Defects In Nuclear Morphology Research Articles

Uncovering the Role of the Yeast Lysine Acetyltransferase NuA4 in the Regulation of Nuclear Shape and Lipid Metabolism.

Here, we report a novel role for the yeast lysine acetyltransferase NuA4 in regulating phospholipid availability for organelle morphology. Disruption of the NuA4 complex results in 70% of cells displaying nuclear deformations and nearly 50% of cells exhibiting vacuolar fragmentation. Cells deficient in NuA4 also show severe defects in the formation of nuclear-vacuole junctions (NJV), as well as a decrease in piecemeal microautophagy of the nucleus (PMN). To determine the cause of these defects we focused on Pah1, an enzyme that converts phosphatidic acid into diacylglycerol, favoring accumulation of lipid droplets over phospholipids that are used for membrane expansion. NuA4 subunit Eaf1 was required for Pah1 localization to the inner nuclear membrane and artificially tethering of Pah1 to the nuclear membrane rescued nuclear deformation and vacuole fragmentation defects, but not defects related to the formation of NVJs. Mutation of a NuA4-dependent acetylation site on Pah1 also resulted in aberrant Pah1 localization and defects in nuclear morphology and NVJ. Our work suggests a critical role for NuA4 in organelle morphology that is partially mediated through the regulation of Pah1 subcellular localization.

Caenorhabditis elegans models for striated muscle disorders caused by missense variants of human LMNA.

Striated muscle laminopathies caused by missense mutations in the nuclear lamin gene LMNA are characterized by cardiac dysfunction and often skeletal muscle defects. Attempts to predict which LMNA variants are pathogenic and to understand their physiological effects lag behind variant discovery. We created Caenorhabditis elegans models for striated muscle laminopathies by introducing pathogenic human LMNA variants and variants of unknown significance at conserved residues within the lmn-1 gene. Severe missense variants reduced fertility and/or motility in C. elegans. Nuclear morphology defects were evident in the hypodermal nuclei of many lamin variant strains, indicating a loss of nuclear envelope integrity. Phenotypic severity varied within the two classes of missense mutations involved in striated muscle disease, but overall, variants associated with both skeletal and cardiac muscle defects in humans lead to more severe phenotypes in our model than variants predicted to disrupt cardiac function alone. We also identified a separation of function allele, lmn-1(R204W), that exhibited normal viability and swimming behavior but had a severe nuclear migration defect. Thus, we established C. elegans avatars for striated muscle laminopathies and identified LMNA variants that offer insight into lamin mechanisms during normal development.

The LMNA p.R541C mutation causes dilated cardiomyopathy in human and mice

Dilated cardiomyopathy (DCM) is a major cause of heart failure. LMNA variants contribute to 6–10% DCM cases, but the underlying mechanisms remain incompletely understood. Here, we reported two patients carrying the LMNA c.1621C > T/ p.R541C variant and generated a knock-in mouse model (LmnaRC) to study the role of this variant in DCM pathogenesis. We found LmnaRC/RC mice exhibited ventricular dilation and reduced systolic functions at 6 months after birth. The LmnaRC/RC cardiomyocytes increased in size but no nuclear morphology defects were detected. Transcriptomic and microscopic analyses revealed suppressed gene expression and perturbed ultrastructure in LmnaRC/RC mitochondria. These defects were associated with increased heterochromatin structures and epigenetic markers including H3K9me2/3. Together, these data implied that the LMNA c.1621C > T/ p.R541C variant enhanced heterochromatic gene suppression and disrupted mitochondria functions as a cause of DCM.

Lamin B1 deletion in myeloid neoplasms causes nuclear anomaly and altered hematopoietic stem cell function.

Abnormal nuclear morphology is a hallmark of malignant cells widely used in cancer diagnosis. Pelger-Huët anomaly (PHA) is a common abnormality of neutrophil nuclear morphology of unknown molecular etiology in myeloid neoplasms (MNs). We show that loss of nuclear lamin B1 (LMNB1) encoded on chromosome 5q, which is frequently deleted in MNs, induces defects in nuclear morphology and human hematopoietic stem cell (HSC) function associated with malignancy. LMNB1 deficiency alters genome organization inducing invitro and invivo expansion of HSCs, myeloid-biased differentiation with impaired lymphoid commitment, and genome instability due to defective DNA damage repair. Nuclear dysmorphology of neutrophils in patients with MNs is associated with 5q deletions spanning the LMNB1 locus, and lamin B1 loss is both necessary and sufficient to cause PHA in normal and 5q-deleted neutrophils. LMNB1 loss thus causes acquired PHA and links abnormal nuclear morphology with HSCs and progenitor cell fate determination via genome organization.

Abf1 Is an Essential Protein That Participates in Cell Cycle Progression and Subtelomeric Silencing in Candida glabrata.

Accurate DNA replication and segregation is key to reproduction and cell viability in all organisms. Autonomously replicating sequence-binding factor 1 (Abf1) is a multifunctional protein that has essential roles in replication, transcription, and regional silencing in the model yeast Saccharomyces cerevisiae. In the opportunistic pathogenic fungus Candida glabrata, which is closely related to S. cerevisiae, these processes are important for survival within the host, for example, the regulation of transcription of virulence-related genes like those involved in adherence. Here, we describe that CgABF1 is an essential gene required for cell viability and silencing near the telomeres, where many adhesin-encoding genes reside. CgAbf1 mediated subtelomeric silencing depends on the 43 C-terminal amino acids. We also found that abnormal expression, depletion, or overexpression of Abf1, results in defects in nuclear morphology, nuclear segregation, and transit through the cell cycle. In the absence of ABF1, cells are arrested in G2 but start cycling again after 9 h, coinciding with the loss of cell viability and the appearance of cells with higher DNA content. Overexpression of CgABF1 causes defects in nuclear segregation and cell cycle progression. We suggest that these effects could be due to the deregulation of DNA replication.

Functional recovery of the germ line following splicing collapse.

Splicing introns from precursor-messenger RNA (pre-mRNA) transcripts is essential for translating functional proteins. Here, we report that the previously uncharacterized Caenorhabditis elegans protein MOG-7 acts as a pre-mRNA splicing factor. Depleting MOG-7 from the C. elegans germ line causes intron retention in most germline-expressed genes, impeding the germ cell cycle, and causing defects in nuclear morphology, germ cell identity and sterility. Despite the deleterious consequences caused by MOG-7 loss, the adult germ line can functionally recover to produce viable and fertile progeny when MOG-7 is restored. Germline recovery is dependent on a burst of apoptosis that likely clears defective germ cells, and viable gametes generated from the proliferation of germ cells in the progenitor zone. Together, these findings reveal that MOG-7 is essential for germ cell development, and that the germ line can functionally recover after a collapse in RNA splicing.

CRISPR/Cas9-engineered Drosophila knock-in models to study VCP diseases.

ABSTRACTMutations in Valosin Containing Protein (VCP) are associated with several degenerative diseases, including multisystem proteinopathy (MSP-1) and amyotrophic lateral sclerosis. However, patients with VCP mutations vary widely in their pathology and clinical penetrance, making it difficult to devise effective treatment strategies. A deeper understanding of how each mutation affects VCP function could enhance the prediction of clinical outcomes and design of personalized treatment options. The power of a genetically tractable model organism coupled with well-established in vivo assays and a relatively short life cycle make Drosophila an attractive system to study VCP disease pathogenesis. Using CRISPR/Cas9, we have generated individual Drosophila knock-in mutants that include nine hereditary VCP disease mutations. Our models display many hallmarks of VCP-mediated degeneration, including progressive decline in mobility, protein aggregate accumulation and defects in lysosomal and mitochondrial function. We also made some novel and unexpected findings, including nuclear morphology defects and sex-specific phenotypic differences in several mutants. Taken together, the Drosophila VCP disease models generated in this study will be useful for studying the etiology of individual VCP patient mutations and testing potential genetic and/or pharmacological therapies.

Spatiotemporal Mislocalization of Nuclear Membrane-Associated Proteins in γ-Irradiation-Induced Senescent Cells.

Cellular senescence, induced by genotoxic or replication stress, is accompanied by defects in nuclear morphology and nuclear membrane-heterochromatin disruption. In this work, we analyzed cytological and molecular changes in the linker of nucleoskeleton and cytoskeleton (LINC) complex proteins in senescence triggered by γ-irradiation. We used human mammary carcinoma and osteosarcoma cell lines, both original and shRNA knockdown clones targeting lamin B receptor (LBR) and leading to LBR and lamin B (LB1) reduction. The expression status and integrity of LINC complex proteins (nesprin-1, SUN1, SUN2), lamin A/C, and emerin were analyzed by immunodetection using confocal microscopy and Western blot. The results show frequent mislocalization of these proteins from the nuclear membrane to cytoplasm and micronuclei and, in some cases, their fragmentation and amplification. The timing of these changes clearly preceded the onset of senescence. The LBR deficiency triggered neither senescence nor changes in the LINC protein distribution before irradiation. However, the cytological changes following irradiation were more pronounced in shRNA knockdown cells compared to original cell lines. We conclude that mislocalization of LINC complex proteins is a significant characteristic of cellular senescence phenotypes and may influence complex events at the nuclear membrane, including trafficking and heterochromatin attachment.

Enhanced nuclear protein export in premature aging and rescue of the progeria phenotype by modulation of CRM1 activity.

The study of Hutchinson–Gilford progeria syndrome (HGPS) has provided important clues to decipher mechanisms underlying aging. Progerin, a mutant lamin A, disrupts nuclear envelope structure/function, with further impairment of multiple processes that culminate in senescence. Here, we demonstrate that the nuclear protein export pathway is exacerbated in HGPS, due to progerin‐driven overexpression of CRM1, thereby disturbing nucleocytoplasmic partitioning of CRM1‐target proteins. Enhanced nuclear export is central in HGPS, since pharmacological inhibition of CRM1 alleviates all aging hallmarks analyzed, including senescent cellular morphology, lamin B1 downregulation, loss of heterochromatin, nuclear morphology defects, and expanded nucleoli. Exogenous overexpression of CRM1 on the other hand recapitulates the HGPS cellular phenotype in normal fibroblasts. CRM1 levels/activity increases with age in fibroblasts from healthy donors, indicating that altered nuclear export is a common hallmark of pathological and physiological aging. Collectively, our findings provide novel insights into HGPS pathophysiology, identifying CRM1 as potential therapeutic target in HGPS.

Are There Common Mechanisms Between the Hutchinson-Gilford Progeria Syndrome and Natural Aging?

The Hutchinson–Gilford progeria syndrome (HGPS) is a premature aging disease caused by mutations of the LMNA gene leading to increased production of a partially processed form of the nuclear fibrillar protein lamin A – progerin. Progerin acts as a dominant factor that leads to multiple morphological anomalies of cell nuclei and disturbances in heterochromatin organization, mitosis, DNA replication and repair, and gene transcription. Progerin-positive cells are present in primary fibroblast cultures obtained from the skin of normal donors at advanced ages. These cells display HGPS-like defects in nuclear morphology, decreased H3K9me3 and HP1, and increased histone H2AX phosphorylation marks of the DNA damage loci. Inhibition of progerin production in cells of aged non-HGPS donors in vivo increases the proliferative activity, H3K9me3, and HP1, and decreases the senescence markers p21, IGFBP3, and GADD45B to the levels of young donor cells. Thus, progerin-dependent mechanisms act in natural aging. Excessive activity of the same mechanisms may well be the cause of premature aging in HGPS. Telomere attrition is widely regarded to be one of the primary hallmarks of aging. Progerin expression in normal human fibroblasts accelerates the loss of telomeres. Changes in lamina organization may directly affect telomere attrition resulting in accelerated replicative senescence and progeroid phenotypes. The chronological aging in normal individuals and the premature aging in HGPS patients are mediated by similar changes in the activity of signaling pathways, including downregulation of DNA repair and chromatin organization, and upregulation of ERK, mTOR, GH-IGF1, MAPK, TGFβ, and mitochondrial dysfunction. Multiple epigenetic changes are common to premature aging in HGPS and natural aging. Recent studies showed that epigenetic systems could play an active role as drivers of both forms of aging. It may be suggested that these systems translate the effects of various internal and external factors into universal molecular hallmarks, largely common between natural and accelerated forms of aging. Drugs acting at both natural aging and HGPS are likely to exist. For example, vitamin D3 reduces the progerin production and alleviates most HGPS features, and also slows down epigenetic aging in overweight and obese non-HGPS individuals with suboptimal vitamin D status.

ANGUSTIFOLIA Regulates Actin Filament Alignment for Nuclear Positioning in Leaves.

During dark adaptation, plant nuclei move centripetally toward the midplane of the leaf blade; thus, the nuclei on both the adaxial and abaxial sides become positioned at the inner periclinal walls of cells. This centripetal nuclear positioning implies that a characteristic cell polarity exists within a leaf, but little is known about the mechanism underlying this process. Here, we show that ANGUSTIFOLIA (AN) and ACTIN7 regulate centripetal nuclear positioning in Arabidopsis (Arabidopsis thaliana) leaves. Two mutants defective in the positioning of nuclei in the dark were isolated and designated as unusual nuclear positioning1 (unp1) and unp2 In the dark, nuclei of unp1 were positioned at the anticlinal walls of adaxial and abaxial mesophyll cells and abaxial pavement cells, whereas the nuclei of unp2 were positioned at the anticlinal walls of mesophyll and pavement cells on both the adaxial and abaxial sides. unp1 was caused by a dominant-negative mutation in ACTIN7, and unp2 resulted from a recessive mutation in AN Actin filaments in unp1 were fragmented and reduced in number, which led to pleiotropic defects in nuclear morphology, cytoplasmic streaming, and plant growth. The mutation in AN caused aberrant positioning of nuclei-associated actin filaments at the anticlinal walls. AN was detected in the cytosol, where it interacted physically with plant-specific dual-specificity tyrosine phosphorylation-regulated kinases (DYRKPs) and itself. The DYRK inhibitor (1Z)-1-(3-ethyl-5-hydroxy-2(3H)-benzothiazolylidene)-2-propanone significantly inhibited dark-induced nuclear positioning. Collectively, these results suggest that the AN-DYRKP complex regulates the alignment of actin filaments during centripetal nuclear positioning in leaf cells.

Mapping the contact surfaces in the Lamin A:AIMP3 complex by hydrogen/deuterium exchange FT-ICR mass spectrometry.

Aminoacyl-tRNA synthetases-interacting multifunctional protein3 (AIMP3/p18) is involved in the macromolecular tRNA synthetase complex via its interaction with several aminoacyl-tRNA synthetases. Recent reports reveal a novel function of AIMP3 as a tumor suppressor by accelerating cellular senescence and causing defects in nuclear morphology. AIMP3 specifically mediates degradation of mature Lamin A (LmnA), a major component of the nuclear envelope matrix; however, the mechanism of how AIMP3 interacts with LmnA is unclear. Here we report solution-phase hydrogen/deuterium exchange (HDX) for AIMP3, LmnA, and AIMP3 in association with the LmnA C-terminus. Reversed-phase LC coupled with LTQ 14.5 T Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) results in high mass accuracy and resolving power for comparing the D-uptake profiles for AIMP3, LmnA, and their complex. The results show that the AIMP3-LmnA interaction involves one of the two putative binding sites and an adjacent novel interface on AIMP3. LmnA binds AIMP3 via its extreme C-terminus. Together these findings provide a structural insight for understanding the interaction between AIMP3 and LmnA in AIMP3 degradation.

Novel nesprin-1 mutations associated with dilated cardiomyopathy cause nuclear envelope disruption and defects in myogenesis.

Nesprins-1 and -2 are highly expressed in skeletal and cardiac muscle and together with SUN (Sad1p/UNC84)-domain containing proteins and lamin A/C form the LInker of Nucleoskeleton-and-Cytoskeleton (LINC) bridging complex at the nuclear envelope (NE). Mutations in nesprin-1/2 have previously been found in patients with autosomal dominant Emery–Dreifuss muscular dystrophy (EDMD) as well as dilated cardiomyopathy (DCM). In this study, three novel rare variants (R8272Q, S8381C and N8406K) in the C-terminus of the SYNE1 gene (nesprin-1) were identified in seven DCM patients by mutation screening. Expression of these mutants caused nuclear morphology defects and reduced lamin A/C and SUN2 staining at the NE. GST pull-down indicated that nesprin-1/lamin/SUN interactions were disrupted. Nesprin-1 mutations were also associated with augmented activation of the ERK pathway in vitro and in hearts in vivo. During C2C12 muscle cell differentiation, nesprin-1 levels are increased concomitantly with kinesin light chain (KLC-1/2) and immunoprecipitation and GST pull-down showed that these proteins interacted via a recently identified LEWD domain in the C-terminus of nesprin-1. Expression of nesprin-1 mutants in C2C12 cells caused defects in myoblast differentiation and fusion associated with dysregulation of myogenic transcription factors and disruption of the nesprin-1 and KLC-1/2 interaction at the outer nuclear membrane. Expression of nesprin-1α2 WT and mutants in zebrafish embryos caused heart developmental defects that varied in severity. These findings support a role for nesprin-1 in myogenesis and muscle disease, and uncover a novel mechanism whereby disruption of the LINC complex may contribute to the pathogenesis of DCM.

LEM2 recruits CHMP7 for ESCRT-mediated nuclear envelope closure in fission yeast and human cells

Endosomal sorting complexes required for transport III (ESCRT-III) proteins have been implicated in sealing the nuclear envelope in mammals, spindle pole body dynamics in fission yeast, and surveillance of defective nuclear pore complexes in budding yeast. Here, we report that Lem2p (LEM2), a member of the LEM (Lap2-Emerin-Man1) family of inner nuclear membrane proteins, and the ESCRT-II/ESCRT-III hybrid protein Cmp7p (CHMP7), work together to recruit additional ESCRT-III proteins to holes in the nuclear membrane. In Schizosaccharomyces pombe, deletion of the ATPase vps4 leads to severe defects in nuclear morphology and integrity. These phenotypes are suppressed by loss-of-function mutations that arise spontaneously in lem2 or cmp7, implying that these proteins may function upstream in the same pathway. Building on these genetic interactions, we explored the role of LEM2 during nuclear envelope reformation in human cells. We found that CHMP7 and LEM2 enrich at the same region of the chromatin disk periphery during this window of cell division and that CHMP7 can bind directly to the C-terminal domain of LEM2 in vitro. We further found that, during nuclear envelope formation, recruitment of the ESCRT factors CHMP7, CHMP2A, and IST1/CHMP8 all depend on LEM2 in human cells. We conclude that Lem2p/LEM2 is a conserved nuclear site-specific adaptor that recruits Cmp7p/CHMP7 and downstream ESCRT factors to the nuclear envelope.

167 The pathogenic implications of prelamin a accumulation in cardiomyocytes: a model of premature cardiac senescence?

Introduction Mutations in the LMNA gene, which encodes the nuclear intermediate filament proteins lamins A and C, lead to a number of premature ageing syndromes, including Hutchinson Gilford Progeria syndrome (HGPS) and Emery Dreifuss muscular dystrophy (EDMD) as well as dilated cardiomyopathy (DCM). Some causal mutations disrupt lamin A processing, resulting in the accumulation of the lamin A precursor, prelamin A, however it is not clear to what extent accumulated prelamin A contributes to DCM. Here we identify DCM patients with cardiomyocyte accumulation of prelamin A and explore the impact of prelamin A accumulation in vivo . Methods We generated a novel line of targeted transgenic mice that accumulate prelamin A specifically in cardiomyocytes (PLA Tg mice), by expressing an uncleavable form of prelamin A, driven by Cre expression from the myosin light chain 2 ventricular (MLC2v) promoter. Results Immunofluorescence staining of human DCM biopsies showed the presence of nuclear prelamin A in cardiomyocytes. PLA Tg mice were born without any obvious phenotype but manifested retarded growth by 3 weeks of age and succumbed to heart failure at ˜5 weeks. At 4 weeks, MRI showed a marked dilatation of the cardiac chambers and a decline in cardiac function in vivo . Ejection fraction (EF) was substantially depressed in PLA Tg micecompared with wildtype indicating DCM and heart failure. Cardiac histology showed marked cardiomyocyte disarray and profound fibrosis. Biochemical and microscopic analyses indicated disruption of the linkers of nucleoskeleton to cytoskeleton complex and perinuclear intermediate filament network and was supported by electron micrographs showing nuclear morphology defects. Myocardial infiltration of CD45 positive cells coincided with the expression of γ-H2AX, phosphorylated ATM and increased NF-κB signalling, which suggested an inflammatory response initiated by DNA damage. This may be related to the senescence associated secretory phenotype (SASP) as senescence associated β-galactosidase was also expressed in PLA Tg myocardium. Conclusion We have demonstrated prelamin A accumulation in the nuclei of human DCM biopsies and show that overexpression of prelamin A in a transgenic mouse model leads to an early decline in cardiac function and premature myocardial senescence.

Condensins exert force on chromatin-nuclear envelope tethers to mediate nucleoplasmic reticulum formation in Drosophila melanogaster.

Although the nuclear envelope is known primarily for its role as a boundary between the nucleus and cytoplasm in eukaryotes, it plays a vital and dynamic role in many cellular processes. Studies of nuclear structure have revealed tissue-specific changes in nuclear envelope architecture, suggesting that its three-dimensional structure contributes to its functionality. Despite the importance of the nuclear envelope, the factors that regulate and maintain nuclear envelope shape remain largely unexplored. The nuclear envelope makes extensive and dynamic interactions with the underlying chromatin. Given this inexorable link between chromatin and the nuclear envelope, it is possible that local and global chromatin organization reciprocally impact nuclear envelope form and function. In this study, we use Drosophila salivary glands to show that the three-dimensional structure of the nuclear envelope can be altered with condensin II-mediated chromatin condensation. Both naturally occurring and engineered chromatin-envelope interactions are sufficient to allow chromatin compaction forces to drive distortions of the nuclear envelope. Weakening of the nuclear lamina further enhanced envelope remodeling, suggesting that envelope structure is capable of counterbalancing chromatin compaction forces. Our experiments reveal that the nucleoplasmic reticulum is born of the nuclear envelope and remains dynamic in that they can be reabsorbed into the nuclear envelope. We propose a model where inner nuclear envelope-chromatin tethers allow interphase chromosome movements to change nuclear envelope morphology. Therefore, interphase chromatin compaction may be a normal mechanism that reorganizes nuclear architecture, while under pathological conditions, such as laminopathies, compaction forces may contribute to defects in nuclear morphology.

Nuclear localization of the dystrophin-associated protein α-dystrobrevin through importin α2/β1 is critical for interaction with the nuclear lamina/maintenance of nuclear integrity.

Although α-dystrobrevin (DB) is assembled into the dystrophin-associated protein complex, which is central to cytoskeletal organization, it has also been found in the nucleus. Here we delineate the nuclear import pathway responsible for nuclear targeting of α-DB for the first time, together with the importance of nuclear α-DB in determining nuclear morphology. We map key residues of the nuclear localization signal of α-DB within the zinc finger domain (ZZ) using various truncated versions of the protein, and site-directed mutagenesis. Pulldown, immunoprecipitation, and AlphaScreen assays showed that the importin (IMP) α2/β1 heterodimer interacts with high affinity with the ZZ domain of α-DB. In vitro nuclear import assays using antibodies to specific importins, as well as in vivo studies using siRNA or a dominant negative importin construct, confirmed the key role of IMPα2/β1 in α-DB nuclear translocation. Knockdown of α-DB expression perturbed cell cycle progression in C2C12 myoblasts, with decreased accumulation of cells in S phase and, significantly, altered localization of lamins A/C, B1, and B2 with accompanying gross nuclear morphology defects. Because α-DB interacts specifically with lamin B1 in vivo and in vitro, nuclear α-DB would appear to play a key role in nuclear shape maintenance through association with the nuclear lamina.

LITTLE NUCLEI 1 and 4 Regulate Nuclear Morphology in Arabidopsis thaliana

The morphology of plant nuclei varies among different species, organs, tissues and cell types. However, mechanisms and factors involved in the maintenance of nuclear morphology are poorly understood. Because nuclei retain their shapes even after cytoskeletal inhibitor treatments both in vivo and in vitro, we assumed involvement of the nuclear lamina, which plays a critical role in the regulation of nuclear morphology in animals. The crude nuclear lamina fraction isolated from Arabidopsis thaliana leaves was analyzed by mass spectrometry, and putative nuclear lamina proteins were identified. Among their T-DNA insertion lines, nuclei of little nuclei1 (linc1) and linc4 disruptants were more spherical than those of wild-type plants. Because A. thaliana harbors four LINC genes, we prepared all single and linc1/4 and linc2/3 double disruptants. In leaf epidermal cells, the circularity index of the nucleus in all linc disruptants except linc3 was significantly higher than that in the wild-type plants. The extent of the effects of LINC1 and/or LINC4 disruption was significantly higher than that of the effects of LINC2 disruption. The nuclear area was significantly smaller in the linc1, linc4 and linc1/4 disruptants than in the wild-type plants. Regardless of the defects in nuclear morphology, all linc disruptants exhibited a normal ploidy level. In interphase cells, LINC1 and LINC4 were mainly localized to the nuclear periphery, whereas LINC2 was in the nucleoplasm and LINC3 was detected in both regions. From prometaphase to anaphase in mitotic root tip cells, LINC1 was co-localized with chromosomes, whereas other LINCs were dispersed in the cytoplasm.

The role of Candida albicans homologous recombination factors Rad54 and Rdh54 in DNA damage sensitivity

BackgroundThe fungal pathogen Candida albicans is frequently seen in immune suppressed patients, and resistance to one of the most widely used antifungals, fluconazole (FLC), can evolve rapidly. In recent years it has become clear that plasticity of the Candida albicans genome contributes to drug resistance through loss of heterozygosity (LOH) at resistance genes and gross chromosomal rearrangements that amplify gene copy number of resistance associated genes. This study addresses the role of the homologous recombination factors Rad54 and Rdh54 in cell growth, DNA damage and FLC resistance in Candida albicans.ResultsThe data presented here support a role for homologous recombination in cell growth and DNA damage sensitivity, as Candida albicans rad54Δ/rad54Δ mutants were hypersensitive to MMS and menadione, and had an aberrant cell and nuclear morphology. The Candida albicans rad54Δ/rad54Δ mutant was defective in invasion of Spider agar, presumably due to the altered cellular morphology. In contrast, mutation of the related gene RDH54 did not contribute significantly to DNA damage resistance and cell growth, and deletion of either Candida albicans RAD54 or Candida albicans RDH54 did not alter FLC susceptibility.ConclusionsTogether, these results support a role for homologous recombination in genome stability under nondamaging conditions. The nuclear morphology defects in the rad54Δ/rad54Δ mutants show that Rad54 performs an essential role during mitotic growth and that in its absence, cells arrest in G2. The viability of the single mutant rad54Δ/rad54Δ and the inability to construct the double mutant rad54Δ/rad54Δ rdh54Δ/rdh54Δ suggests that Rdh54 can partially compensate for Rad54 during mitotic growth.

Downregulation of lamin A by tumor suppressor AIMP3/p18 leads to a progeroid phenotype in mice

Although AIMP3/p18 is normally associated with the macromolecular tRNA synthetase complex, recent reports have revealed a new role of AIMP3 in tumor suppression. In this study, we generated a transgenic mouse that overexpresses AIMP3 and characterized the associated phenotype in vivo and in vitro. Surprisingly, the AIMP3 transgenic mouse exhibited a progeroid phenotype, and the cells that overexpressed AIMP3 showed accelerated senescence and defects in nuclear morphology. We found that overexpression of AIMP3 resulted in proteasome-dependent degradation of mature lamin A, but not of lamin C, prelamin A, or progerin. The resulting imbalance in the protein levels of lamin A isoforms, namely altered stoichiometry of prelamin A and progerin to lamin A, appeared to be responsible for a phenotype that resembled progeria. An increase in the level of endogenous AIMP3 has been observed in aged human tissues and cells. The findings in this report suggest that AIMP3 is a specific regulator of mature lamin A and imply that enhanced expression of AIMP3 might be a factor driving cellular and/or organismal aging.