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.

Mitochondrial genotype and risk for Alzheimer's disease: cross-sectional data from the Vienna-Transdanube-Aging "VITA" study.

The Vienna Transdanube Aging (VITA) study searches for early markers of Alzheimer's disease (AD) by examining the mental status in a community-based cohort of 606, 75-years old volunteers that are then related to various clinical and genetic analyses. To determine whether mutations in mtDNA are involved in expression of AD, the mtDNA of 79 "control" participants is screened for alterations by sequencing of "hot-spot-regions". This study on mtDNA mutations has eliminated the influence of aging on the occurrence of mtDNA alterations by sequencing samples from persons at the age of exactly 75 years. Thus, our cohort reveals a snap-shot of mitochondrial sequences of elderly persons. So far, a high percentage (56%) of persons with known or unknown mutations in the fragments analyzed were found. These data will be compared in due time to a cohort of participants with proven late-onset AD.

Nuclear envelope and nuclear matrix: interactions and dynamics.

The peripheral nuclear lamina is located near the nuclear inner membrane and consists of lamin filaments and integral membrane proteins, including the lamin B receptor and various isoforms of lamina-associated polypeptides (LAP) 1 and 2. Several nuclear membrane proteins also interact with chromatin proteins BAF and Hp1. Lamins in the nuclear interior associate with at least one soluble (non-membrane-bound) LAP2 isoform named LAP2alpha. The internal lamins, together with Tpr-based filaments that connect to nuclear pore complexes, are proposed to be major structural elements of the internal nuclear matrix. We describe the structural links between the peripheral lamina and the internal nuclear matrix that are thought to be mediated by LAP2 family members, filament protein Tpr and nucleoporin Nup153. These findings are discussed in relation to human diseases that arise from mutations in nuclear lamina proteins.

Lamina-associated polypeptide 2alpha binds intranuclear A-type lamins.

The nucleoskeletal protein lamina-associated polypeptide 2(&agr;) (LAP2*) contains a large, unique C terminus and differs significantly from its alternatively spliced, mostly membrane-integrated isoforms, such as LAP2beta. Unlike lamin B-binding LAP2beta, LAP2alpha was found by confocal immunofluorescence microscopy to colocalize preferentially with A-type lamins in the newly formed nuclei assembled after mitosis. While only a subfraction of lamins A and C (lamin A/C) was associated with the predominantly nuclear LAP2alpha in telophase, the majority of lamin A/C colocalized with LAP2alpha in G(1)-phase nuclei. Furthermore, selective disruption of A-type lamin structures by overexpression of lamin mutants in HeLa cells caused a redistribution of LAP2alpha. Coimmunoprecipitation experiments revealed that a fraction of lamin A/C formed a stable, SDS-resistant complex with LAP2alpha in interphase cells and in postmetaphase cell extracts. Blot overlay binding studies revealed a direct binding of LAP2alpha to exclusively A-type lamins and located the interaction domains to the C-terminal 78 amino acids of LAP2alpha and to residues 319-566 in lamin A/C, which include the C terminus of the rod and the entire tail common to lamin A/C. These findings suggest that LAP2alpha and A-type lamins cooperate in the organization of internal nuclear structures.

Review: lamina-associated polypeptide 2 isoforms and related proteins in cell cycle-dependent nuclear structure dynamics.

The lamina-associated polypeptide (LAP) 2 family comprises up to six alternatively spliced proteins in mammalian cells and three isoforms in Xenopus. LAP2beta is a type II integral protein of the inner nuclear membrane, which binds to lamin B and the chromosomal protein BAF, and may link the nuclear membrane to the underlying lamina and provide docking sites for chromatin. LAP2alpha shares only the N-terminus with the other isoforms and contains a unique C-terminus. It is a nonmembrane protein associated with the nucleoskeleton and may help to organize higher order chromatin structure by interacting with A-lamins and chromosomes. Recent studies using mutant proteins have just begun to unravel functions of LAP2 isoforms during postmitotic nuclear reassembly. LAP2alpha associates with chromosomes via an alpha-specific domain at early stages of assembly, possibly providing a structural framework for chromosome reorganization. The subsequent interaction of both LAP2alpha and LAP2beta with the chromosomal BAF may stabilize chromatin structure and target membranes to the chromosomes. At later stages LAP2 may regulate the assembly of lamins. LAP2 isoforms have been found to share a homologous approximately 40 amino acid long region, the LEM domain, with nuclear membrane proteins MAN1 and emerin, which has been implicated in Emery-Dreifuss muscular dystrophy.

Differential nuclear localization and nuclear matrix association of the splicing factors PSF and PTB.

A monoclonal antibody raised against nuclear matrix proteins detected a protein of basic pI in human nuclear matrix protein samples of various cellular origin. The ubiquitously occurring (common) nuclear matrix protein was identified as splicing factor PSF (PTB associated splicing factor). The interaction between the splicing factors PSF and PTB/hnRNP I was confirmed by co-immunoprecipitation from nuclear salt extracts. However, the nuclear localization of PSF and PTB and their distribution in subnuclear fractions differed markedly. Isolated nuclear matrices contained the bulk of PSF, but only minor amounts of PTB. In confocal microscopy both proteins appeared in speckles, the majority of which did not co-localize. Removing a large fraction of the soluble PTB structures by salt extraction revealed some colocalization of the more stable PTB fraction with PSF. These PTB/PSF complexes as well as the observed PSF-PTB interaction may reflect the previously reported presence of PTB and PSF in spliceosomal complexes during RNA processing. The present data, however, point to different cellular distribution and nuclear matrix association of the majority of PSF and PTB.

Functional diversity of LAP2alpha and LAP2beta in postmitotic chromosome association is caused by an alpha-specific nuclear targeting domain.

Lamina-associated polypeptide 2alpha (LAP2alpha) is a non-membrane-bound isoform of the LAP2 family implicated in nuclear structure organization. We show that during postmitotic nuclear assembly LAP2alpha associates with chromosomes prior to accumulation of the membrane-bound isoform LAP2beta, although both proteins contain the same putative chromatin interaction domains located in their common N-terminal regions. By transient and stable expression of various N- and C-terminal LAP2alpha deletion mutants in HeLa cells, we identified an approximately 350-amino-acid-long region in the C-terminal alpha-specific domain of the protein that is required for retention of LAP2alpha in interphase nuclei and for association with mitotic chromosomes, while the N-terminal domain seemed to be dispensable for these interactions. In vitro chromosome binding studies using recombinant LAP2alpha mutants revealed that this LAP2alpha-specific 'nuclear targeting domain' was essential and sufficient for association with chromosomes. These data suggested a functional diversity of chromosome binding properties of LAP2 isoforms.

Detergent-salt resistance of LAP2alpha in interphase nuclei and phosphorylation-dependent association with chromosomes early in nuclear assembly implies functions in nuclear structure dynamics.

Lamina-associated polypeptide (LAP) 2 of the inner nuclear membrane (now LAP2beta) and LAP2alpha are related proteins produced by alternative splicing, and contain a common 187 amino acid N-terminal domain. We show here that, unlike LAP2beta, LAP2alpha behaved like a nuclear non-membrane protein in subcellular fractionation studies and was localized throughout the nuclear interior in interphase cells. It co-fractionated with LAP2beta in nuclear lamina/matrix-enriched fractions upon extraction of nuclei with detergent, salt and nucleases. During metaphase LAP2alpha dissociated from chromosomes and became concentrated around the spindle poles. Furthermore, LAP2alpha was mitotically phosphorylated, and phosphorylation correlated with increased LAP2alpha solubility upon extraction of cells in physiological buffers. LAP2alpha relocated to distinct sites around chromosomes at early stages of nuclear reassembly and intermediarily co-localized with peripheral lamin B and intranuclear lamin A structures at telophase. During in vitro nuclear assembly LAP2alpha was dephosphorylated and assembled into insoluble chromatin-associated structures, and recombinant LAP2alpha was found to interact with chromosomes in vitro. Some LAP2alpha may also associate with membranes prior to chromatin attachment. Altogether the data suggest a role of LAP2alpha in post-mitotic nuclear assembly and in the dynamic structural organization of the nucleus.