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.

Nuclear lamins promote protrusion dynamics and collective, confined migration in vivo

Cells migrate collectively through confined environments during development and cancer metastasis. While the nucleus, a large and stiff organelle, impedes cell migration between non-deformable pillars in vitro, its function in vivo may vary depending on the microenvironment. Further, it is unknown how nuclei contribute to collective migration in vivo and whether nuclei in different positions within cell collectives experience different forces. Here, we use border cell migration in the fly ovary as an in vivo model to investigate the effects of confined, collective migration on nuclei and the contribution of nuclear lamins to migration. We found severe yet transient nuclear deformations occur, particularly in the leading cell, as border cells squeeze through tiny crevices between germline cells, termed nurse cells. Leading cells extend protrusions between nurse cells, which may pry open space to allow the cluster to advance. Here we report that the leading cell nuclei deformed as they moved into leading protrusions. Then as protrusions widened, the nucleus recovered a more circular shape. These data suggest that lead cell nuclei may help protrusions expand and thereby enlarge the migration path. To test how nuclei might promote or impede border cell migration, we investigated nuclear lamins, proteins that assemble into intermediate filaments and structurally support the nuclear envelope. Depletion of the Drosophila B-type lamin, Lam, from the outer, motile border cells, but not the inner, nonmotile polar cells, impeded border cell migration, whereas perturbations of the A-type lamin, LamC, did not. While wild type border cell clusters typically have one large leading protrusion as they delaminate from the anterior follicular epithelium, clusters depleted of B-type lamin had multiple, short-lived protrusions, resulting in unproductive cluster movement and failure to progress along the migration path. Further, border cell nuclei depleted of B-type lamins were small, formed blebs, and ruptured. Together, these data indicate that B-type lamin is requied for nuclear integrity, which in turn stabilizes the leading protrusion and promotes overall cluster polarization and collective movement through confined spaces.

Progerin-Expressing Endothelial Cells are Unable to Adapt to Shear Stress

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare premature aging disease caused by a single-point mutation in the lamin A gene, resulting in a truncated and farnesylated form of lamin A. This mutant lamin A protein, known as progerin, accumulates at the periphery of the nuclear lamina, resulting in both an abnormal nuclear morphology and nuclear stiffening. HGPS patients experience rapid onset of atherosclerosis, with death from heart attack or stroke as teenagers. Progerin expression has been shown to cause dysfunction in both vascular smooth muscle cells and endothelial cells (ECs). In this study we examined how progerin-expressing ECs adapt to fluid shear stress, the principal mechanical force from blood flow. We compared the response to shear stress for progerin-expressing, wild-type lamin A overexpressing, and control ECs to physiological levels of fluid shear stress. Additionally, we also knocked down ZMPSTE24 in ECs, which results in increased farnesylation of lamin A and similar phenotypes to HGPS. Our results showed that ECs either expressing progerin or with ZMPSTE24 knockdown were unable to adapt to shear stress, experiencing significant cell loss at a longer duration of exposure to shear stress (3 days). ECs overexpressing wild-type lamin A also exhibited similar impairments in adaptation to shear stress, including similar levels of cell loss. Quantification of nuclear morphology showed that progerin-expressing ECs had similar nuclear abnormalities in both static and shear conditions. Treatment of progerin-expressing cells and ZMPSTE24 KD cells with lonafarnib and methystat, drugs previously shown to improve HGPS nuclear morphology, resulted in improvements in adaptation to shear stress. Additionally, pre-alignment of cells to shear stress prior to progerin-expression prevented cell loss. Our results demonstrate that changes in nuclear lamins can affect the ability of EC to properly adapt to shear stress.

Bend, Push, Stretch: Remarkable Structure and Mechanics of Single Intermediate Filaments and Meshworks

The cytoskeleton of the eukaryotic cell provides a structural and functional scaffold enabling biochemical and cellular functions. While actin and microtubules form the main framework of the cell, intermediate filament networks provide unique mechanical properties that increase the resilience of both the cytoplasm and the nucleus, thereby maintaining cellular function while under mechanical pressure. Intermediate filaments (IFs) are imperative to a plethora of regulatory and signaling functions in mechanotransduction. Mutations in all types of IF proteins are known to affect the architectural integrity and function of cellular processes, leading to debilitating diseases. The basic building block of all IFs are elongated α-helical coiled-coils that assemble hierarchically into complex meshworks. A remarkable mechanical feature of IFs is the capability of coiled-coils to metamorphize into β-sheets under stress, making them one of the strongest and most resilient mechanical entities in nature. Here, we discuss structural and mechanical aspects of IFs with a focus on nuclear lamins and vimentin.

LAP1 regulates nuclear plasticity to enable constrained migration

Metastatic spread involves the dissemination of cancer cells from a primary tumour and their colonisation of distal sites. During this process, cancer cells must negotiate multiple physical constraints imposed by the microenvironment and tissue structure, and the biophysical properties of the nucleus place a physical challenge on this form of migration. By analysing nuclear genes upregulated during the acquisition of metastatic potential, we discovered increased expression of the inner nuclear membrane protein LAP1 in metastatic cell lines and at the leading edge of human primary tumours and in metastatic lesions. Human cells express two LAP1 isoforms (LAP1B and LAP1C), which differ in their amino terminus. We found that the longer isoform, LAP1B, binds more strongly to nuclear lamins and enhances nuclear mechanocoupling, whilst the shorter isoform, LAP1C, favours nuclear envelope blebbing and permits migration through physical constraints. Thus, we propose that LAP1B and LAP1C act together to support a permissive nucleus which overcomes the physical constraints that cancer cells face during metastatic spread.

HSV-1 UL34 mutants that affect membrane budding regulation and nuclear lamina disruption

Nuclear envelope budding in herpesvirus nuclear egress may be negatively regulated, since the pUL31/pUL34 nuclear egress complex heterodimer can induce membrane budding without capsids when expressed ectopically or on artificial membranes in vitro, but not in the infected cell. We have previously described a pUL34 mutant that contained alanine substitutions at R158 and R161, and that showed impaired growth, impaired pUL31/pUL34 interaction, and unregulated budding. Here we determine the phenotypic contributions of the individual substitutions to these phenotypes. Neither substitution alone was able to reproduce the impaired growth or NEC interaction phenotypes. Either substitution, however, could fully reproduce the unregulated budding phenotype, suggesting that mis-regulated budding may not substantially impair virus replication. Additionally, the R158A substitution caused re-localization of the NEC to intranuclear punctate structures and recruited lamin A/C to those structures, suggesting that this residue might be important for recruitment of kinases for dispersal of nuclear lamins. Importance Herpesvirus nuclear egress is a complex, regulated process coordinated by two virus proteins that are conserved among the herpesviruses that form a heterodimeric nuclear egress complex (NEC). The NEC drives budding of capsids at the inner nuclear membrane, and recruits other viral and host cell proteins for disruption of the nuclear lamina, membrane scission and fusion. The structural basis of individual activities of the NEC, apart from membrane budding, are not clear, nor is the basis of the regulation of membrane budding. Here we explore the properties of NEC mutants that have an unregulated budding phenotype, determine the significance of that regulation for virus replication, and also characterize a structural requirement for nuclear lamina disruption.

Increased expression of LAP2β eliminates nuclear membrane ruptures in nuclear lamin–deficient neurons and fibroblasts

Defects or deficiencies in nuclear lamins cause pathology in many cell types, and recent studies have implicated nuclear membrane (NM) ruptures as a cause of cell toxicity. We previously observed NM ruptures and progressive cell death in the developing brain of lamin B1–deficient mouse embryos. We also observed frequent NM ruptures and DNA damage in nuclear lamin–deficient fibroblasts. Factors modulating susceptibility to NM ruptures remain unclear, but we noted low levels of LAP2β, a chromatin-binding inner NM protein, in fibroblasts with NM ruptures. Here, we explored the apparent link between LAP2β and NM ruptures in nuclear lamin–deficient neurons and fibroblasts, and we tested whether manipulating LAP2β expression levels would alter NM rupture frequency. In cortical plate neurons of lamin B1–deficient embryos, we observed a strong correlation between low LAP2β levels and NM ruptures. We also found low LAP2β levels and frequent NM ruptures in neurons of cultured Lmnb1−/− neurospheres. Reducing LAP2β expression in Lmnb1−/− neurons with an siRNA markedly increased the NM rupture frequency (without affecting NM rupture duration), whereas increased LAP2β expression eliminated NM ruptures and reduced DNA damage. Consistent findings were observed in nuclear lamin–deficient fibroblasts. Reduced LAP2β expression increased NM ruptures, whereas increased LAP2β expression virtually abolished NM ruptures. Increased LAP2β expression nearly abolished NM ruptures in cells subjected to mechanical stress (an intervention that increases NM ruptures). Our studies showed that increasing LAP2β expression bolsters NM integrity in nuclear lamin–deficient cells and markedly reduces NM rupture frequency.

Characterization of a Plant Nuclear Matrix Constituent Protein in Liverwort

The nuclear lamina (NL) is a complex network of nuclear lamins and lamina-associated nuclear membrane proteins, which scaffold the nucleus to maintain structural integrity. In animals, type V intermediate filaments are the main constituents of NL. Plant genomes do not encode any homologs of these intermediate filaments, yet plant nuclei contain lamina-like structures that are present in their nuclei. In Arabidopsis thaliana, CROWDED NUCLEI (CRWN), which are required for maintaining structural integrity of the nucleus and specific perinuclear chromatin anchoring, are strong candidates for plant lamin proteins. Recent studies revealed additional roles of Arabidopsis Nuclear Matrix Constituent Proteins (NMCPs) in modulating plants’ response to pathogen and abiotic stresses. However, detailed analyses of Arabidopsis NMCP activities are challenging due to the presence of multiple homologs and their functional redundancy. In this study, we investigated the sole NMCP gene in the liverwort Marchantia polymorpha (MpNMCP). We found that MpNMCP proteins preferentially were localized to the nuclear periphery. Using CRISPR/Cas9 techniques, we generated an MpNMCP loss-of-function mutant, which displayed reduced growth rate and curly thallus lobes. At an organelle level, MpNMCP mutants did not show any alteration in nuclear morphology. Transcriptome analyses indicated that MpNMCP was involved in regulating biotic and abiotic stress responses. Additionally, a highly repetitive genomic region on the male sex chromosome, which was preferentially tethered at the nuclear periphery in wild-type thalli, decondensed in the MpNMCP mutants and located in the nuclear interior. This perinuclear chromatin anchoring, however, was not directly controlled by MpNMCP. Altogether, our results unveiled that NMCP in plants have conserved functions in modulating stress responses.

Nuclear organization and regulation of the differentiated state

Regulation of the differentiated identity requires active and continued supervision. Inability to maintain the differentiated state is a hallmark of aging and aging-related disease. To maintain cellular identity, a network of nuclear regulators is devoted to silencing previous and non-relevant gene programs. This network involves transcription factors, epigenetic regulators, and the localization of silent genes to heterochromatin. Together, identity supervisors mold and maintain the unique nuclear environment of the differentiated cell. This review describes recent discoveries regarding mechanisms and regulators that supervise the differentiated identity and protect from de-differentiation, tumorigenesis, and attenuate forced somatic cell reprograming. The review focuses on mechanisms involved in H3K9me3-decorated heterochromatin and the importance of nuclear lamins in cell identity. We outline how the biophysical properties of these factors are involved in self-compartmentalization of heterochromatin and cell identity. Finally, we discuss the relevance of these regulators to aging and age-related disease.