Nuclear Membrane Protein Lamin Research Articles

Phosphorylation Promotes the Accumulation of PERIOD Protein Foci.

Circadian clock drives the 24-h rhythm in our behavior and physiology. The molecular clock consists of a series of transcriptional/translational feedback loops operated by a number of clock genes. A very recent study reported that the clock protein PERIOD (PER) is organized into discrete foci at the nuclear envelope in fly circadian neurons, which is believed to be important for controlling the subcellular localization of clock genes. Loss of inner nuclear membrane protein lamin B receptor (LBR) leads to disruption of these foci, but how they are regulated is yet unknown. Here, we found that PER foci are likely phase-separated condensates, the formation of which is mediated by intrinsically disordered region in PER. Phosphorylation promotes the accumulation of these foci. Protein phosphatase 2A, which is known to dephosphorylate PER, hampers the accumulation of the foci. On the other hand, the circadian kinase DOUBLETIME (DBT) which phosphorylates PER enhances the accumulation of the foci. LBR likely facilitates PER foci accumulation by destabilizing the catalytic subunit of protein phosphatase 2A, MICROTUBULE STAR (MTS). In conclusion, here, we demonstrate a key role for phosphorylation in promoting the accumulation of PER foci, while LBR modulates this process by impinging on the circadian phosphatase MTS.

Exosomes as Key Regulators of Neutrophil Chemotaxis

Neutrophils, the most abundant leukocytes in human blood, are the first line of defense at sites of injury or infection. Upon activation by primary chemoattractants such as the formyl peptide N‐Formyl‐methionine‐leucyl‐phenylalanine (fMLF), neutrophils secrete the secondary chemoattractant leukotriene B4 (LTB4) which serves to relay primary chemotactic signals and increase the range and persistence of migration. LTB4 is synthesized from arachidonic acid (AA) through the sequential action of the 5‐lipoxygenase (5‐LO), its associated activating protein (FLAP) and leukotriene A4 hydrolase (LTA4H). We previously demonstrated that LTB4 and its synthesizing enzymes are packaged and released in extracellular vesicles called exosomes. Conventional exosome biogenesis originates from the plasma membrane. However, the LTB4 synthesizing machinery resides on the nuclear envelope (NE). We found that the localized activation of membrane curvature inducing proteins facilitates the selective sorting of the LTB4‐synthesis machinery on the NE and its packaging within NE‐derived exosomes. We show that chemoattractant stimulation leads to the generation of 5‐LO‐ and FLAP‐positive NE buds and cytosolic vesicles, which are also positive for the inner nuclear membrane protein lamin B receptor (LBR). Furthermore, we found that the generation of ceramide from sphingomyelin through the action of the neutral sphingomyelinase 1 (nSMase1) is essential for the generation of these NE buds and cytosolic vesicles by generating ordered lipid microdomains on the NE. Together, our findings show that the spatiotemporal orchestration of chemoattractant‐initiated events leads to the recruitment of the LTB4 biogenesis machinery on the NE followed by NE‐budding and subsequent release of cytosolic vesicles containing exosomes. We envision that this novel mechanism will represent an important means of exosome biogenesis for the release of NE‐associated components in other cells, including immune cells and invasive cancer cells.

Spatial distribution of lamin A/C determines nuclear stiffness and stress-mediated deformation.

ABSTRACTWhile diverse cellular components have been identified as mechanotransduction elements, the deformation of the nucleus itself is a critical mechanosensory mechanism, implying that nuclear stiffness is essential in determining responses to intracellular and extracellular stresses. Although the nuclear membrane protein lamin A/C is known to contribute to nuclear stiffness, bulk moduli of nuclei have not been reported for various levels of lamin A/C. Here, we measure the nuclear bulk moduli as a function of lamin A/C expression and applied osmotic stress, revealing a linear dependence within the range of 2–4 MPa. We also find that the nuclear compression is anisotropic, with the vertical axis of the nucleus being more compliant than the minor and major axes in the substrate plane. We then related the spatial distribution of lamin A/C with submicron 3D nuclear envelope deformation, revealing that local areas of the nuclear envelope with higher density of lamin A/C have correspondingly lower local deformations. These findings describe the complex dispersion of nuclear deformations as a function of lamin A/C expression and distribution, implicating a lamin A/C role in mechanotransduction.This article has an associated First Person interview with the first author of the paper.

Nuclear softening is essential for protease-independent migration

During amoeboidal migration, cancer cells migrate in a protease-independent manner by squeezing through pre-existing gaps in the extracellular matrix (ECM). However, the extent to which cells alter their physical properties in order to sustain this mode of migration remains unclear. Here, we address this question by documenting biophysical changes in the properties of highly invasive MDA-MB-231 and HT-1080 cells upon inhibition of pericellular proteolysis. Remarkably, treatment with the broad spectrum MMP inhibitor GM6001 not only induces cell rounding and loss of actomyosin contractility, but also induces nuclear softening via increased phosphorylation of the nuclear membrane protein lamin A/C. Though nuclear softening is necessary for sustaining migration through sub-nuclear sized transwell pores, it is not sufficient. In addition, baseline levels of contractility mediating pore entry and peri-nuclear actin inside the pores mediating pore migration are also required. Taken together, our results suggest that protease-independent migration through sub-nuclear sized pre-existing tracks is enabled by deformation of a softened nucleus by contractility and the peri-nuclear actin network.

Lamin A/C might be involved in the EMT signalling pathway

We have previously reported a heterogeneous expression pattern of the nuclear membrane protein lamin A/C in low- and high-Gleason score (GS) prostate cancer (PC) tissues, and we have now found that this change is not associated with LMNA mutations. This expression pattern appears to be similar to the process of epithelial to mesenchymal transition (EMT) or to that of mesenchymal to epithelial transition (MET). The role of lamin A/C in EMT or MET in PC remains unclear. Therefore, we first investigated the expression levels of and the associations between lamin A/C and several common EMT markers, such as E-cadherin, N-cadherin, β-catenin, snail, slug and vimentin in PC tissues with different GS values and in different cell lines with varying invasion abilities. Our results suggest that lamin A/C might constitute a type of epithelial marker that better signifies EMT and MET in PC tissue, since a decrease in lamin A/C expression in GS 4 + 5 cases is likely associated with the EMT process, while the re-expression of lamin A/C in GS 5 + 4 cases is likely linked with MET. The detailed GS better exhibited the changes in lamin A/C and the EMT markers examined. Lamin A/C overexpression or knockdown had an impact on EMT biomarkers in a cell model by direct regulation of β-catenin. Hence, we suggest that lamin A/C might serve as a reliable epithelial biomarker for the distinction of PC cell differentiation and might also be a fundamental factor in the occurrence of EMT or MET in PC.

Suppression of Activated FOXO Transcription Factors in the Heart Prolongs Survival in a Mouse Model of Laminopathies.

Mutations in the LMNA gene, encoding nuclear inner membrane protein lamin A/C, cause distinct phenotypes, collectively referred to as laminopathies. Heart failure, conduction defects, and arrhythmias are the common causes of death in laminopathies. The objective of this study was to identify and therapeutically target the responsible mechanism(s) for cardiac phenotype in laminopathies. Whole-heart RNA sequencing was performed before the onset of cardiac dysfunction in the Lmna-/- and matched control mice. Differentially expressed transcripts and their upstream regulators were identified, validated, and targeted by adeno-associated virus serotype 9-short hairpin RNA constructs. A total of 576 transcripts were upregulated and 233 were downregulated in the Lmna-/- mouse hearts (q<0.05). Forkhead box O (FOXO) transcription factors (TFs) were the most activated while E2 factors were the most suppressed transcriptional regulators. Transcript levels of FOXO targets were also upregulated in the isolated Lmna-/- cardiac myocytes and in the myocardium of human heart failure patients. Nuclear localization of FOXO1 and 3 was increased, whereas phosphorylated (inactive) FOXO1 and 3 levels were reduced in the Lmna-/- hearts. Gene set enrichment analysis and gene ontology showed activation of apoptosis and inflammation and suppression of cell cycle, adipogenesis, and oxidative phosphorylation in the Lmna-/- hearts. Adeno-associated virus serotype 9-short hairpin RNA-mediated suppression of FOXO TFs rescued selected molecular signatures, improved apoptosis, and prolonged survival by ≈2-fold. FOXO TFs are activated and contribute to the pathogenesis of cardiac phenotype in laminopathies. Suppression of the FOXO TFs in cardiac myocytes partially rescues the phenotype and prolongs survival. The findings identify FOXO TFs as potential therapeutic targets for cardiac phenotype in laminopathies.

STEM-29. LAMINA ORGANIZATION PROMOTES MAINTENANCE OF BRAIN TUMOR INITIATING CELLS IN GLIOMA

AbstractGlioblastoma is one of the most aggressive tumors, containing self-renewing glioma stem cells (GSCs) at the apex of the tumor hierarchy that drive tumor growth. Global chromatin regulation contributes to development of the cellular hierarchy, suggesting that targeting chromatin regulators may disrupt GSCs. Nuclear peripheral anchoring of heterochromatin is mainly mediated by interactions with lamina structure Lamin A/C, Lamin B, and the inner nuclear membrane protein Lamin B Receptor (LBR). LBR mediates peripheral localization at early stages of development, and then Lamin A/C assumes tethering during terminal differentiation. Here, we report that GSCs preferentially express LBR at both the transcriptional and translational levels, whereas differentiated progeny preferentially express Lamin A/C, phenocopying developmental regulation. Interrogation of chromatin marks of active enhancer and promoters through histone 3 lysine 27 acetyl (H3K27ac) ChIP-Seq revealed stem cell specific activation peaks at LBR locus. Targeting LBR expression by RNA interference potently reduced GSC cellular proliferation, self-renewal in vitro, and tumor growth in vivo. Leveraging an in silico discovery approach based on promoter sequence analysis, we found that GABPB1 mediates upstream transcriptional regulation of LBR in GSCs. We are currently defining the specific anchored chromatin domains at the nuclear peripheral tethered by LBR required for GSC maintenance to better understand the lamina organization dependency in glioblastoma, potentially informing the development of novel targeted therapeutics.

Nuclear envelope localization of LEMD2 is developmentally dynamic and lamin A/C dependent yet insufficient for heterochromatin tethering

Peripheral heterochromatin in mammalian nuclei is tethered to the nuclear envelope by at least two mechanisms here referred to as the A- and B-tethers. The A-tether includes lamins A/C and additional unknown components presumably INM protein(s) interacting with both lamins A/C and chromatin. The B-tether includes the inner nuclear membrane (INM) protein Lamin B-receptor, which binds B-type lamins and chromatin. Generally, at least one of the tethers is always present in the nuclear envelope of mammalian cells. Deletion of both causes the loss of peripheral heterochromatin and consequently inversion of the entire nuclear architecture, with this occurring naturally in rod photoreceptors of nocturnal mammals. The tethers are differentially utilized during development, regulate gene expression in opposite manners, and play an important role during cell differentiation. Here we aimed to identify the unknown chromatin binding component(s) of the A-tether. We analyzed 10 mouse tissues by immunostaining with antibodies against 7 INM proteins and found that every cell type has specific, although differentially and developmentally regulated, sets of these proteins. In particular, we found that INM protein LEMD2 is concomitantly expressed with A-type lamins in various cell types but is lacking in inverted nuclei of rod cells. Truncation or deletion of Lmna resulted in the downregulation and mislocalization of LEMD2, suggesting that the two proteins interact and pointing at LEMD2 as a potential chromatin binding mediator of the A-tether. Using nuclei of mouse rods as an experimental model lacking peripheral heterochromatin, we expressed a LEMD2 transgene alone or in combination with lamin C in these cells and observed no restoration of peripheral heterochromatin in either case. We conclude that in contrary to the B-tether, the A-tether has a more intricate composition and consists of multiple components that presumably vary, at differing degrees of redundancy, between cell types and differentiation stages.

Alterations in nuclear structure promote lupus autoimmunity in a mouse model.

ABSTRACTSystemic lupus erythematosus (SLE) is an autoimmune disorder characterized by the development of autoantibodies that recognize components of the cell nucleus. The vast majority of lupus research has focused on either the contributions of immune cell dysfunction or the genetics of the disease. Because granulocytes isolated from human SLE patients had alterations in neutrophil nuclear morphology that resembled the Pelger–Huet anomaly, and had prominent mis-splicing of mRNA encoding the nuclear membrane protein lamin B receptor (LBR), consistent with their Pelger–Huet-like nuclear morphology, we used a novel mouse model system to test the hypothesis that a disruption in the structure of the nucleus itself also contributes to the development of lupus autoimmunity. The lupus-prone mouse strain New Zealand White (NZW) was crossed with c57Bl/6 mice harboring a heterozygous autosomal dominant mutation in Lbr (B6.Lbric/+), and the (NZW×B6.Lbric)F1 offspring were evaluated for induction of lupus autoimmunity. Only female (NZW×B6.Lbric)F1 mice developed lupus autoimmunity, which included splenomegaly, kidney damage and autoantibodies. Kidney damage was accompanied by immune complex deposition, and perivascular and tubule infiltration of mononuclear cells. The titers of anti-chromatin antibodies exceeded those of aged female MRL-Faslpr mice, and were predominantly of the IgG2 subclasses. The anti-nuclear antibody staining profile of female (NZW×B6.Lbric)F1 sera was complex, and consisted of an anti-nuclear membrane reactivity that colocalized with the A-type lamina, in combination with a homogeneous pattern that was related to the recognition of histones with covalent modifications that are associated with gene activation. An anti-neutrophil IgM recognizing calreticulin, but not myeloperoxidase (MPO) or proteinase 3 (PR3), was also identified. Thus, alterations in nuclear structure contribute to lupus autoimmunity when expressed in the context of a lupus-prone genetic background, suggesting a mechanism for the development of lupus autoimmunity in genetically predisposed individuals that is induced by the disruption of nuclear architecture.

Nuclear localization of bradykinin B2 receptors reflects binding to the nuclear envelope protein lamin C

The mechanism of action of bradykinin (BK), a pro-inflammatory mediator, is thought to be mediated by specific cell surface membrane bradykinin B2 receptors. Some evidence suggests that there are both intracellular and nuclear bradykinin B2 receptors. This study identified proteins that interact with the C-terminus of the bradykinin B2 receptor (in particular, the nuclear membrane protein lamin C), using the yeast two-hybrid system. The motif of the C-terminal domain (CT) mutant 303–320 in bradykinin B2 receptor was identified as a lamin C protein binding motif. Immunohistochemistry revealed colocalization of FLAG- bradykinin B2 receptor with HA-lamin C in the nucleus of HEK 293T cells. In situ proximity ligation assay (PLA) showed that FLAG-bradykinin B2 receptor formed heterodimers with HA-lamin C in the nucleus. In addition, live cell fluorescence imaging showed that bradykinin B2 receptor-EGFP was located in the nucleus and co-localized with HcRed-lamin C. Interestingly, neither BK addition nor bradykinin B2 receptor CT mutation reduced the binding to lamin C or changed the distribution of bradykinin B2 receptor. Taken together, these findings demonstrate that bradykinin B2 receptor-lamin C heterodimers form in the nucleus independent of BK stimulation and CT mutation. We propose that heterodimerization of bradykinin B2 receptor with lamin C is essential to nuclear localization of bradykinin B2 receptor and plays an important role in cell signaling and function.

The N and C Termini of ZO-1 Are Surrounded by Distinct Proteins and Functional Protein Networks

Biotin ligase tagging with ZO-1 was applied to identify a more complete tight junction proteome. Identical but also different proteins and functional networks were identified near the N and C ends of ZO-1. The ends of ZO-1 are embedded in different functional subcompartments of the tight junction. Biotin tagging with ZO-1 expands the tight junction proteome and defines subcompartments of the junction. The proteins and functional protein networks of the tight junction remain incompletely defined. Among the currently known proteins are barrier-forming proteins like occludin and the claudin family; scaffolding proteins like ZO-1; and some cytoskeletal, signaling, and cell polarity proteins. To define a more complete list of proteins and infer their functional implications, we identified the proteins that are within molecular dimensions of ZO-1 by fusing biotin ligase to either its N or C terminus, expressing these fusion proteins in Madin-Darby canine kidney epithelial cells, and purifying and identifying the resulting biotinylated proteins by mass spectrometry. Of a predicted proteome of ∼9000, we identified more than 400 proteins tagged by biotin ligase fused to ZO-1, with both identical and distinct proteins near the N- and C-terminal ends. Those proximal to the N terminus were enriched in transmembrane tight junction proteins, and those proximal to the C terminus were enriched in cytoskeletal proteins. We also identified many unexpected but easily rationalized proteins and verified partial colocalization of three of these proteins with ZO-1 as examples. In addition, functional networks of interacting proteins were tagged, such as the basolateral but not apical polarity network. These results provide a rich inventory of proteins and potential novel insights into functions and protein networks that should catalyze further understanding of tight junction biology. Unexpectedly, the technique demonstrates high spatial resolution, which could be generally applied to defining other subcellular protein compartmentalization.

DIDS (4,4-diisothiocyanatostilbenedisulphonic acid) induces apoptotic cell death in a hippocampal neuronal cell line and is not neuroprotective against ischemic stress.

DIDS is a commonly used anion channel antagonist that is putatively cytoprotective against ischemic insult. However, recent reports indicate potentially deleterious secondary effects of DIDS. To assess the impact of DIDS on cellular viability comprehensively we examined neuronal morphology and function through 24 hours treatment with ACSF ± DIDS (40 or 400 µM). Control cells were unchanged, whereas DIDS induced an apoptotic phenotype (chromatin condensation, nuclear fragmentation and cleavage of the nuclear membrane protein lamin A, expression of pro-apoptotic proteins c-Jun N-terminal kinase 3, caspase 3, and cytochrome C, Annexin V staining, RNA degradation, and oligonucleosomal DNA cleavage). These deleterious effects were mediated by DIDS in a dose- and time-dependant manner, such that higher [DIDS] induced apoptosis more rapidly while apoptosis was observed at lower [DIDS] with prolonged exposure. In an apparent paradox, despite a clear overall apoptotic phenotype, certain hallmarks of apoptosis were not present in DIDS treated cells, including mitochondrial fission and loss of plasma membrane integrity. We conclude that DIDS induces apoptosis in cultured hippocampal neurons, in spite of the fact that some common hallmarks of cell death pathways are prevented. These contradictory effects may cause false-positive results in certain assays and future evaluations of DIDS as a neuroprotective agent should incorporate multiple viability assays.

Temporal Control of Nuclear Envelope Assembly by Phosphorylation of Lamin B Receptor

The nuclear envelope of metazoans disassembles during mitosis and reforms in late anaphase after sister chromatids have well separated. The coordination of these mitotic events is important for genome stability, yet the temporal control of nuclear envelope reassembly is unknown. Although the steps of nuclear formation have been extensively studied in vitro using the reconstitution system from egg extracts, the temporal control can only be studied in vivo. Herein, we use time‐lapse microscopy to investigate this process in living HeLa cells. We demonstrate that Cdk1 activity prevents premature nuclear envelope assembly and that phosphorylation of the inner nuclear membrane protein lamin B receptor (LBR) by Cdk1 contributes to the temporal control. We further identify a region in the nucleoplasmic domain of LBR that inhibits premature chromatin binding of the protein. We propose that this inhibitory effect is partly mediated by Cdk1 phosphorylation. Furthermore, we show that the reduced chromatin‐binding ability of LBR together with Aurora B activity contribute to nuclear envelope breakdown. Our studies reveal for the first time a mechanism that controls the timing of nuclear envelope reassembly through modification of an integral nuclear membrane protein.

Lamin A/C protein is overexpressed in tissue-invading prostate cancer and promotes prostate cancer cell growth, migration and invasion through the PI3K/AKT/PTEN pathway

Prostate cancer (PC) remains the second most common cause of cancer-related death in Western countries. A previous proteomics study suggested that the nuclear membrane protein lamin A/C to be a maker to discriminate low- and high-Gleason score tumors and to identify high-risk cancers. To characterize its function in PC cells, we performed a detailed expression analysis in PC tissue and explored the consequences of down or upregulation of lamin A/C in PC cells. Our results confirm an increased lamin A/C protein expression in high-risk cancers and show association of expression with tumor cell formations at the invasion fronts of tumors and in invasion 'spearheading' tumor cell clusters. In the prostate tumor cell lines, LNCaP, DU145, and PC3 small hairpin RNA knockdown or overexpression of lamin A/C resulted in inhibition or stimulation, respectively, of cell growth, colony formation, migration and invasion. Further mechanism studies suggested that the lamin A/C-related malignant behavior is regulated through modulation of the phosphoinositide 3-kinase (PI3K)/AKT/PTEN signaling pathway. Western blot results indicated that knockdown or overexpression of lamin A/C decreased or increased, respectively, protein levels of the PI3K subunits p110 and p85 in all three cell lines; phosphor-AKT in the PTEN-negative cell lines LNCaP and PC3, and, increased or decreased, respectively, PTEN protein levels in PTEN-positive DU145 cells. Together, our data suggest that lamin A/C proteins are positively involved in malignant behavior of PC cells through the PI3K/AKT/PTEN pathway. Lamin A/C may represent a new oncogenic factor and a novel therapeutic target for PC.

Temporal control of nuclear envelope assembly by phosphorylation of lamin B receptor

The nuclear envelope of metazoans disassembles during mitosis and reforms in late anaphase after sister chromatids have well separated. The coordination of these mitotic events is important for genome stability, yet the temporal control of nuclear envelope reassembly is unknown. Although the steps of nuclear formation have been extensively studied in vitro using the reconstitution system from egg extracts, the temporal control can only be studied in vivo. Here, we use time-lapse microscopy to investigate this process in living HeLa cells. We demonstrate that Cdk1 activity prevents premature nuclear envelope assembly and that phosphorylation of the inner nuclear membrane protein lamin B receptor (LBR) by Cdk1 contributes to the temporal control. We further identify a region in the nucleoplasmic domain of LBR that inhibits premature chromatin binding of the protein. We propose that this inhibitory effect is partly mediated by Cdk1 phosphorylation. Furthermore, we show that the reduced chromatin-binding ability of LBR together with Aurora B activity contributes to nuclear envelope breakdown. Our studies reveal for the first time a mechanism that controls the timing of nuclear envelope reassembly through modification of an integral nuclear membrane protein.

Clinical Features and Genetic Basis of Progressive Cardiac Conduction Defect: Japanese PCCD Registry

Progressive cardiac conduction disturbance (PCCD) is a hereditary cardiac bundle branch disorder that often processes to complete heart block. We evaluated the clinical features and genetic basis in 61 individuals from 44 unrelated families affected by PCCD. Individuals with structural heart abnormalities at the time of diagnosis were excluded. Despite the assumption that PCCD predominates in elderly population, the age of onset showed a wide-range distribution (6–95 years). Conduction disturbance was progressively aggravated in 29 individuals (48%) and the cardiac dysfunction was manifested in 17 individuals (28%). Pacemaker or ICD was implanted in 43 individuals (70%). However, 3 patients who received pacemaker or ICD and 3 individuals who declined the device therapy died suddenly. Genetic screening revealed mutations in cardiac ion channel genes: SCN5A (n=6), KCNH2 (n=1), and GJA5 (n=1) encoding a gap junction isoform connexin 40 predominantly expressed in the conduction system. Two probands were heterozygous mutation carries. Moreover, the screening identified mutations in LMNA (n=8), the most prevalent causative genes of dilated cardiomyopathy that encodes an inner nuclear membrane protein lamin A/C. Although mutations remain unknown in most cases (n=38, 62%), mutation carries of SCN5A (57%) and LMNA (55%) progressively manifested heart failure. In conclusion, clinical features and genetic basis underlying PCCD are heterogenous.

Identification of mutational hot spots in LMNA encoding lamin A/C in patients with familial dilated cardiomyopathy

The familial form of dilated cardiomyopathy (DCM) occurs in about 20%-50% of DCM cases. It is a heterogeneous genetic disease: mutations in more than 20 different genes have been shown to cause familial DCM. LMNA, encoding the nuclear membrane protein lamin A/C, is one of the most important disease gene for that disease. Therefore, we analyzed the LMNA gene in a large cohort of 73 patients with familial DCM. Clinical examination (ECG, echocardiography, and catheterization) was followed by genetic characterization of LMNA by direct sequencing. We detected five heterozygous missense mutations (prevalence 7%) in five different families characterized by severe DCM and heart failure with conduction system disease necessitating pacemaker implantation and heart transplantation. Four of these variants clustered in the protein domain coil 1B, which is important for lamin B interaction and lamin A/C dimerization. Although we identified two novel mutations (E203V, K219T) besides three known ones (E161K, R190Q, R644C), it was remarkable that four mutations represent LMNA hot spots. DCM patients with LMNA mutations show a notable homogenous severe phenotype as we could confirm in our study. Testing LMNA in such families seems to be recommended because genotype information in an individual could definitely be useful for the clinician.

Association of Nuclear Membrane Protein Lamin B1 with Necrosis and Apoptosis in Cell Death Induced by 5-Fluoro-2′-Deoxyuridine

We report that anticancer 5-fluoro-2 ′-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells was performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FUdR-treated original F28-7. Thus, the swelling feature for the necrosis was no longer observable, and instead cell shrinkage typical of apoptosis took place in almost all the cells examined. This finding suggests a new role for lamin B1 as a regulator in cell death.

Myofiber degeneration in autosomal‐dominant Emery‐Dreifuss muscular dystrophy (AD‐EDMD) (LGMD1B)

Autosomal dominant Emery-Dreifuss muscular dystrophy is caused by mutations in the LMNA gene that code for the nuclear membrane protein lamin A/C. We investigated skeletal muscle fibers from several muscles for cytoplasmic degenerative changes in three patients with genetically confirmed Emery-Dreifuss muscular dystrophy. Methods included quantitative light and electron microscopy and PCR-based mutational analysis. The degenerative pathway was characterized by the gradual replacement of individual myofibers by connective tissue. Early stages of degeneration typically involved only a segment of the cross-sectional area of a myofiber. Intermediate stages consisted of myofiber shrinkage due to “shedding” of peripheral cytoplasmic portions into the endomysial space, and fragmentation of the myofibers by interposed collagen fibrils. Empty basement membrane sheaths surrounded by abundant deposits of extracellular matrix marked the end stage of the degenerative process. The pattern of degeneration described herein differs from muscular dystrophies with plasma membrane defects (dystrophinopathy, dysferlinopathy) and explains the frequently found absence of highly elevated serum creatine kinase levels in autosomal dominant Emery-Dreifuss muscular dystrophy.

Myofiber Degeneration in Autosomal Dominant Emery–Dreifuss Muscular Dystrophy (AD‐EDMD) (LGMD1B)

Autosomal dominant Emery-Dreifuss muscular dystrophy is caused by mutations in the LMNA gene that code for the nuclear membrane protein lamin A/C. We investigated skeletal muscle fibers from several muscles for cytoplasmic degenerative changes in three patients with genetically confirmed Emery-Dreifuss muscular dystrophy. Methods included quantitative light and electron microscopy and PCR-based mutational analysis. The degenerative pathway was characterized by the gradual replacement of individual myofibers by connective tissue. Early stages of degeneration typically involved only a segment of the cross-sectional area of a myofiber. Intermediate stages consisted of myofiber shrinkage due to "shedding" of peripheral cytoplasmic portions into the endomysial space, and fragmentation of the myofibers by interposed collagen fibrils. Empty basement membrane sheaths surrounded by abundant deposits of extracellular matrix marked the end stage of the degenerative process. The nuclear number-to-cytoplasmic area in myofibers of one patient increased with increasing cross-sectional area, suggesting that satellite cell fusion with myofibers may have compensated for myofiber shrinkage. The pattern of degeneration described herein differs from muscular dystrophies with plasma membrane defects (dystrophinopathy, dysferlinopathy) and explains the frequently found absence of highly elevated serum creatine kinase levels in autosomal dominant Emery-Dreifuss muscular dystrophy.