Nucleoplasmic Lamin Research Articles

Nuclear Matrix Proteome Analysis of Drosophila melanogaster

The nucleus is a highly structured organelle and contains many functional compartments. Although the structural basis for this complex spatial organization of compartments is unknown, a major component of this organization is likely to be the non-chromatin scaffolding called nuclear matrix (NuMat). Experimental evidence over the past decades indicates that most of the nuclear functions are at least transiently associated with the NuMat, although the components of NuMat itself are poorly known. Here, we report NuMat proteome analysis from Drosophila melanogaster embryos and discuss its links with nuclear architecture and functions. In the NuMat proteome, we found structural proteins, chaperones, DNA/RNA-binding proteins, chromatin remodeling and transcription factors. This complexity of NuMat proteome is an indicator of its structural and functional significance. Comparison of the two-dimensional profile of NuMat proteome from different developmental stages of Drosophila embryos showed that less than half of the NuMat proteome is constant, and the rest of the proteins are stage-specific dynamic components. These NuMat dynamics suggest a possible functional link between NuMat and embryonic development. Finally, we also showed that a subset of NuMat proteins remains associated with the mitotic chromosomes, implicating their role in mitosis and possibly the epigenetic cellular memory. NuMat proteome analysis provides tools and opens up ways to understand nuclear organization and function.

Hypertonicity induces cytidylyltransferaseα redistribution in nucleoplasmic lamin A/C speckles in MDCK cells

Papillary collecting ducts works in high renal interstitial osmolarity, and increase their phosphatidylcholine (PtdCho) synthesis that occurs by Kennedy pathway. CCTα is the regulatory enzyme which is an amphitropic enzyme, but in most cell lines it has been localized predominantly in the nucleus. We have demonstrated that hypertonicity induces lamin A/C distribution to nucleoplasm speckle. In the present work we examined the nuclear distribution of CCTα in MDCK cells. We evaluated the relationship between the lamin A/C speckles and intranuclear CCTα. Confocal microscopy showed that under hypertonic conditions CCTα colocalize with speckles and colocalization involves protein interaction. ClNa‐TritonX‐100 extraction and immunoprecipitation assays were performed and demonstrated that CCTα interacts with Lamin A/C. Silencing of Lamin A/C causes decreases in lamin A/C speckles number and CCTα redistribution to soluble nuclear matrix. Results herein demonstrate that hypertonicity induces CCTα redistribution from soluble nuclear matrix to nuclear speckles, and this distribution dependent on lamin A/C scaffold. We propose that under hypertonic condition CCTα interact with lamin A/C‐speckles thus establishing two different intranuclear enzyme pools. This work was supported by the University of Buenos Aires (UBACyT B076), CONICET (PIP 5557) and ANPCyT (PICT 33154) grants.

Lamina-Independent Lamins in the Nuclear Interior Serve Important Functions

Nuclear lamins were originally described as the main constituents of the nuclear lamina, a filamentous meshwork closely associated with the inner nuclear membrane. However, within recent years, it has become increasingly evident that a fraction of lamins also resides throughout the nuclear interior. As intermediate-filament-type proteins, lamins have been suggested to fulfill mainly structural functions such as providing shape and mechanical stability to the nucleus. But recent findings show that both peripheral and nucleoplasmic lamins also have important roles in essential cellular processes such as transcription, DNA replication, cell cycle progression, and chromatin organization. Furthermore, more than 300 mutations in the gene encoding A-type lamins have been associated with several human diseases now generally termed laminopathies and comprising muscular dystrophies, lipodystrophies, cardiomyopathies, and premature aging diseases. This review focuses on the lamina-independent pool of lamins in the nuclear interior, which surprisingly has not been studied in much detail so far. We discuss the properties and regulation of nucleoplasmic lamins during the cell cycle, their interaction partners, and their potential involvement in cellular processes and the development of laminopathies.

The A- and B-type nuclear lamin networks: microdomains involved in chromatin organization and transcription

The nuclear lamins function in the regulation of replication, transcription, and epigenetic modifications of chromatin. However, the mechanisms responsible for these lamin functions are poorly understood. We demonstrate that A- and B-type lamins form separate, but interacting, stable meshworks in the lamina and have different mobilities in the nucleoplasm as determined by fluorescence correlation spectroscopy (FCS). Silencing lamin B1 (LB1) expression dramatically increases the lamina meshwork size and the mobility of nucleoplasmic lamin A (LA). The changes in lamina mesh size are coupled to the formation of LA/C-rich nuclear envelope blebs deficient in LB2. Comparative genomic hybridization (CGH) analyses of microdissected blebs, fluorescence in situ hybridization (FISH), and immunofluorescence localization of modified histones demonstrate that gene-rich euchromatin associates with the LA/C blebs. Enrichment of hyperphosphorylated RNA polymerase II (Pol II) and histone marks for active transcription suggest that blebs are transcriptionally active. However, in vivo labeling of RNA indicates that transcription is decreased, suggesting that the LA/C-rich microenvironment induces promoter proximal stalling of Pol II. We propose that different lamins are organized into separate, but interacting, microdomains and that LB1 is essential for their organization. Our evidence suggests that the organization and regulation of chromatin are influenced by interconnections between these lamin microdomains.

141st ENMC International Workshop Inaugural Meeting of the EURO-Laminopathies Project Nuclear Envelope-linked Rare Human Diseases: From Molecular Pathophysiology towards Clinical Applications 10–12 March 2006, Naarden, The Netherlands

EURO-Laminopathies is a European Commissionfunded research project, including 13 scientific group leaders in the fields of human genetics, clinical research, structural biology, molecular cell biology, and pharmaceutical industry, and one administrative manager from eight countries (Austria, France, Germany, Italy Israel, Spain, Switzerland and the United Kingdom). The consortium held its kick-off meeting as an International ENMC Workshop in Naarden on the weekend of March 10–12, 2006. The EURO-Laminopathies project aims at understanding the molecular mechanisms of laminopathies, which are rare human diseases linked tomutations in genes encoding nuclear envelope proteins, such as A-type lamins (LMNA), proteins involved in the post-translational processing of A-type lamins (ZMPSTE24), and lamin-binding proteins (EMD, LBR, LAP2). Laminopathies are clinically manifested after birth, can affect different tissues andprogressively develop during childhood or adolescence, often leading to early death. Efficient therapies have been hampered by the lack

Nucleoplasmic lamins and their interaction partners, LAP2α, Rb, and BAF, in transcriptional regulation

Lamins are major structural components of the nuclear envelope in multicellular eukaryotes. Particularly A-type lamins are also located in the nucleoplasm, likely involving a specific binding partner, lamina-associated polypeptide 2alpha (LAP2alpha). LAP2alpha-lamins A/C complexes in the nucleoplasm have been implicated in the regulation of gene expression by various means. They bind chromatin proteins and chromatin modifying enzymes, and can thus participate in epigenetic control pathways. Furthermore, binding of lamins A/C complexes to specific transcription factors and repressors may directly affect their transcriptional activity. LAP2alpha-lamins A/C also regulate retinoblastoma protein and influence cell cycle progression and differentiation, which could have important implications for molecular mechanisms of laminopathic diseases, linked to lamins A/C mutations.

Thymopoietin (lamina-associated polypeptide 2) gene mutation associated with dilated cardiomyopathy

Thymopoietin or TMPO (indicated by its alternative gene symbol, LAP2, in this work) has been proposed as a candidate disease gene for dilated cardiomyopathy (DCM), since a LAP2 product associates with nucleoplasmic lamins A/C, which are encoded by the DCM gene LMNA. We developed a study to screen for genetic mutations in LAP2 in a large collection of DCM patients and families. A total of 113 subjects from 88 families (56 with familial DCM (FDC) and 32 with sporadic DCM) were screened for LAP2 mutations using denaturing high-performance liquid chromatography and sequence analysis. We found a single putative mutation affecting the LAP2alpha isoform in one FDC pedigree. The mutation predicts an Arg690Cys substitution (c.2068C>T; p.R690C) located in the C-terminal domain of the LAP2alpha protein, a region that is known to interact with lamin A/C. RT-PCR, Western blot analyses, and immunolocalization revealed low-level LAP2alpha expression in adult cardiac muscle, and localization to a subset of nuclei. Mutated Arg690Cys LAP2alpha expressed in HeLa cells localized to the nucleoplasm like wild-type LAP2alpha, with no effect on peripheral and nucleoplasmic lamin A distribution. However, the in vitro interaction of mutated LAP2alpha with the pre-lamin A C-terminus was significantly compromised compared to the wild-type protein. LAP2 mutations may represent a rare cause of DCM. The Arg690Cys mutation altered the observed LAP2alpha interaction with A-type lamins. Our finding implicates a novel nuclear lamina-associated protein in the pathogenesis of genetic forms of dilated cardiomyopathy.

Immunolocalization of detergent‐susceptible nucleoplasmic lamin A/C foci by a novel monoclonal antibody

The A-type lamins are localized in the interior of the nucleus as well as on the nuclear periphery. In this study, we have characterized a monoclonal antibody LA-2F9 produced against recombinant rat lamin A which stains a subpopulation of various cell types in a pattern of small nucleoplasmic foci that are unusually susceptible to mild detergent/salt extraction. The specific reactivity of mAb LA-2F9 towards lamins was confirmed by immunoblotting of HeLa and C3H10T(1/2) whole cell lysates and nuclear lysates. The epitope for LA-2F9 was narrowed down to amino acid residues 268-278 (SAKLDNARQSA). To check whether the appearance of lamin foci was cell-cycle-dependent, C3H10T(1/2) cells were serum-starved and then refed to trigger cells to enter the G(1) phase of the cell-cycle. The intensity of staining increased 3.5-fold within 6 h of refeeding, when the maximum number of cells were labeled with LA-2F9. We also checked whether the LA-2F9 foci colocalized with nuclear proteins known to be distributed in small foci such as hnRNPs, snRNPs, SC-35, and p80 coilin, but did not find evidence of colocalization. Our studies suggest that LA-2F9 has a novel and specific reactivity towards detergent-susceptible lower order lamin structures that are likely to be assembly intermediates.