in p53KOs. We believe these puncta were IINMs. In the absence of p53, UL53 expression was inhibited, disrupting formation of the NEC/IINMs, and reducing functional virion secretion.

The telomeric protein AKTIP interacts with A- and B-type lamins and is involved in regulation of cellular senescence.

AKTIP is a shelterin-interacting protein required for replication of telomeric DNA. Here, we show that AKTIP biochemically interacts with A- and B-type lamins and affects lamin A, but not lamin C or B, expression. In interphase cells, AKTIP localizes at the nuclear rim and in discrete regions of the nucleoplasm just like lamins. Double immunostaining revealed that AKTIP partially co-localizes with lamin B1 and lamin A/C in interphase cells, and that proper AKTIP localization requires functional lamin A. In mitotic cells, AKTIP is enriched at the spindle poles and at the midbody of late telophase cells similar to lamin B1. AKTIP-depleted cells show senescence-associated markers and recapitulate several aspects of the progeroid phenotype. Collectively, our results indicate that AKTIP is a new player in lamin-related processes, including those that govern nuclear architecture, telomere homeostasis and cellular senescence.

Cytoplasmic localization of SBR (Dm NXF1) protein and its zonal distribution in the ganglia of Drosophila melanogaster larvae.

The Drosophila gene Dm nxf1 (nuclear export factor 1) previously known as small bristles (sbr) controls nuclear export of various mRNA transcripts. We found that Dm NXF1 is present not only in nucleoplasm or at the nuclear rim but also in the cytoplasm. On the spatiotemporal level, anti-SBR antibodies labeled some neuroblasts and their lineages in the brains of Drosophila larvae. The number of Dm NXF1-rich lineages increased during larval development, but Dm NXF1 expression was not evident in all lineages. In all larval stages, Dm NXF1 concentrated in the midline cells of the ventral nerve cord, which reflects a specific status of those cells. In neurites, Dm NXF1 was present in the form of cytoplasmic granules, which is similar to the behavior of another RNA-binding protein, dFMR. Interestingly, though, the granule expression pattern of Dm NXF1 and dFMR did not always overlap, as some granules stained exclusively for one or the other protein. It suggests the existence of specific mRNA partners for Dm NXF1 in neurites.