The nuclear lamina, an intermediate filament meshwork lining the inner nuclear membrane, is formed by the nuclear lamins (lamins A, C, B1, and B2). Defects or deficiencies in individual nuclear lamin ...proteins have been reported to elicit nuclear blebs (protrusions or outpouchings of the nuclear envelope) and increase susceptibility for nuclear membrane ruptures. It is unclear, however, howa complete absence of nuclear lamins would affect nuclear envelope morphology and nuclear membrane integrity (i.e., whether nuclear membrane blebs or protrusions would occur and, if not, whether cells would be susceptible to nuclear membrane ruptures). To address these issues, we generated mouse embryonic fibroblasts (MEFs) lacking all nuclear lamins. The nuclear lamin-deficient MEFs had irregular nuclear shapes but no nuclear blebs or protrusions. Despite a virtual absence of nuclear blebs, MEFs lacking nuclear lamins had frequent, prolonged, and occasionally nonhealing nuclear membrane ruptures. By transmission electron microscopy, the inner nuclear membrane in nuclear lamin-deficient MEFs have a “wavy” appearance, and there were discrete discontinuities in the inner and outer nuclear membranes. Nuclear membrane ruptures were accompanied by a large increase in DNA damage, as judged by γ-H2AX foci. Mechanical stress increased both nuclear membrane ruptures and DNA damage, whereas minimizing transmission of cytoskeletal forces to the nucleus had the opposite effects.
The nuclear envelope (NE) is a protective barrier to the genome, yet its membranes undergo highly dynamic remodeling processes that are necessary for cell growth and maintenance. While mechanisms by ...which proteins promote NE remodeling are emerging, the types of bilayer lipids and the lipid‐protein interactions that define and sculpt nuclear membranes remain elusive. The NE is continuous with the endoplasmic reticulum (ER) and recent evidence suggests that lipids produced in the ER are harnessed to remodel nuclear membranes. In this review, we examine new roles for lipid species made proximally within the ER and locally at the NE to control NE dynamics. We further explore how the biosynthesis of lipids coordinates NE remodeling to ensure genome protection.
The genome is surrounded by the nuclear envelope (NE), a sealed double membrane sheet that is contiguous with the endoplasmic reticulum (ER).The NE is highly dynamic and undergoes membrane remodeling such as breakdown, reformation, sealing, and growth.New data suggest that remodeling of the NE involves crosstalk and coordination with lipid synthesis, which occurs primarily within the ER.Some lipid synthesis enzymes also act at the NE and may locally regulate the biogenesis and remodeling of nuclear membranes.Insights into the specific chemistry and types of lipids required to remodel nuclear membranes are also emerging.Research into how the regulation of bilayer lipid production and composition is coupled to NE remodeling is key to understanding mechanisms underlying diseases related to loss of NE integrity.
Mutations in the LMNA gene, which encodes the nuclear envelope (NE) proteins lamins A/C, cause Emery-Dreifuss muscular dystrophy, congenital muscular dystrophy and other diseases collectively known ...as laminopathies. The mechanisms responsible for these diseases remain incompletely understood. Using three mouse models of muscle laminopathies and muscle biopsies from individuals with LMNA-related muscular dystrophy, we found that Lmna mutations reduced nuclear stability and caused transient rupture of the NE in skeletal muscle cells, resulting in DNA damage, DNA damage response activation and reduced cell viability. NE and DNA damage resulted from nuclear migration during skeletal muscle maturation and correlated with disease severity in the mouse models. Reduction of cytoskeletal forces on the myonuclei prevented NE damage and rescued myofibre function and viability in Lmna mutant myofibres, indicating that myofibre dysfunction is the result of mechanically induced NE damage. Taken together, these findings implicate mechanically induced DNA damage as a pathogenic contributor to LMNA skeletal muscle diseases.
Although mutations leading to a compromised nuclear envelope cause diseases such as muscular dystrophies or accelerated aging, the consequences of mechanically induced nuclear envelope ruptures are ...less known. Here, we show that nuclear envelope ruptures induce DNA damage that promotes senescence in non-transformed cells and induces an invasive phenotype in human breast cancer cells. We find that the endoplasmic reticulum (ER)-associated exonuclease TREX1 translocates into the nucleus after nuclear envelope rupture and is required to induce DNA damage. Inside the mammary duct, cellular crowding leads to nuclear envelope ruptures that generate TREX1-dependent DNA damage, thereby driving the progression of in situ carcinoma to the invasive stage. DNA damage and nuclear envelope rupture markers were also enriched at the invasive edge of human tumors. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.
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•Deformed nuclei and DNA damage are enriched at micro-invasive foci of breast cancer•TREX1 causes DNA damage in deformed nuclei upon nuclear envelope rupture events•TREX1 drives senescence in normal cells and an invasive phenotype in cancer cells•Nuclear envelope rupture events and DNA damage are observed in human tumors
Extreme nuclear deformations in dense microenvironments lead to repeated nuclear envelope rupture followed by TREX1-dependent chronic DNA damage. This triggers a partial EMT in malignant cells which might underlie tumor progression.
Yeast Fld1 and Ldb16 resemble mammalian seipin, implicated in neutral lipid storage. Both proteins form a complex at the endoplasmic reticulum-lipid droplet (LD) interface. Malfunction of this ...complex either leads to LD clustering or to the generation of supersized LD (SLD) in close vicinity to the nuclear envelope, in response to altered phospholipid (PL) composition. We show that similar to mutants lacking Fld1, deletion of LDB16 leads to abnormal proliferation of a subdomain of the nuclear envelope, which is tightly associated with clustered LD. The human lipin-1 ortholog, the PAH1 encoded phosphatidic acid (PA) phosphatase, and its activator Nem1 are highly enriched at this site. The specific accumulation of PA-binding marker proteins indicates a local enrichment of PA in the fld1 and ldb16 mutants. Furthermore, we demonstrate that clustered LD in fld1 or ldb16 mutants are transformed to SLD if phosphatidylcholine synthesis is compromised by additional deletion of the phosphatidylethanolamine methyltransferase, Cho2. Notably, treatment of wild-type cells with oleate induced a similar LD clustering and nuclear membrane proliferation phenotype as observed in fld1 and ldb16 mutants. These data suggest that the Fld1–Ldb16 complex affects PA homeostasis at an LD-forming subdomain of the nuclear envelope. Lack of Fld1–Ldb16 leads to locally elevated PA levels that induce an abnormal proliferation of nER membrane structures and the clustering of associated LD. We suggest that the formation of SLD is a consequence of locally altered PL metabolism at this site.
•Fld1–Ldb16 modulates the balance between neutral lipid and phospholipid synthesis at a LD-forming subdomain of the nuclear ER.•Fld1–Ldb16 affects the distribution of enzymes involved in PA to DAG conversion.•Formation of giant LD is a pleiotropic effect caused by locally altered phospholipid metabolism.
The nuclear pore complex (NPC) mediates nucleocytoplasmic transport through the nuclear envelope. How the NPC assembles into this double membrane boundary has remained enigmatic. Here, we captured ...temporally staged assembly intermediates by correlating live cell imaging with high-resolution electron tomography and super-resolution microscopy. Intermediates were dome-shaped evaginations of the inner nuclear membrane (INM), that grew in diameter and depth until they fused with the flat outer nuclear membrane. Live and super-resolved fluorescence microscopy revealed the molecular maturation of the intermediates, which initially contained the nuclear and cytoplasmic ring component Nup107, and only later the cytoplasmic filament component Nup358. EM particle averaging showed that the evagination base was surrounded by an 8-fold rotationally symmetric ring structure from the beginning and that a growing mushroom-shaped density was continuously associated with the deforming membrane. Quantitative structural analysis revealed that interphase NPC assembly proceeds by an asymmetric inside-out extrusion of the INM.
During differentiation, thousands of genes are repositioned toward or away from the nuclear envelope. These movements correlate with changes in transcription and replication timing. Using synthetic ...(TALE) transcription factors, we found that transcriptional activation of endogenous genes by a viral trans-activator is sufficient to induce gene repositioning toward the nuclear interior in embryonic stem cells. However, gene relocation was also induced by recruitment of an acidic peptide that décondenses chromatin without affecting transcription, indicating that nuclear reorganization is driven by chromatin remodeling rather than transcription. We identified an epigenetic inheritance of chromatin decondensation that maintained central nuclear positioning through mitosis even after the TALE transcription factor was lost. Our results also demonstrate that transcriptional activation, but not chromatin decondensation, is sufficient to change replication timing.
Abstract Although its properties have long been used for both typing and prognosis of various tumors, the nuclear envelope (NE) itself and its potential roles in tumorigenesis are only beginning to ...be understood. Historically viewed as merely a protective barrier, the nuclear envelope is now linked to a wide range of functions. Nuclear membrane proteins connect the nucleus to the cytoskeleton on one side and to chromatin on the other. Several newly identified nuclear envelope functions associated with these connections intersect with cancer pathways. For example, the nuclear envelope could affect genome stability by tethering chromatin. Some nuclear envelope proteins affect cell cycle regulation by directly binding to the master regulator pRb, others by interacting with TGF-ß and Smad signaling cascades, and others by affecting the mitotic spindle. Finally, the NE directly affects cytoskeletal organization and can also influence cell migration in metastasis. In this review we discuss the link between the nuclear envelope and cellular defects that are common in cancer cells, and we show that NE proteins are often aberrantly expressed in tumors. The NE represents a potential reservoir of diagnostic and prognostic markers in cancer.
Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the ...inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM.
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•Multimodal imaging reveals mechanism of vesicle formation at inner nuclear membrane•Nucleo-cytoplasmic cargo vesicle coat in situ comprises two distinct lattices•Lattices are formed by hexameric building blocks made of the nuclear egress complex•Induction of membrane curvature based solely on heterodimeric interactions
A multi-modal live-cell and cryo-imaging approach reveals how vesicles assemble at the inner nuclear membrane for transport to the cytoplasm during herpesvirus maturation. This also suggests a functional model for counterparts in uninfected cells that mediate nuclear egress of large cargo like ribonucleoprotein particles.
In eukaryotic cells, the nuclear envelope (NE) is a membrane partition between the nucleus and the cytoplasm to compartmentalize nuclear contents. It plays an important role in facilitating nuclear ...functions including transcription, DNA replication and repair. In mammalian cells, the NE breaks down and then reforms during cell division, and in interphase it is restored shortly after the NE rupture induced by mechanical force. In this way, the partitioning effect is regulated through dynamic processes throughout the cell cycle. A failure in rebuilding the NE structure triggers the mixing of nuclear and cytoplasmic contents, leading to catastrophic consequences for the nuclear functions. Whereas the precise details of molecular mechanisms for NE reformation during cell division and NE restoration in interphase are still being investigated, here, we mostly focus on mammalian cells to describe key aspects that have been identified and to discuss the crosstalk between them.
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