SUMOylation of the nuclear pore complex basket is involved in sensing cellular stresses.

The nuclear pore complex (NPC) is the major conduit for nucleocytoplasmic transport and serves as a platform for gene regulation and DNA repair. Several nucleoporins undergo ubiquitylation and SUMOylation, and these modifications play an important role in nuclear pore dynamics and plasticity. Here, we perform a detailed analysis of these post-translational modifications of yeast nuclear basket proteins under normal growth conditions as well as upon cellular stresses, with a focus on SUMOylation. We find that the balance between the dynamics of SUMOylation and deSUMOylation of Nup60 and Nup2 at the NPC differs substantially, particularly in G1 and S phase. While Nup60 is the unique target of genotoxic stress within the nuclear basket that probably belongs to the SUMO-mediated DNA damage response pathway, both Nup2 and Nup60 show a dramatic increase in SUMOylation upon osmotic stress, with Nup2 SUMOylation being enhanced in Nup60 SUMO-deficient mutant yeast strains. Taken together, our data reveal that there are several levels of crosstalk between nucleoporins, and that the post-translational modifications of the NPC serve in sensing cellular stress signals.

INO80 Chromatin Remodeler Facilitates Release of RNA Polymerase II from Chromatin for Ubiquitin-Mediated Proteasomal Degradation.

Stalling of RNA Polymerase II (RNAPII) on chromatin during transcriptional stress results in polyubiquitination and degradation of the largest subunit of RNAPII, Rpb1, by the ubiquitin proteasome system (UPS). Here, we report that the ATP-dependent chromatin remodeling complex INO80 is required for turnover of chromatin-bound RNAPII in yeast. INO80 interacts physically and functionally with Cdc48/p97/VCP, a component of UPS required for degradation of RNAPII. Cells lacking INO80 are defective in Rpb1 degradation and accumulate tightly bound ubiquitinated Rpb1 on chromatin. INO80 forms a ternary complex with RNAPII and Cdc48 and targets Rpb1 primed for degradation. The function of INO80 in RNAPII turnover is required for cell growth and survival during genotoxic stress. Our results identify INO80 as a bona fide component of the proteolytic pathway for RNAPII degradation and suggest that INO80 nucleosome remodeling activity promotes the dissociation of ubiquitinated Rpb1 from chromatin to protect the integrity of the genome.

Nesprin interchain associations control nuclear size.

Nesprins-1/-2/-3/-4 are nuclear envelope proteins, which connect nuclei to the cytoskeleton. The largest nesprin-1/-2 isoforms (termed giant) tether F-actin through their N-terminal actin binding domain (ABD). Nesprin-3, however, lacks an ABD and associates instead to plectin, which binds intermediate filaments. Nesprins are integrated into the outer nuclear membrane via their C-terminal KASH-domain. Here, we show that nesprin-1/-2 ABDs physically and functionally interact with nesprin-3. Thus, both ends of nesprin-1/-2 giant are integrated at the nuclear surface: via the C-terminal KASH-domain and the N-terminal ABD-nesprin-3 association. Interestingly, nesprin-2 ABD or KASH-domain overexpression leads to increased nuclear areas. Conversely, nesprin-2 mini (contains the ABD and KASH-domain but lacks the massive nesprin-2 giant rod segment) expression yields smaller nuclei. Nuclear shrinkage is further enhanced upon nesprin-3 co-expression or microfilament depolymerization. Our findings suggest that multivariate intermolecular nesprin interactions with the cytoskeleton form a lattice-like filamentous network covering the outer nuclear membrane, which determines nuclear size.

LINC complex alterations in DMD and EDMD/CMT fibroblasts.

Emery-Dreifuss muscular dystrophy (EDMD) is a late onset-disease characterized by skeletal muscle wasting and heart defects with associated risk of sudden death. The autosomal dominant form of the disease is caused by mutations in the LMNA gene encoding LaminA and C, the X-linked form results from mutations in the gene encoding the inner nuclear membrane protein Emerin (STA). Both Emerin and LaminA/C interact with the nuclear envelope proteins Nesprin-1 and -2 and mutations in genes encoding C-terminal isoforms of Nesprin-1 and -2 have also been implicated in EDMD. Here we analyse primary fibroblasts from patients affected by either Duchenne muscular dystrophy (DMD) or Emery-Dreifuss muscular dystrophy/Charcot-Marie-Tooth syndrome (EDMD/CMT) that in addition to the disease causing mutations harbour mutations in the Nesprin-1 gene and in the SUN1 and SUN2 gene, respectively. SUN proteins together with the Nesprins form the core of the LINC complex which connects the nucleus with the cytoskeleton. The mutations are accompanied by changes in cell adhesion, cell migration, senescence, and stress response, as well as in nuclear shape and nuclear envelope composition which are changes characteristic for laminopathies. Our results point to a potential influence of mutations in components of the LINC complex on the clinical outcome and the molecular pathology in the patients.

Cytoskeletal interactions at the nuclear envelope mediated by nesprins.

Nesprin-1 is a giant tail-anchored nuclear envelope protein composed of an N-terminal F-actin binding domain, a long linker region formed by multiple spectrin repeats and a C-terminal transmembrane domain. Based on this structure, it connects the nucleus to the actin cytoskeleton. Earlier reports had shown that Nesprin-1 binds to nuclear envelope proteins emerin and lamin through C-terminal spectrin repeats. These repeats can also self-associate. We focus on the N-terminal Nesprin-1 sequences and show that they interact with Nesprin-3, a further member of the Nesprin family, which connects the nucleus to the intermediate filament network. We show that upon ectopic expression of Nesprin-3 in COS7 cells, which are nearly devoid of Nesprin-3 in vitro, vimentin filaments are recruited to the nucleus and provide evidence for an F-actin interaction of Nesprin-3 in vitro. We propose that Nesprins through interactions amongst themselves and amongst the various Nesprins form a network around the nucleus and connect the nucleus to several cytoskeletal networks of the cell.