Nuclear Cytoskeleton in Virus Infection

The nuclear lamina is the main component of the nuclear cytoskeleton that maintains the integrity of the nucleus. However, it represents a natural barrier for viruses replicating in the cell nucleus. The lamina blocks viruses from being trafficked to the nucleus for replication, but it also impedes the nuclear egress of the progeny of viral particles. Thus, viruses have evolved mechanisms to overcome this obstacle. Large viruses induce the assembly of multiprotein complexes that are anchored to the inner nuclear membrane. Important components of these complexes are the viral and cellular kinases phosphorylating the lamina and promoting its disaggregation, therefore allowing virus egress. Small viruses also use cellular kinases to induce lamina phosphorylation and the subsequent disruption in order to facilitate the import of viral particles during the early stages of infection or during their nuclear egress. Another component of the nuclear cytoskeleton, nuclear actin, is exploited by viruses for the intranuclear movement of their particles from the replication sites to the nuclear periphery. This study focuses on exploitation of the nuclear cytoskeleton by viruses, although this is just the beginning for many viruses, and promises to reveal the mechanisms and dynamic of physiological and pathological processes in the nucleus.

Nucleoplasmic Lamin C Rapidly Accumulates at Sites of Nuclear Envelope Rupture with BAF and cGAS

In mammalian cell nuclei, the nuclear lamina (NL) underlies the nuclear envelope (NE) to maintain nuclear structure. The nuclear lamins, the major structural components of the NL, are involved in the protection against NE rupture induced by mechanical stress. However, the specific role of the lamins in repair of NE ruptures has not been fully determined. Our analyses using immunofluorescence and live-cell imaging revealed that lamin C but not the other lamin isoforms rapidly accumulated at sites of NE rupture induced by laser microirradiation in mouse embryonic fibroblasts. The immunoglobulin-like fold domain and the NLS were required for the recruitment from the nucleoplasm to the rupture sites with the Barrier-to-autointegration factor (BAF). The accumulation of nuclear BAF and cytoplasmic cyclic GMP-AMP (cGAMP) synthase (cGAS) at the rupture sites was in part dependent on lamin A/C. These results suggest that nucleoplasmic lamin C, BAF and cGAS concertedly accumulate at sites of NE rupture for repair.

Deformation of the nucleus by TGFβ1 via the remodeling of nuclear envelope and histone isoforms

The cause of nuclear shape abnormalities which are often seen in pre-neoplastic and malignant tissues is not clear. In this study we report that deformation of the nucleus can be induced by TGFβ1 stimulation in several cell lines including Huh7. In our results, the upregulated histone H3.3 expression downstream of SMAD signaling contributed to TGFβ1-induced nuclear deformation, a process of which requires incorporation of the nuclear envelope (NE) proteins lamin B1 and SUN1. During this process, the NE constitutively ruptured and reformed. Contrast to lamin B1 which was relatively stationary around the nucleus, the upregulated lamin A was highly mobile, clustering at the nuclear periphery and reintegrating into the nucleoplasm. The chromatin regions that lost NE coverage formed a supra-nucleosomal structure characterized by elevated histone H3K27me3 and histone H1, the formation of which depended on the presence of lamin A. These results provide evidence that shape of the nucleus can be modulated through TGFβ1-induced compositional changes in the chromatin and nuclear lamina.

Checkpoint activation drives global gene expression changes in Drosophila nuclear lamina mutants

The nuclear lamina (NL) lines the inner nuclear membrane. This extensive protein network organizes chromatin and contributes to the regulation of transcription, DNA replication and repair. Lap2-emerin-MAN1 domain (LEM-D) proteins are key members of the NL, representing proteins that connect the NL to the genome through shared interactions with the chromatin binding protein Barrier-to-autointegration factor (BAF). Functions of the LEM-D protein emerin and BAF are essential during Drosophila melanogaster oogenesis. Indeed, loss of either emerin or BAF blocks germ cell development and causes loss of germline stem cells, defects linked to deformation of NL structure and non-canonical activation of Checkpoint kinase 2 (Chk2). Here, we investigate contributions of emerin and BAF to gene expression in the ovary. Profiling RNAs from emerin and baf mutant ovaries revealed that nearly all baf mis-regulated genes were shared with emerin mutants, defining a set of NL-regulated genes. Strikingly, loss of Chk2 restored expression of most NL-regulated genes, identifying a large class of Chk2-dependent genes (CDGs). Nonetheless, some genes remained mis-expressed upon Chk2 loss, identifying a smaller class of emerin-dependent genes (EDGs). Properties of EDGs suggest a shared role for emerin and BAF in repression of developmental genes. Properties of CDGs demonstrate that Chk2 activation drives global mis-expression of genes in the emerin and baf mutant backgrounds. Notably, CDGs were found up-regulated in lamin-B mutant backgrounds. These observations predict that Chk2 activation might have a general role in gene expression changes found in NL-associated diseases, such as laminopathies.

Nuclear Architecture Disruption During Aging Causes Misexpression of Genes Lacking CpG Islands

Global disorganization of chromatin architecture, characterized by disrupted nuclear lamina and associated heterochromatin, is commonly observed in various aging contexts, including premature aging diseases, cellular senescence, and normative aging. Although these conserved structural changes have been reported for over two decades, their impact on transcription and contribution to age-related degenerative changes remain unclear. Here we show that genes not associated with CpG islands (CGI- genes), which form heterochromatin when transcriptionally silent, are globally misexpressed in aged nuclei with disrupted chromatin architectures. Our data also show that CGI- gene misexpression is a direct outcome of nuclear architecture disruption. Notably, CGI- gene misexpression explains the molecular basis of various defects observed during aging, including loss of cellular identity and increased noises in transcription. We also show that uncontrolled secretory phenotypes commonly observed during aging are largely attributable to CGI- gene misexpression, which drives disruption of intercellular communication and fuel chronic inflammation in aged tissues. Our large-scale meta-analysis further demonstrates that CGI- gene misexpression is a common feature of mammalian aging and age-associated diseases. Interestingly, CGI- gene misexpression can be suppressed by anti-aging interventions. Our study suggests that age-associated CGI- gene misexpression is a novel biomarker of physiological aging which offers an effective therapeutic target for delaying or ameliorating degenerative changes associated with aging.

Senescent stroma induces nuclear deformations in cancer cells via the inhibition of RhoA/ROCK/myosin II-based cytoskeletal tension

The presence of senescent cells within tissues has been functionally linked to malignant transformations. Here, using tension-gauge tethers technology, particle-tracking microrheology, and quantitative microscopy, we demonstrate that senescent associated secretory phenotype (SASP) derived from senescent fibroblasts impose nuclear lobulations and volume shrinkage on malignant cells, which stems from the loss of RhoA/ROCK/myosin II-based cortical tension. This loss in cytoskeletal tension induces decreased cellular contractility, adhesion, and increased mechanical compliance. These SASP-induced morphological changes are in part mediated by lamin A/C. These findings suggest that SASP induces a defective outside-in mechanotransduction, from actomyosin fibers in the cytoplasm to the nuclear lamina, thereby triggering a cascade of biophysical and biomolecular changes in cells that associate with malignant transformations.

Chromosome length and gene density contribute to micronuclear membrane stability

Micronuclei are derived from missegregated chromosomes and frequently lose membrane integrity, leading to DNA damage, innate immune activation, and metastatic signaling. Here, we demonstrate that two characteristics of the trapped chromosome, length and gene density, are key contributors to micronuclei membrane stability and determine the timing of micronucleus rupture. We demonstrate that these results are not due to chromosome-specific differences in spindle position or initial protein recruitment during post-mitotic nuclear envelope assembly. Micronucleus size strongly correlates with lamin B1 levels and nuclear pore density in intact micronuclei, but, unexpectedly, lamin B1 levels do not completely predict nuclear lamina organization or membrane stability. Instead, small gene-dense micronuclei have decreased nuclear lamina gaps compared to large micronuclei, despite very low levels of lamin B1. Our data strongly suggest that nuclear envelope composition defects previously correlated with membrane rupture only partly explain membrane stability in micronuclei. We propose that an unknown factor linked to gene density has a separate function that inhibits the appearance of nuclear lamina gaps and delays membrane rupture until late in the cell cycle.

Lamin C is required to establish genome organization after mitosis

Background The dynamic 3D organization of the genome is central to gene regulation and development. The nuclear lamina influences genome organization through the tethering of lamina-associated domains (LADs) to the nuclear periphery. Evidence suggests that lamins A and C are the predominant lamins involved in the peripheral association of LADs, potentially serving different roles. Results Here, we examine chromosome architecture in mouse cells in which lamin A or lamin C are downregulated. We find that lamin C, and not lamin A, is required for the 3D organization of LADs and overall chromosome organization. Striking differences in localization are present as cells exit mitosis and persist through early G1 and are linked to differential phosphorylation. Whereas lamin A associates with the nascent nuclear envelope (NE) during telophase, lamin C remains in the interior, surrounding globular LAD aggregates enriched on euchromatic regions. Lamin C association with the NE is delayed until several hours into G1 and correlates temporally and spatially with the post-mitotic NE association of LADs. Post-mitotic LAD association with the NE, and global 3D genome organization, is perturbed only in cells depleted of lamin C, and not lamin A. Conclusions Lamin C regulates LAD dynamics during exit from mitosis and is a key regulator of genome organization in mammalian cells. This reveals an unexpectedly central role for lamin C in genome organization, including inter-chromosomal LAD-LAD segregation and LAD scaffolding at the NE, raising intriguing questions about the individual and overlapping roles of lamin A/C in cellular function and disease.

Genome Compartmentalization with Nuclear Landmarks: Random yet Precise

The three-dimensional (3D) organization of eukaryotic genomes plays an important role in genome function. While significant progress has been made in deciphering the folding mechanisms of individual chromosomes, the principles of the dynamic large-scale spatial arrangement of all chromosomes inside the nucleus are poorly understood. We use polymer simulations to model the diploid human genome compartmentalization relative to nuclear bodies such as nuclear lamina, nucleoli, and speckles. We show that a self-organization process based on a co-phase separation between chromosomes and nuclear bodies can capture various features of genome organization, including the formation of chromosome territories, phase separation of A/B compartments, and the liquid property of nuclear bodies. The simulated 3D structures quantitatively reproduce both sequencing-based genomic mapping and imaging assays that probe chromatin interaction with nuclear bodies. Importantly, our model captures the heterogeneous distribution of chromosome positioning across cells, while simultaneously producing well-defined distances between active chromatin and nuclear speckles. Such heterogeneity and preciseness of genome organization can coexist due to the non-specificity of phase separation and the slow chromosome dynamics. Together, our work reveals that the co-phase separation provides a robust mechanism for encoding functionally important 3D contacts without requiring thermodynamic equilibration that can be difficult to achieve.