Human papillomavirus (HPV) infection is a primary cause of cervical and head-and-neck cancers. The HPV genome enters the nucleus during mitosis when the nuclear envelope disassembles. Given that lamins maintain nuclear integrity during interphase, we asked to what extent their loss would affect early HPV infection. To address this question, we infected human cervical cancer cells and keratinocytes lacking the major lamins with a HPV16 pseudovirus (HP-PsV) encoding an EGFP reporter. We found that a sustained reduction or complete loss of lamin B1 significantly increased HP-PsV infection rate. A corresponding greater nuclear HP-PsV load in LMNB1 knockout cells was directly related to their prolonged mitotic window and extensive nuclear rupture propensity. Despite the increased HP-PsV presence, EGFP transcript levels remained virtually unchanged, indicating an additional defect in protein turnover. Further investigation revealed that LMNB1 knockout led to a substantial decrease in autophagic capacity, possibly linked to the persistent activation of cGAS by cytoplasmic chromatin exposure. Thus, the attrition of lamin B1 increases nuclear perviousness and attenuates autophagic capacity, creating an environment conducive to unrestrained accumulation of HPV capsids. Our identification of lower lamin B1 levels and nuclear BAF foci in the basal epithelial layer of several human cervix samples suggests that this pathway may contribute to an increased individual susceptibility to HPV infection.

The nuclear envelope (NE) creates a barrier between the cytosol and nucleus during interphase that is key for cellular compartmentalization and protecting genomic DNA. NE rupture can expose genomic DNA to the cytosol and allow admixture of the nuclear and cytosolic constituents, a proposed mechanism of cancer and NE-associated diseases. Barrier-to-autointegration factor (BAF) is a DNA-binding protein that localizes to NE ruptures where it recruits LEM-domain proteins, A-type lamins, and participates in rupture repair. To further reveal the mechanisms by which BAF responds to and aids in repairing NE ruptures, we investigated known properties of BAF including LEM domain binding, lamin binding, compartmentalization, phosphoregulation of DNA binding, and BAF dimerization. We demonstrate that it is the cytosolic population of BAF that functionally repairs NE ruptures, and phosphoregulation of BAF\'s DNA-binding that enables its ability to facilitate that repair. Interestingly, BAF\'s LEM or lamin binding activity appears dispensable for its role in functional repair. Furthermore, we demonstrate that BAF functions to reduce the extent of leakage though NE ruptures, suggesting that BAF effectively forms a diffusion barrier prior to NE repair. Collectively, these results enhances our knowledge of the mechanisms by which BAF responds to NE ruptures and facilitates their repair.

The glycocalyx-that forms a protective barrier around cells-has been implicated in cancer cell proliferation, survival, and metastasis. However, its role in maintaining the integrity of DNA/nucleus during migration through dense matrices remains unexplored. In this study, we address this question by first documenting heterogeneity in glycocalyx expression in highly invasive MDA-MB-231 breast cancer cells, and establishing a negative correlation between cell size and glycocalyx levels. Next, we set-up transwell migration through 3 µm pores, to isolate two distinct sub-populations and to show that the early migrating cell sub-population possesses a bulkier glycocalyx and undergoes less DNA damage and nuclear rupture, assessed using γH2AX foci formation and nuclear/cytoplasmic distribution of Ku70/80. Interestingly, enzymatic removal of glycocalyx led to disintegration of the nuclear membrane indicated by increased cytoplasmic localisation of Ku70/80, increased nuclear blebbing and reduction in nuclear area. Together, these results illustrate an inverse association between bulkiness of the glycocalyx and nuclear stresses, and highlights the mechanical role of the glycocalyx in shielding migration associated stresses.

Mutations in the genes LMNA and BANF1 can lead to accelerated aging syndromes called progeria. The protein products of these genes, A-type lamins and BAF, respectively, are nuclear envelope (NE) proteins that interact and participate in various cellular processes, including nuclear envelope rupture and repair. BAF localizes to sites of nuclear rupture and recruits NE-repair machinery, including the LEM-domain proteins, ESCRT-III complex, A-type lamins, and membranes. Here, we show that it is a mobile, nucleoplasmic population of A-type lamins that is rapidly recruited to ruptures in a BAF-dependent manner via BAF\'s association with the Ig-like β fold domain of A-type lamins. These initially mobile lamins become progressively stabilized at the site of rupture. Farnesylated prelamin A and lamin B1 fail to localize to nuclear ruptures, unless that farnesylation is inhibited. Progeria-associated LMNA mutations inhibit the recruitment affected A-type lamin to nuclear ruptures, due to either permanent farnesylation or inhibition of BAF binding. A progeria-associated BAF mutant targets to nuclear ruptures but is unable to recruit A-type lamins. Together, these data reveal the mechanisms that determine how lamins respond to nuclear ruptures and how progeric mutations of LMNA and BANF1 impair recruitment of A-type lamins to nuclear ruptures.

The nuclear envelope (NE) is a critical barrier between the cytosol and nucleus that is key for compartmentalization within the cell and serves an essential role in organizing and protecting genomic DNA. Rupturing of the NE through loss of constitutive NE proteins and/or mechanical force applied to the nucleus results in the unregulated mixing of cytosolic and nuclear compartments, leading to DNA damage and genomic instability. Nuclear rupture has recently gained interest as a mechanism that may participate in various NE-associated diseases as well as cancer. Remarkably, these rupturing events are often transient, with cells being capable of rapidly repairing nuclear ruptures. Recently, we identified Barrier-to-Autointegration Factor (BAF), a DNA-binding protein involved in post-mitotic NE reformation and cytosolic viral regulation, as an essential protein for nuclear rupture repair. During interphase, the highly mobile cytosolic BAF is primed to monitor for a compromised NE by rapidly binding to newly exposed nuclear DNA and subsequently recruiting the factors necessary for NE repair. This review highlights the recent findings of BAF\'s roles in rupture repair, and offers perspectives on how regulatory factors that control BAF activity may potentially alter the cellular response to nuclear ruptures and how BAF may participate in human disease.

Barrier-to-autointegration factor (BAF; encoded by BANF1) is a small highly conserved, ubiquitous and self-associating protein that coordinates with numerous binding partners to accomplish several key cellular processes. By interacting with double-stranded DNA, histones and various other nuclear proteins, including those enriched at the nuclear envelope, BAF appears to be essential for replicating cells to protect the genome and enable cell division. Cellular processes, such as innate immunity, post-mitotic nuclear reformation, repair of interphase nuclear envelope rupture, genomic regulation, and the DNA damage and repair response have all been shown to depend on BAF. This Review focuses on the regulation of the numerous interactions of BAF, which underlie the mechanisms by which BAF accomplishes its essential cellular functions. We will also discuss how perturbation of BAF function may contribute to human disease.

The physical connections between the cytoskeletal system and the nucleus provide a route for the nucleus to sense the mechanical stress both inside and outside of the cell. Failure to withstand such stress leads to nuclear rupture, which is observed in human diseases. In this review, we will go through the recent findings and our current understandings of nuclear rupture. Starting with the triggers of nuclear rupture, including the aberrant nuclear lamina composition and the elevated actomyosin contractility. We will also discuss the role of ESCRT-III in nuclear rupture repair and the biological consequences of nuclear rupture, including the negative impacts on cellular compartmentalization, DNA damage, and cellular differentiation. Recent studies on nuclear rupture provide further insights into the direct mechanistic link between nuclear rupture and several pathological conditions. Such knowledge can guide us in developing potential therapeutic solutions for the patients.

The nuclear lamins are the major components of the nuclear lamina in the nuclear envelope. Lamins are involved in numerous functions, including a role in providing structural support to the cell and the mechanosensing of the cell. Mutations in the genes encoding for lamins lead to the rare diseases termed laminopathies. However, not only laminopathies show alterations in the nuclear lamina. Deregulation of lamin expression is reported in multiple cancers and several viral infections lead to a disrupted nuclear lamina. The structural and mechanical effects of alterations in the nuclear lamina can partly explain the phenotypes seen in disease, such as muscular weakness in certain laminopathies and transmigration of cancer cells. However, a lot of answers to questions about the relation between changes in the nuclear lamina and disease development remain elusive. Here, we review the current understandings of the contribution of the nuclear lamina in the structural support and mechanosensing of healthy and diseased cells.

The nuclear envelope (NE) serves as a central organizing unit for the eukaryotic cell. By virtue of its highly selective, semipermeable barrier function, the NE shields the enclosed genetic material, while at the same time ensuring its regulated transcription, replication, and repair. The NE has long been considered to only dismantle during mitosis. However, in recent years it has become clear that in a variety of pathologies, NE integrity becomes compromised during interphase as well. Loss of NE integrity, or briefly NE stress, is manifested in various ways, ranging from a gradual reduction in nucleocytoplasmic transport function, to selective loss and degradation of NE components, and finally to catastrophic rupture events that provoke abhorrent molecular fluxes between the nucleus and cytoplasm. Although cells manage to cope with such forms of NE stress, the different insults to nuclear compartmentalization alter gene regulation and jeopardize genome stability. Hence, loss of NE integrity is emerging as a broad-spectrum pathogenic mechanism. In this review, we discuss the relevance of nuclear compartmentalization and the loss thereof in aging and disease development.

Laminopathies, mainly caused by mutations in the LMNA gene, are a group of inherited diseases with a highly variable penetrance; i.e., the disease spectrum in persons with identical LMNA mutations range from symptom-free conditions to severe cardiomyopathy and progeria, leading to early death. LMNA mutations cause nuclear abnormalities and cellular fragility in response to cellular mechanical stress, but the genotype/phenotype correlations in these diseases remain unclear. Consequently, tools such as mutation analysis are not adequate for predicting the course of the disease.   Here, we employ growth substrate stiffness to probe nuclear fragility in cultured dermal fibroblasts from a laminopathy patient with compound progeroid syndrome. We show that culturing of these cells on substrates with stiffness higher than 10 kPa results in malformations and even rupture of the nuclei, while culture on a soft substrate (3 kPa) protects the nuclei from morphological alterations and ruptures. No malformations were seen in healthy control cells at any substrate stiffness. In addition, analysis of the actin cytoskeleton organization in this laminopathy cells demonstrates that the onset of nuclear abnormalities correlates to an increase in cytoskeletal tension. Together, these data indicate that culturing of these LMNA mutated cells on substrates with a range of different stiffnesses can be used to probe the degree of nuclear fragility. This assay may be useful in predicting patient-specific phenotypic development and in investigations on the underlying mechanisms of nuclear and cellular fragility in laminopathies.