Christopher J. Hutchison

Genomic instability in laminopathy-based premature aging

Premature aging syndromes often result from mutations in nuclear proteins involved in the maintenance of genomic integrity. Lamin A is a major component of the nuclear lamina and nuclear skeleton. Truncation in lamin A causes Hutchinson-Gilford progerial syndrome (HGPS), a severe form of early-onset premature aging. Lack of functional Zmpste24, a metalloproteinase responsible for the maturation of prelamin A, also results in progeroid phenotypes in mice and humans. We found that Zmpste24-deficient mouse embryonic fibroblasts (MEFs) show increased DNA damage and chromosome aberrations and are more sensitive to DNA-damaging agents. Bone marrow cells isolated from Zmpste24−/− mice show increased aneuploidy and the mice are more sensitive to DNA-damaging agents. Recruitment of p53 binding protein 1 (53BP1) and Rad51 to sites of DNA lesion is impaired in Zmpste24−/− MEFs and in HGPS fibroblasts, resulting in delayed checkpoint response and defective DNA repair. Wild-type MEFs ectopically expressing unprocessible prelamin A show similar defects in checkpoint response and DNA repair. Our results indicate that unprocessed prelamin A and truncated lamin A act dominant negatively to perturb DNA damage response and repair, resulting in genomic instability which might contribute to laminopathy-based premature aging.

Lamins: building blocks or regulators of gene expression?

Intermediate filament (IF) proteins are the building blocks of cytoskeletal filaments, the main function of which is to maintain cell shape and integrity. The lamins are thought to be the evolutionary progenitors of IF proteins and they have profound influences on both nuclear structure and function. These influences require the lamins to have dynamic properties and dual identities — as building blocks and transcriptional regulators. Which one of these identities underlies a myriad of genetic diseases is a topic of intense debate.

A-type lamins: Guardians of the soma?

The gene LMNA encodes the proteins lamins A and C and is implicated in nine different laminopathies — inherited diseases that are linked to premature ageing. Recent evidence has demonstrated that lamins A and C have essential functions in protecting cells from physical damage, as well as in maintaining the function of transcription factors required for the differentiation of adult stem cells. Thus, the degenerative nature of laminopathies is explained because these lamins are essential for maintenance of somatic tissues in adulthood.

The inner nuclear membrane protein Emerin regulates β‐catenin activity by restricting its accumulation in the nucleus

Emerin is a type II inner nuclear membrane (INM) protein of unknown function. Emerin function is likely to be important because, when it is mutated, emerin promotes both skeletal muscle and heart defects. Here we show that one function of Emerin is to regulate the flux of β‐catenin, an important transcription coactivator, into the nucleus. Emerin interacts with β‐catenin through a conserved adenomatous polyposis coli (APC)‐like domain. When GFP‐emerin is expressed in HEK293 cells, β‐catenin is restricted to the cytoplasm and β‐catenin activity is inhibited. In contrast, expression of an emerin mutant, lacking its APC‐like domain (GFP‐emerinΔ), dominantly stimulates β‐catenin activity and increases nuclear accumulation of β‐catenin. Human fibroblasts that are null for emerin have an autostimulatory growth phenotype. This unusual growth phenotype arises through enhanced nuclear accumulation and activity of β‐catenin and can be replicated in wild‐type fibroblasts by transfection with constitutively active β‐catenin. Our results support recent findings that suggest that INM proteins can influence signalling pathways by restricting access of transcription coactivators to the nucleus.

A novel role for the nuclear membrane protein emerin in association of the centrosome to the outer nuclear membrane

The type II inner nuclear membrane protein emerin is a component of the LINC complex that connects the nuclear lamina to the actin cytoskeleton. In emerin-null or -deficient human dermal fibroblasts we find that the centrosome is detached from the nucleus. Moreover, following siRNA knockdown of emerin in wild-type fibroblasts, the centrosome also becomes detached from the nucleus. We show that emerin interacts with tubulin, and that nocadozole-treated wild-type cells phenocopy the detached centrosome characteristic of emerin-null/deficient cells. We also find that a significant fraction of emerin is located at the outer nuclear membrane and peripheral ER, where it interacts directly with the centrosome. Our data provide the first evidence in mammalian cells as to the nature of the linkage of the centrosome, and therefore the tubulin cytoskeleton, with the outer nuclear membrane.

Lamin A/C is a risk biomarker in colorectal cancer

Background A-type lamins are type V intermediate filament proteins encoded by the gene LMNA. Mutations in LMNA give rise to diverse degenerative diseases related to premature ageing. A-type lamins also influence the activity of the Retinoblastoma protein (pRb) and oncogenes such a β-catenin. Consequently, it has been speculated that expression of A-type lamins may also influence tumour progression. Methodology/Principal Findings An archive of colorectal cancer (CRC) and normal colon tissue was screened for expression of A-type lamins. We used the Cox proportional hazard ratio (HR) method to investigate patient survival. Using CRC cell lines we investigated the effects of lamin A expression on other genes by RT-PCR; on cell growth by FACS analysis; and on invasiveness by cell migration assays and siRNA knockdown of targeted genes. We found that lamin A is expressed in colonic stem cells and that patients with A-type lamin-expressing tumours have significantly worse prognosis than patients with A-type lamin negative tumours (HR = 1.85, p = 0.005). To understand this finding, we established a model system based upon expression of GFP-lamin A in CRC cells. We found that expression of GFP-lamin A in these cells did not affect cell proliferation but did promote greatly increased cell motility and invasiveness. The reason for this increased invasiveness was that expression of lamin A promoted up-regulation of the actin bundling protein T-plastin, leading to down regulation of the cell adhesion molecule E-cadherin. Conclusions Expression of A-type lamins increases the risk of death from CRC because its presence gives rise to increased invasiveness and potentially a more stem cell-like phenotype. This report directly links A-type lamin expression to tumour progression and raises the profile of LMNA from one implicated in multiple but rare genetic conditions to a gene involved in one of the commonest diseases in the Western World.

Incorporation of the nuclear pore basket protein Nup153 into nuclear pore structures is dependent upon lamina assembly: evidence from cell‐free extracts of Xenopus eggs

In cell‐free extracts of Xenopus eggs that support the assembly of replication‐competent nuclei, we found that lamin B3 specifically associates with four polypeptides (termed SLAPs, soluble lamin associated proteins). Here, one SLAP is identified as the nuclear pore complex protein Nup153, one member of the F/GXFG motif‐containing nucleoporins. In vitro translated Nup153 and lamin B3 co‐immunoprecipitate, and lamin B3 interacts specifically with the C‐terminal domain of Nup153. During nuclear envelope assembly, other F/GXFG‐containing nucleoporins are incorporated into the nuclear envelope preceding lamina assembly. Incorporation of Nup153 occurs at the same time as lamina assembly. When lamina assembly is prevented using the dominant‐negative mutant XlaminBΔ2+, Nup153 does not appear at the nuclear envelope, while other F/GXFG‐containing nucleoporins and Nup93 are recruited normally. When the lamina of pre‐assembled nuclei is disrupted using the same dominant‐negative mutant, the distribution of other nucleoporins is unaffected. However, Nup153 recruitment at the nuclear envelope is lost. Our results indicate that both the recruitment and maintenance of Nup153 at the pore are dependent upon the integrity of the lamina.

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

Lamina-associated polypeptide (LAP) 2α is a nonmembrane-bound LAP2 isoform that forms complexes with nucleoplasmic A-type lamins. In this study, we show that the overexpression of LAP2α in fibroblasts reduced proliferation and delayed entry into the cell cycle from a G0 arrest. In contrast, stable down-regulation of LAP2α by RNA interference accelerated proliferation and interfered with cell cycle exit upon serum starvation. The LAP2α-linked cell cycle phenotype is mediated by the retinoblastoma (Rb) protein because the LAP2α COOH terminus directly bound Rb, and overexpressed LAP2α inhibited E2F/Rb-dependent reporter gene activity in G1 phase in an Rb-dependent manner. Furthermore, LAP2α associated with promoter sequences in endogenous E2F/Rb-dependent target genes in vivo and negatively affected their expression. In addition, the expression of LAP2α in proliferating preadipocytes caused the accumulation of hypophosphorylated Rb, which is reminiscent of noncycling cells, and initiated partial differentiation into adipocytes. The effects of LAP2α on cell cycle progression and differentiation may be highly relevant for the cell- and tissue-specific phenotypes observed in laminopathic diseases.

Filaments made from A- and B-type lamins differ in structure and organization.

Lamins are intermediate filament proteins and the major component of the nuclear lamina. Current views of the lamina are based on the remarkably regular arrangement of lamin LIII in amphibian oocyte nuclei. We have re-examined the LIII lamina and propose a new interpretation of its organization. Rather than consisting of two perpendicular arrays of parallel filaments, we suggest that the oocyte lamina consists of parallel filaments that are interconnected in register to give the impression of a second set of perpendicular filaments. We have also used the oocyte system to investigate the organization of somatic lamins. Currently, it is not feasible to examine the organization of somatic lamins in situ because of their tight association with chromatin. It is also difficult to assemble vertebrate lamin filaments in vitro. Therefore, we have used the oocyte system, where exogenously expressed somatic B-type and A-type lamins assemble into filaments. Expression of B-type lamins induces the formation of intranuclear membranes that are covered by single filament layers. LIII filaments appear identical to the endogenous lamina, whereas lamin B2 assembles into filaments that are organized less precisely. Lamin A induces sheets of thicker filaments on the endogenous lamina and significantly increases the rigidity of the nuclear envelope.

Nucleoplasmic LAP2alpha-lamin A complexes are required to maintain a proliferative state in human fibroblasts

In human diploid fibroblasts (HDFs), expression of lamina-associated polypeptide 2 α (LAP2α) upon entry and exit from G0 is tightly correlated with phosphorylation and subnuclear localization of retinoblastoma protein (Rb). Phosphoisoforms of Rb and LAP2α are down-regulated in G0. Although RbS780 phosphoform and LAP2α are up-regulated upon reentry into G1 and colocalize in the nucleoplasm, RbS795 migrates between nucleoplasmic and speckle compartments. In HDFs, which are null for lamins A/C, LAP2α is mislocalized within nuclear aggregates, and this is correlated with cell cycle arrest and accumulation of Rb within speckles. Nuclear retention of nucleoplasmic Rb during G1 phase but not of speckle-associated Rb depends on lamin A/C. siRNA knock down of LAP2α or lamin A/C in HDFs leads to accumulation of Rb in speckles and G1 arrest, probably because of activation of a cell cycle checkpoint. Our results suggest that LAP2α and lamin A/C are involved in controlling Rb localization and phosphorylation, and a lack or mislocalization of either protein leads to cell cycle arrest in HDFs.