Lamins and lamin-associated proteins in aging and disease.

Lamins, together with the lamin-associated proteins of the inner nuclear membrane, are structural proteins in the nucleus that mediate mechanical stress resistance. Novel findings show that lamin complexes also have scaffolding functions in the formation and regulation of higher order chromatin and in epigenetic regulatory pathways. Furthermore, lamins serve as scavenging complexes and regulators of signaling molecules in diverse pathways. Lamin complexes in the nuclear interior contribute to retinoblastoma-mediated cell cycle regulation. Because of their multiple and diverse roles, lamins are linked to an increasing number of human diseases. The molecular mechanisms of these diseases, which are just beginning to emerge, may involve cell cycle and differentiation defects in adult stem cells and genomic instability.

Lamina-associated polypeptide 2alpha regulates cell cycle progression and differentiation via the retinoblastoma-E2F pathway.

Lamina-associated polypeptide (LAP) 2alpha is a nonmembrane-bound LAP2 isoform that forms complexes with nucleoplasmic A-type lamins. In this study, we show that the overexpression of LAP2alpha in fibroblasts reduced proliferation and delayed entry into the cell cycle from a G0 arrest. In contrast, stable down-regulation of LAP2alpha by RNA interference accelerated proliferation and interfered with cell cycle exit upon serum starvation. The LAP2alpha-linked cell cycle phenotype is mediated by the retinoblastoma (Rb) protein because the LAP2alpha COOH terminus directly bound Rb, and overexpressed LAP2alpha inhibited E2F/Rb-dependent reporter gene activity in G1 phase in an Rb-dependent manner. Furthermore, LAP2alpha associated with promoter sequences in endogenous E2F/Rb-dependent target genes in vivo and negatively affected their expression. In addition, the expression of LAP2alpha in proliferating preadipocytes caused the accumulation of hypophosphorylated Rb, which is reminiscent of noncycling cells, and initiated partial differentiation into adipocytes. The effects of LAP2alpha on cell cycle progression and differentiation may be highly relevant for the cell- and tissue-specific phenotypes observed in laminopathic diseases.

A-type lamin networks in light of laminopathic diseases.

Lamins are major structural components of the lamina providing mechanical support for the nuclear envelope in vertebrates. A subgroup of lamins, the A-type lamins, are only expressed in differentiated cells and serve important functions both at the nuclear envelope and in the nucleoplasm in higher order chromatin organization and gene regulation. Mutations in A-type lamins cause a variety of diseases from muscular dystrophy and lipodystrophy to systemic diseases such as premature ageing syndromes. The molecular basis of these diseases is still unknown. Here we summarize known interactions of A-type lamins with components of the nuclear envelope and the nucleoplasm and discuss their potential involvement in the etiology and molecular mechanisms of the diseases. Lamin binding partners involve chromatin proteins potentially involved in higher order chromatin organization, transcriptional regulators controlling gene expression during cell cycle progression, differentiation and senescence, and several enzymes involved in a multitude of functions.

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.

Lamina-associated polypeptide 2-alpha forms homo-trimers via its C terminus, and oligomerization is unaffected by a disease-causing mutation.

The nucleoplasmic protein, Lamina-associated polypeptide (LAP) 2alpha, is one of six alternatively spliced products of the LAP2gene, which share a common N-terminal region. In contrast to the other isoforms, which also share most of their C termini, LAP2alpha has a large unique C-terminal region that contains binding sites for chromatin, A-type lamins, and retinoblastoma protein. By immunoprecipitation analyses of LAP2alpha complexes from cells expressing differently tagged LAP2alpha proteins and fragments, we demonstrate that LAP2alpha forms higher order structures containing multiple LAP2alpha molecules in vivo and that complex formation is mediated by the C terminus. Solid phase binding assays using recombinant and in vitro translated LAP2alpha fragments showed direct interactions of LAP2alpha C termini. Cross-linking of LAP2alpha complexes and multiangle light scattering of purified LAP2alpha revealed the existence of stable homo-trimers in vivo and in vitro. Finally, we show that, in contrast to the LAP2alpha-lamin A interaction, its self-association is not affected by a disease-linked single point mutation in the LAP2alpha C terminus.

Breaking and making of the nuclear envelope.

During mitosis, a single nucleus gives rise to two nuclei that are identical to the parent nucleus. Mitosis consists of a continuous sequence of events that must be carried out once and only once. Two such important events are the disassembly of the nuclear envelope (NE) during the first stages of mitosis, and its accurate reassembly during the last stages of mitosis. NE breakdown (NEBD) is initiated when maturation-promoting factor (MPF) enters the nucleus and starts phosphorylating nuclear pore complexes (NPCs) and nuclear lamina proteins, followed by NPC and lamina breakdown. Nuclear reassembly starts when nuclear membranes assemble onto the chromatin. This article focuses on the different models of NEBD and reassembly with emphasis on recent data.

Distinct functions of the unique C terminus of LAP2alpha in cell proliferation and nuclear assembly.

The non-membrane-bound lamina-associated polypeptide 2 isoform, LAP2alpha, forms nucleoskeletal structures with A-type lamins and interacts with chromosomes in a cell cycle-dependent manner. LAP2alpha contains a LEM (LAP2, emerin, and MAN1) domain in the constant N terminus that binds to chromosomal barrier-to-autointegration factor, and a C-terminal unique region that is essential for chromosome binding. Here we show that C-terminal LAP2alpha fragment efficiently bound to mitotic chromosomes and inhibited assembly of endogenous LAP2alpha, nuclear membranes, and lamins A/C in in vitro nuclear assembly assays. Full-length recombinant LAP2alpha, which bound to chromosomes, and N-terminal fragment, which did not bind, had no effect on assembly. This suggested an essential role for the LAP2alpha C terminus in chromosome association and for the N-terminal LEM domain in subsequent assembly stages. In vivo analysis upon transient expression of GFP-tagged LAP2alpha fragments confirmed that, unlike the N-terminal fragment, the C-terminal fragment was able to bind to chromosomes during mitosis, if expressed weakly. At higher expression levels, C-terminal LAP2alpha fragment and full-length protein led to cell cycle arrest in interphase and apoptosis, as shown by fluorescence-activated cell sorter analysis, time lapse microscopy, and BrdUrd incorporation assays. These data indicated distinct functions of LAP2alpha in cell cycle progression during interphase and in nuclear reassembly during mitosis.

Proteins that bind A-type lamins: integrating isolated clues.

What do such diverse molecules as DNA, actin, retinoblastoma protein and protein kinase Calpha all have in common? They and additional partners bind 'A-type' lamins, which form stable filaments in animal cell nuclei. Mutations in A-type lamins cause a bewildering range of tissue-specific diseases, termed 'laminopathies', including Emery-Dreifuss muscular dystrophy and the devastating Hutchinson-Gilford progeria syndrome, which mimics premature aging. Considered individually and collectively, partners for A-type lamins form four loose groups: architectural partners, chromatin partners, gene-regulatory partners and signaling partners. We describe 16 partners in detail, summarize their binding sites in A-type lamins, and sketch portraits of ternary complexes and functional pathways that might depend on lamins in vivo. On the basis of our limited current knowledge, we propose lamin-associated complexes with multiple components relevant to nuclear structure (e.g. emerin, nesprin 1alpha, actin) or signaling and gene regulation (e.g. LAP2alpha, retinoblastoma, E2F-DP heterodimers, genes) as 'food for thought'. Testing these ideas will deepen our understanding of nuclear function and human disease.

Lco1 is a novel widely expressed lamin-binding protein in the nuclear interior.

A-type lamins are localized at the nuclear envelope and in the nucleoplasm, and are implicated in human diseases called laminopathies. In a yeast two-hybrid screen with lamin C, we identified a novel widely expressed 171-kDa protein that we named Lamin companion 1 (Lco1). Three independent biochemical assays showed direct binding of Lco1 to the C-terminal tail of A-type lamins with an affinity of 700 nM. Lco1 also bound the lamin B1 tail with lower affinity (2 microM). Ectopic Lco1 was found primarily in the nucleoplasm and colocalized with endogenous intranuclear A-type lamins in HeLa cells. Overexpression of prelamin A caused redistribution of ectopic Lco1 to the nuclear rim together with ectopic lamin A, confirming association of Lco1 with lamin A in vivo. Whereas the major C-terminal lamin-binding fragment of Lco1 was cytoplasmic, the N-terminal Lco1 fragment localized in the nucleoplasm upon expression in cells. Furthermore, full-length Lco1 was nuclear in cells lacking A-type lamins, showing that A-type lamins are not required for nuclear targeting of Lco1. We conclude that Lco1 is a novel intranuclear lamin-binding protein. We hypothesize that Lco1 is involved in organizing the internal lamin network and potentially relevant as a laminopathy disease gene or modifier.