Role Of Lamin Research Articles

Lamins in development, tissue maintenance and stress

Lamins are nuclear intermediate filament proteins. They provide mechanical stability, organize chromatin and regulate transcription, replication, nuclear assembly and nuclear positioning. Recent studies provide new insights into the role of lamins in development, differentiation and tissue response to mechanical, reactive oxygen species and thermal stresses. These studies also propose the existence of separate filament networks for A- and B-type lamins and identify new roles for the different networks. Furthermore, they show changes in lamin composition in different cell types, propose explanations for the more than 14 distinct human diseases caused by lamin A and lamin C mutations and propose a role for lamin B1 in these diseases.

Suppression of lamin A/C by short hairpin RNAs promotes adipocyte lineage commitment in mesenchymal progenitor cell line, ROB-C26

Lamin A/C gene encodes a nuclear membrane protein, and mutations in this gene are associated with diverse degenerative diseases that are linked to premature aging. While lamin A/C is involved in the regulation of tissue homeostasis, the distinct expression patterns are poorly understood in the mesenchymal cells differentiating into adipocytes. Here, we examined the expression of lamin A/C in a rat mesenchymal progenitor cell-line, ROB-C26 (C26). Immunocytochemical analysis showed that lamin A/C was transiently down-regulated in immature adipocytes, but its expression increased with terminal differentiation. To elucidate the role of lamin A/C expression on mesenchymal cell differentiation, lamin A/C expression was suppressed using short hairpin RNA (shRNA) molecules in C26 cells. In the absence of adipogenic stimuli, lamin A/C shRNA decreased alkaline phosphatase (ALP) activity, but induced preadipocyte factor -1 (Pref-1) mRNA expression. In the presence of adipogenic stimuli, lamin A/C knockdown promotes adipocytes differentiation, as assessed by the detection of an increase in Oil Red O staining. RT-PCR analysis showed that lamin A/C shRNA resulted in increased mRNA expression of PPARγ2 and aP2 during adipocyte differentiation. These results suggest that decreased lamin A/C expression levels not only suppress osteoblast phenotypes but also promote adipocyte differentiation in C26 cells.

The role of Lamin A in cytoskeleton organization in colorectal cancer cells

Up-regulated expression of lamin A has been implicated in increased cell invasiveness and mortality in colorectal cancer. Here we use quantitative proteomics to investigate lamin A regulated changes in the cytoskeleton that might underpin increased cell motility. Using siRNA knockdown of lamin A in a model cell line (SW480/lamA) we confirm that the presence of lamin A promotes cell motility. Using an enhanced technique to prepare cytoskeleton fractions in combination with 2D DiGE we were able to accurately and reproducibly detect changes in the representation of protein species within the cytoskeleton as low as 20%. In total 64 protein spots displayed either increased or decreased representation within the cytoskeleton of SW480/lamA cells compared to controls. Of these the identities of 29 spots were determined by mass spectrometry. A majority were multiple forms of three classes of proteins, including components of the actin and IF cytoskeletons, protein chaperones and translation initiation and elongation factors. In particular our data reveal that the representation of tissue transglutaminase 2, which is known to modify elements of the cytoskeleton and is associated with cancer progression, was highly over-represented in the cytoskeleton fraction of SW480/lamA cells. Overall, our data are consistent with changed protein cross-linking and folding that favours the formation of dynamic actin filaments over stress fibres accounting for the altered cell motility properties in SW480/lamA cells.

Lamins, laminopathies and disease mechanisms: Possible role for proteasomal degradation of key regulatory proteins

Lamins are major structural proteins of the nucleus and are essential for nuclear integrity and organization of nuclear functions. Mutations in the human lamin genes lead to highly degenerative genetic diseases that affect a number of different tissues such as muscle, adipose or neuronal tissues, or cause premature ageing syndromes. New findings on the role of lamins in cellular signalling pathways, as well as in ubiquitin-mediated proteasomal degradation, have given important insights into possible mechanisms of pathogenesis.

Reversal of age-dependent nuclear morphology by inhibition of prenylation does not affect lifespan in Caenorhabditis elegans

Fibroblasts derived from Hutchinson-Gilford progeria syndrome (HGPS) patients and dermal cells derived from healthy old humans in culture display age-dependent progressive changes in nuclear architecture due to accumulation of farnesylated lamin A. Treating human HGPS cells or mice expressing farnesylated lamin A with farnesyl transferase inhibitors (FTIs) reverses nuclear phenotypes and extends lifespan. Aging adult Caenorhabditis elegans show changes in nuclear architecture resembling those seen in HGPS fibroblasts, as well as a decline in motility, phenotypes which are also inhibited by the FTI gliotoxin. However, it was not clear whether these effects were due to loss of farnesylation or to side effects of this drug. Here, we used a different FTI, manumycin, or downregulated polyprenyl synthetase with RNAi to test the roles of farnesylation in C. elegans aging. Our results show that the age-dependent changes in nuclear morphology depend on farnesylation. We also demonstrate that inhibition of farnesylation does not affect motility or lifespan, suggesting that the effects of blocking protein prenylation on nuclear morphology could be separated from their effects on motility and lifespan. These results provide further understanding of the role of lamin and farnesylation in the normal aging process and in HGPS.

Muscular laminopathies: Role of prelamin A in early steps of muscle differentiation

Lamin A is a nuclear envelope constituent involved in a group of human disorders, collectively referred to as laminopathies, which include Emery-Dreifuss muscular dystrophy. Because increasing evidence suggests a role of lamin A precursor in nuclear functions, we investigated the processing of prelamin A along muscle differentiation. Both protein levels and cellular localization of prelamin A appears to be modulated during C2C12 mouse myoblasts activation. Similar changes also occur in the expression of two lamin A-binding proteins: emerin and LAP2α. Furthermore prelamin A forms a complex with LAP2α in differentiating myoblasts. Prelamin A accumulation in cycling myoblasts by expressing unprocessable mutants affects LAP2α and PCNA amount and increases caveolin 3 mRNA and protein levels, whilst accumulation of prelamin A in differentiated muscle cells following treatment with a farnesyl transferase inhibitor inhibits caveolin 3 expression. These data provide evidence for a critical role of lamin A precursor in the early steps of muscle cell differentiation. In fact the post-translational processing of prelamin A affects caveolin 3 expression and influences the myoblast differentiation process. Thus, altered lamin A processing could affect myoblast differentiation and/or muscle regeneration and might contribute to the myopathic phenotype.

The role of Lamin A/C mutations in Danish patients with idiopathic dilated cardiomyopathy

The role of Lamin A/C mutations in Danish patients with idiopathic dilated cardiomyopathy

Lamin A: a putative colonic epithelial stem cell biomarker which identifies colorectal tumours with a more aggressive phenotype

Abnormalities in the expression, distribution and structural organization of A-type lamins are most commonly associated with a spectrum of inherited disorders which predominantly affect mesenchymal lineages, collectively known as laminopathies. However, a new role for lamin A has been discovered in the progression of a common epithelial cancer. CRC (colorectal cancer) patients expressing lamin A/C in their tumour tissue were found to have a 2-fold greater risk of CRC-related mortality compared with patients with lamin A/C-negative tumours. Consequently, lamin A/C is a prognostic biomarker in CRC. In vitro studies suggest that lamin A is an upstream regulator of a pathway linking actin dynamics to loss of cell adhesion, leading to enhanced cell motility and consequently increased invasive potential within a tumour. The finding that lamin A is a putative colonic epithelial stem cell biomarker suggests that the poor outcome associated with lamin A/C-positive tumours may be reflective of a more stem-cell-like phenotype. The present review discusses the link between lamin A expression and tumour progression in one of the commonest causes of cancer-related death in the Western world.

The R527H mutation in LMNA gene causes an increased sensitivity to ionizing radiation

Mandibuloacral dysplasia type A (MADA; OMIM # 248370) is a premature ageing disease caused by the homozygous R527H mutation in the LMNA gene. At the cellular level, MADA is characterized by unprocessed prelamin A accumulation, nuclear architecture alterations, chromatin defects and increased incidence of apoptosis. In some progeroid laminopathies (e.g. HGPS) it has been demonstrated that such biochemical and morphological alterations are strongly linked with genomic instability. To test this also in MADA fibroblasts, their response to the ionising radiation- induced damage was analysed. We observed that their ability to repair the damage was significantly impaired, as demonstrated by the increased chromosome damage and the higher percentage of residual γ-H2AX foci, corresponding to unrepaired DNA-damage sites. Moreover, MADA fibroblasts showed a markedly reduced phosphorylation of p53 at Ser15(S15) and a lower induction of p53 and CDKN1A proteins after irradiation, compared to the control cell line. Upon irradiation, we also detected differences in the expression of some p53 downstream target genes. In addition, MADA cells showed partial defects in the checkpoint response, particularly in G1/S transition. Our results indicate that accumulation of the lamin A precursor protein determines a defect in DNA damage response after X-ray exposure, supporting a crucial role of lamin A in regulating DNA repair process and cell cycle control.

Alterations in mitosis and cell cycle progression caused by a mutant lamin A known to accelerate human aging

Mutations in the gene encoding nuclear lamin A (LA) cause the premature aging disease Hutchinson-Gilford Progeria Syndrome. The most common of these mutations results in the expression of a mutant LA, with a 50-aa deletion within its C terminus. In this study, we demonstrate that this deletion leads to a stable farnesylation and carboxymethylation of the mutant LA (LADelta50/progerin). These modifications cause an abnormal association of LADelta50/progerin with membranes during mitosis, which delays the onset and progression of cytokinesis. Furthermore, we demonstrate that the targeting of nuclear envelope/lamina components into daughter cell nuclei in early G(1) is impaired in cells expressing LADelta50/progerin. The mutant LA also appears to be responsible for defects in the retinoblastoma protein-mediated transition into S-phase, most likely by inhibiting the hyperphosphorylation of retinoblastoma protein by cyclin D1/cdk4. These results provide insights into the mechanisms responsible for premature aging and also shed light on the role of lamins in the normal process of human aging.

Les laminopathies : lipodystrophies, insulino-résistance, syndromes de vieillissement accéléré… et les autres

Laminopathies are a group of diseases due to mutations of type A-lamins, a group of proteins lining the inner aspect of cell nuclei. These diseases illustrate the complexity of the genotype-phenotype relationship characteristic of same genetic diseases. Since the discovery of the causal role of LMNA gene mutations in the genesis of Emery Dreifuss muscular dystrophy in 1999, no less than eight other diseases have been associated with mutations of this same gene! The tissue-specific nature of the clinical manifestations, contrasting with the ubiquitous expression of these proteins, has incited much research concerning the physiological role of lamins, considered to be much broader than the structural function initially put forward. Certain laminopathies, which combine insulin resistance, android distribution of adipose tissue, dyslipidemia, early atherosclerosis, and hepatic steatosis, appear very similar though more severe to the frequent dysmetabolism syndrome. The relationships of laminopathies with accelerated aging syndrome, Hutchinson-Gilford progeria, or progeroid syndromes, which are also related to A/C lamin anomalies, could provide new avenues of research on the pathogenesis of the metabolic syndrome. In addition, clinicians have to be aware of atypical and milder forms of laminopathies, that require specific investigations and molecular screening of relatives allowing an adequate medical management.

New roles for lamins, nuclear envelope proteins and actin in the nucleus

New roles for lamins, nuclear envelope proteins and actin in the nucleus

Colocalization of intranuclear lamin foci with RNA splicing factors.

The lamins form a fibrous network underlying the inner nuclear membrane termed the nuclear lamina. In order to gain insights into the role of lamins in nuclear organization, we have characterized a monoclonal antibody (LA-2H10) raised against recombinant rat lamin A that labels nuclei in a speckled pattern in all cells of unsynchronized populations of HeLa and rat F-111 fibroblast cells, unlike the typical nuclear periphery staining by another monoclonal antibody to lamin A, LA-2B3. In immunolocalization studies the lamin A speckles or foci were found to colocalize with the RNA splicing factors SC-35 and U5-116 kD, but not with p80 coilin found in coiled bodies. Lamin B1 was also associated with these foci. These foci dispersed when cells entered mitosis and reformed during anaphase. The differential reactivity of LA-2H10 and LA-2B3 was retained after nuclei were extracted with detergents, nucleases and salt to disrupt interactions of lamins with chromatin and other nuclear proteins. Using deletion fragments of recombinant lamin A, the epitope recognized by LA-2H10 was located between amino acids 171 and 246. Our findings are consistent with a structural role for lamins in supporting nuclear compartments containing proteins involved in RNA splicing.

Lamin B is a prompt heat shock protein.

The cellular response to hyperthermia involves the increased synthesis of heat shock proteins (HSPs) within several hours after treatment. In addition, a subset of proteins has been shown to be increased immediately after heating. These "prompt" HSPs are predominantly found in the nuclear matrix-intermediate filament fraction and are not present or detectable in unheated cells. Since the nuclear matrix has been suggested to be a target for heat-induced cell killing, prompt HSPs may play a prominent role in the heat shock response. Using Western blotting and flow cytometry, we found that an increase in the synthesis of lamin B, one of the major proteins of the nuclear lamina, is induced during heating at 45.5 degrees C but not during heating at 42 degrees C. Since it is an abundant protein which is constitutively expressed in mammalian cells, lamin B appears to be a unique member of the prompt HSP family. The kinetics of induction of lamin B during 45.5 degrees C heating did not correlate with the dose-dependent reduction in cell survival. While increased levels of lamin B during 45.5 degrees C heating do not appear to confer a survival advantage directly, a possible role for lamin B in cellular recovery after heat shock cannot be discounted.

Nuclear Lamins: Their Structure, Assembly, and Interactions

Nuclear lamins are intermediate filament-type proteins that are the major building blocks of the nuclear lamina, a fibrous proteinaceous meshwork underlying the inner nuclear membrane. Lamins can also be localized in the nuclear interior, in a diffuse or spotted pattern. Nuclei assembledin vitroin the absence of lamins are fragile, indicating that lamins mechanically stabilize the cell nucleus. Available evidence also indicates a role for lamins in DNA replication, chromatin organization, spatial arrangement of nuclear pore complexes, nuclear growth, and anchorage of nuclear envelope proteins. In this review we summarize the current state of knowledge on the structure, assembly, and possible functional roles of nuclear lamins, emphasizing the information concerning the ability of nuclear lamins to self-assemble into distinct oligomers and polymers.

Expression of a keratan sulfate proteoglycan during development of the dorsal lateral geniculate nucleus in the ferret

ABAKAN is a keratin sulfate proteoglycan that was identified in rat brain by monoclonal antibody TED15 (Geisert et al. [1992] Brain Res. 571:165-168). It blocks neuronal attachment and neurite outgrowth in culture, is associated with astrocytes, and marks the boundaries of areas in the developing rat brain (Geisert and Bidanset [1993] Dev. Brain Res., 75:163-173). In the present study TED15 was used to examine the distribution of ABAKAN during laminar development of the dorsal lateral geniculate nucleus in ferrets. This distribution was also compared with that of astrocytes as displayed with antibodies to GFAP. In the adult, TED15 and anti-glial fibrillary acidic protein (GFAP) labeling are similar. Both are fairly uniform in the nucleus although somewhat elevated near the optic tract and in the interlaminar zone between laminae A and A1. During development the pattern is quite different. At postnatal day 1 (P1), before lamination is evident, TED15 and anti-GFAP labeling are light in the nucleus. By P10, when laminae are emerging, both are elevated in the A-A1 interlaminar zone and in the C laminae. At P18, when laminae are distinct, TED15 labels the A-A1 interlaminar zone, and it marks the borders between the ON and OFF leaflets within A and A1 (Stryker and Zahs [1983] J. Neurosci. 3:1943-1951). In comparison, anti-GFAP marks the interlaminar zone but not the ON/OFF leaflets. By 6 weeks the nucleus resembles the adult nucleus. These results show that ABAKAN marks the boundaries of the major functional subdivisions of the lateral geniculate nucleus in the developing ferret and suggest that it plays a role in lamination.

Ectopic expression of an A-type lamin does not interfere with differentiation of lamin A-negative embryonal carcinoma cells

The nuclear lamina is an intermediate filament-type network underlying the inner nuclear membrane. It is believed to be important for nuclear envelope integrity and the organization of interphase chromatin. On the basis of biochemical properties and sequence criteria, vertebrate lamin proteins are classified as either A- or B-type. While B-type lamins are expressed in almost all cell types, no A-type lamins are present in early vertebrate embryos or undifferentiated embryonal carcinoma cell lines. Intriguingly, expression of A-type lamins occurs concomitant with cell differentiation and embryonic development. These findings have led to the hypothesis that A-type lamins might play a role in establishing or stabilizing cell-type specific differences in nuclear organization, which in turn might relate to the developmental potential of a cell. To test this hypothesis, we have stably expressed chicken lamin A in undifferentiated murine embryonal carcinoma (P19) cells, and examined the consequences of ectopic lamin A expression for the differentiation state and potential of these cells. Our results demonstrate that the P19 cells, although normally devoid of lamin A, properly incorporate and process chicken lamin A. Moreover, the stably transfected cell lines maintain the properties of undifferentiated cells, demonstrating that expression of lamin A does not directly induce differentiation. Conversely, when exposed to retinoic acid, an inducer of differentiation, lamin A-expressing P19 cells are able to differentiate normally. Taken together, our results suggest that unscheduled expression of A-type lamins is not sufficient to deregulate cell differentiation programs. The implications of these findings for the possible role for lamin A expression during development are discussed.

Expression of nuclear lamins during mouse preimplantation development.

The expression of nuclear lamins during mouse preimplantation development was studied by immunofluorescence, immunoblotting and immunoprecipitation. Two sera were used, specific either for lamin B or lamins A and C. Both sera gave a positive staining of the nuclear periphery throughout preimplantation development (fertilized eggs to late blastocysts). Immunoblots revealed that the three lamins were present in eggs and blastocysts. However, lamin A from eggs was found to have a higher apparent Mr than lamin A from blastocysts and other mouse cells. Using immunoprecipitation, synthesis of lamin A was detected in eggs while synthesis of lamin B was detected in 8-cell embryos and blastocysts, indicating that at least some of the lamins used during early development do not come from a store in the egg. These results are discussed in relation to the possible role of lamins during cell differentiation.