Genetic and pharmacological modulation of lamin A farnesylation determines its function and turnover.

Hutchinson-Gilford Progeria syndrome (HGPS) is a severe premature ageing disorder caused by a 50 amino acid truncated (Δ50AA) and permanently farnesylated lamin A (LA) mutant called progerin. On a cellular level, progerin expression leads to heterochromatin loss, impaired nucleocytoplasmic transport, telomeric DNA damage and a permanent growth arrest called cellular senescence. Although the genetic basis for HGPS has been elucidated 20 years ago, the question whether the Δ50AA or the permanent farnesylation causes cellular defects has not been addressed. Moreover, we currently lack mechanistic insight into how the only FDA-approved progeria drug Lonafarnib, a farnesyltransferase inhibitor (FTI), ameliorates HGPS phenotypes. By expressing a variety of LA mutants using a doxycycline-inducible system, and in conjunction with FTI, we demonstrate that the permanent farnesylation, and not the Δ50AA, is solely responsible for progerin-induced cellular defects, as well as its rapid accumulation and slow clearance. Importantly, FTI does not affect clearance of progerin post-farnesylation and we demonstrate that early, but not late FTI treatment prevents HGPS phenotypes. Collectively, our study unravels the precise contributions of progerin's permanent farnesylation to its turnover and HGPS cellular phenotypes, and how FTI treatment ameliorates these. These findings are applicable to other diseases associated with permanently farnesylated proteins, such as adult-onset autosomal dominant leukodystrophy.

Subcellular Force Imbalance in Actin Bundles Induces Nuclear Repositioning and Durotaxis.

Durotaxis is a phenomenon in which cells migrate toward substrates of increasing stiffness. However, how cells assimilate substrate stiffness as a directional cue remains poorly understood. In this study, we experimentally show that mouse embryonic fibroblasts can discriminate between different substrate stiffnesses and develop higher traction forces at regions of the cell adhering to the stiffer pillars. In this way, the cells generate a force imbalance between adhesion sites. It is this traction force imbalance that drives durotaxis by providing directionality for cell migration. Significantly, we found that traction forces are transmitted via LINC complexes to the cell nucleus, which serves to maintain the global force imbalance. In this way, LINC complexes play an essential role in anterograde nuclear movement and durotaxis. This conclusion is supported by the fact that LINC complex-deficient cells are incapable of durotaxis and instead migrate randomly on substrates featuring a stiffness gradient.

Disrupting the LINC complex by AAV mediated gene transduction prevents progression of Lamin induced cardiomyopathy.

Mutations in the LaminA gene are a common cause of monogenic dilated cardiomyopathy. Here we show that mice with a cardiomyocyte-specific Lmna deletion develop cardiac failure and die within 3-4 weeks after inducing the mutation. When the same Lmna mutations are induced in mice genetically deficient in the LINC complex protein SUN1, life is extended to more than one year. Disruption of SUN1's function is also accomplished by transducing and expressing a dominant-negative SUN1 miniprotein in Lmna deficient cardiomyocytes, using the cardiotrophic Adeno Associated Viral Vector 9. The SUN1 miniprotein disrupts binding between the endogenous LINC complex SUN and KASH domains, displacing the cardiomyocyte KASH complexes from the nuclear periphery, resulting in at least a fivefold extension in lifespan. Cardiomyocyte-specific expression of the SUN1 miniprotein prevents cardiomyopathy progression, potentially avoiding the necessity of developing a specific therapeutic tailored to treating each different LMNA cardiomyopathy-inducing mutation of which there are more than 450.

Trophic interactions between indigenous and non-indigenous species in Lampedusa Island, Mediterranean Sea.

Using stable isotope analysis, we investigated trophic interactions between indigenous benthic taxa and the non-indigenous species (NIS): the green alga Caulerpa cylindracea, the red alga Asparagopsis taxiformis, the crab Percnon gibbesi and the sea hare Aplysia dactylomela. The study was conducted on Lampedusa Island, Mediterranean Sea. We evaluated the trophic positions and isotopic niches of consumers. Using Bayesian mixing models, we quantified the food source contribution to diets of indigenous and non-indigenous herbivores. Isotopic niche of NIS showed no overlap with the ones of indigenous macroinvertebrates and fish. Caulerpa cylindracea provided the largest contribution to the diet of P. gibbesi (0.431-1), while the dietary contribution estimates overlapped considerably for all sources of A. dactylomela and indigenous herbivores. From these results, we conclude that the invasion of C. cylindracea is increasing the diversity of available prey and might facilitate the expansion of other NIS.

Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins.

Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but is enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway.

BST-2/tetherin-mediated restriction of chikungunya (CHIKV) VLP budding is counteracted by CHIKV non-structural protein 1 (nsP1).

Chikungunya virus (CHIKV) is a re-emerging alphavirus transmitted by Aedes mosquitoes. Infection with CHIKV elicits a type I interferon response that facilities virus clearance, probably through the action of down-stream effectors such as antiviral IFN-stimulated genes (ISGs). Bone marrow stromal antigen 2 (BST-2) is an ISG shown to restrict HIV-1 replication by preventing the infection of bystander cells by tethering progeny virions on the surface of infected cells. Here we show that enrichment of cell surface BST-2 results in retention of CHIKV virus like particles (VLPs) on the cell membrane. BST-2 was found to co-localize with CHIKV structural protein E1 in the context of VLPs without any noticeable effect on BST-2 level. However, CHIKV nonstructural protein 1 (nsP1) overcomes BST-2-mediated VLPs tethering by down-regulating BST-2 expression. We conclude that BST-2 tethers CHIKV VLPs on the host cell plasma membrane and identify CHIKV nsP1 as a novel BST-2 antagonist.

Bone marrow stromal cell antigen 2 (BST-2) restricts mouse mammary tumor virus (MMTV) replication in vivo.

BACKGROUND: Bone marrow stromal cell antigen 2 (BST-2) is a cellular factor that restricts the egress of viruses such as human immunodeficiency virus (HIV-1) from the surface of infected cells, preventing infection of new cells. BST-2 is variably expressed in most cell types, and its expression is enhanced by cytokines such as type I interferon alpha (IFN-α). In this present study, we used the beta-retrovirus, mouse mammary tumor virus (MMTV) as a model to examine the role of mouse BST-2 in host infection in vivo. RESULTS: By using RNA interference, we show that loss of BST-2 enhances MMTV replication in cultured mammary tumor cells and in vivo. In cultured cells, BST-2 inhibits virus accumulation in the culture medium, and co-localizes at the cell surface with virus structural proteins. Furthermore, both scanning electron micrograph (SEM) and transmission electron micrograph (TEM) show that MMTV accumulates on the surface of IFNα-stimulated cells. CONCLUSIONS: Our data provide evidence that BST-2 restricts MMTV release from naturally infected cells and that BST-2 is an antiviral factor in vivo.

A functional role for TorsinA in herpes simplex virus 1 nuclear egress.

Herpes simplex virus 1 (HSV-1) capsids leave the nucleus by a process of envelopment and de-envelopment at the nuclear envelope (NE) that is accompanied by structural alterations of the NE. As capsids translocate across the NE, transient primary enveloped virions form in the perinuclear space. Here, we provide evidence that torsinA (TA), a ubiquitously expressed ATPase, has a role in HSV-1 nuclear egress. TA resides within the lumen of the endoplasmic reticulum (ER)/NE and functions in maintaining normal NE architecture. We show that perturbation of TA normal function by overexpressing torsinA wild type (TAwt) inhibits HSV-1 production. Ultrastructural analysis of infected cells overexpressing TAwt revealed reduced levels of surface virions in addition to accumulation of novel, double-membrane structures called virus-like vesicles (VLVs). Although mainly found in the cytoplasm, VLVs resemble primary virions in their size, by the appearance of the inner membrane, and by the presence of pUL34, a structural component of primary virions. Collectively, our data suggest a model in which interference of TA normal function by overexpression impairs de-envelopment of the primary virions leading to their accumulation in a cytoplasmic membrane compartment. This implies novel functions for TA at the NE.