Epigenetic plasticity safeguards heterochromatin configuration in mammals.

Heterochromatin is a key architectural feature of eukaryotic chromosomes critical for cell type-specific gene expression and genome stability. In the mammalian nucleus, heterochromatin segregates from transcriptionally active genomic regions and exists in large, condensed, and inactive nuclear compartments. However, the mechanisms underlying the spatial organization of heterochromatin need to be better understood. Histone H3 lysine 9 trimethylation (H3K9me3) and lysine 27 trimethylation (H3K27me3) are two major epigenetic modifications that enrich constitutive and facultative heterochromatin, respectively. Mammals have at least five H3K9 methyltransferases (SUV39H1, SUV39H2, SETDB1, G9a and GLP) and two H3K27 methyltransferases (EZH1 and EZH2). In this study, we addressed the role of H3K9 and H3K27 methylation in heterochromatin organization using a combination of mutant cells for five H3K9 methyltransferases and an EZH1/2 dual inhibitor, DS3201. We showed that H3K27me3, which is normally segregated from H3K9me3, was redistributed to regions targeted by H3K9me3 after the loss of H3K9 methylation and that the loss of both H3K9 and H3K27 methylation resulted in impaired condensation and spatial organization of heterochromatin. Our data demonstrate that the H3K27me3 pathway safeguards heterochromatin organization after the loss of H3K9 methylation in mammalian cells.

The molecular architecture of lamins in somatic cells.

The nuclear lamina is a fundamental constituent of metazoan nuclei. It is composed mainly of lamins, which are intermediate filament proteins that assemble into a filamentous meshwork, bridging the nuclear envelope and chromatin. Besides providing structural stability to the nucleus, the lamina is involved in many nuclear activities, including chromatin organization, transcription and replication. However, the structural organization of the nuclear lamina is poorly understood. Here we use cryo-electron tomography to obtain a detailed view of the organization of the lamin meshwork within the lamina. Data analysis of individual lamin filaments resolves a globular-decorated fibre appearance and shows that A- and B-type lamins assemble into tetrameric filaments of 3.5 nm thickness. Thus, lamins exhibit a structure that is remarkably different from the other canonical cytoskeletal elements. Our findings define the architecture of the nuclear lamin meshworks at molecular resolution, providing insights into their role in scaffolding the nuclear lamina.

RNA polymerase II condensate formation and association with Cajal and histone locus bodies in living human cells.

In eukaryotic nuclei, a number of phase-separated nuclear bodies (NBs) are present. RNA polymerase II (Pol II) is the main player in transcription and forms large condensates in addition to localizing at numerous transcription foci. Cajal bodies (CBs) and histone locus bodies (HLBs) are NBs that are involved in transcriptional and post-transcriptional regulation of small nuclear RNA and histone genes. By live-cell imaging using human HCT116 cells, we here show that Pol II condensates (PCs) nucleated near CBs and HLBs, and the number of PCs increased during S phase concomitantly with the activation period of histone genes. Ternary PC-CB-HLB associates were formed via three pathways: nucleation of PCs and HLBs near CBs, interaction between preformed PC-HLBs with CBs and nucleation of PCs near preformed CB-HLBs. Coilin knockout increased the co-localization rate between PCs and HLBs, whereas the number, nucleation timing and phosphorylation status of PCs remained unchanged. Depletion of PCs did not affect CBs and HLBs. Treatment with 1,6-hexanediol revealed that PCs were more liquid-like than CBs and HLBs. Thus, PCs are dynamic structures often nucleated following the activation of gene clusters associated with other NBs.

Adaptive multiorientation resolution analysis of complex filamentous network images.

MOTIVATION: Microscopy images of cytoskeletal, nucleoskeletal and other structures contain complex junctions of overlapping filaments with arbitrary geometry. Yet, state-of-the-art algorithms generally perform single orientation analysis to segment these structures, resulting in gaps near junctions, or assume particular junction geometries to detect them. RESULTS: We developed a fully automated image analysis approach to address the challenge of determining the number of orientations and their values at each point in space to detect both lines and their junctions. Our approach does not assume any fixed number of orientations or any particular geometry in the case of multiple coincident orientations. It is based on analytically resolving coincident orientations revealed by steerable ridge filtering in an adaptive manner that balances orientation resolution and spatial localization. Combining this multiorientation resolution information with a generalization of the concept of non-maximum suppression allowed us to then identify the centers of lines and their junctions in an image. We validated our approach using a wide array of synthetic junctions and by comparison to manual segmentation. We also applied it to light microscopy images of cytoskeletal and nucleoskeletal networks. AVAILABILITY AND IMPLEMENTATION: https://github.com/mkitti/AdaptiveResolutionOrientationSpace. SUPPLEMENTARY INFORMATION: Supplementary information is available at Bioinformatics online.

Variable immersion microscopy with a high numerical aperture.

Three-dimensional (3D) optical microscopy with a high numerical aperture (NA) remains challenging for thick biological specimens owing to aberrations arising from interface refractions. We developed a variable immersion lens (VIL) to passively minimize these aberrations. A VIL is a high-NA concentric meniscus lens and was used in combination with an aberration-corrected high-NA reflecting objective (TORA-FUJI mirror). Wave-optics simulation at a wavelength of 488 nm showed that a VIL microscope enables diffraction-limited 1.2-NA imaging in water (refractive index of 1.34) at a depth of 0.3 mm by minimizing aberrations due to refraction of a sample interface. Another aberration due to the refractive index mismatching between a mounting medium, and an object can also be corrected by the VIL system, because various fluids with different refractive indices can be used as mounting media for the VIL. As a result of correcting the two aberrations at the same time, we experimentally demonstrated that a 6 µm diameter fluorescent bead can be imaged to the true dimensions in 3D.

Autophagy mediates degradation of nuclear lamina.

Macroautophagy (hereafter referred to as autophagy) is a catabolic membrane trafficking process that degrades a variety of cellular constituents and is associated with human diseases. Although extensive studies have focused on autophagic turnover of cytoplasmic materials, little is known about the role of autophagy in degrading nuclear components. Here we report that the autophagy machinery mediates degradation of nuclear lamina components in mammals. The autophagy protein LC3/Atg8, which is involved in autophagy membrane trafficking and substrate delivery, is present in the nucleus and directly interacts with the nuclear lamina protein lamin B1, and binds to lamin-associated domains on chromatin. This LC3-lamin B1 interaction does not downregulate lamin B1 during starvation, but mediates its degradation upon oncogenic insults, such as by activated RAS. Lamin B1 degradation is achieved by nucleus-to-cytoplasm transport that delivers lamin B1 to the lysosome. Inhibiting autophagy or the LC3-lamin B1 interaction prevents activated RAS-induced lamin B1 loss and attenuates oncogene-induced senescence in primary human cells. Our study suggests that this new function of autophagy acts as a guarding mechanism protecting cells from tumorigenesis.

The role of nuclear lamin B1 in cell proliferation and senescence.

Nuclear lamin B1 (LB1) is a major structural component of the nucleus that appears to be involved in the regulation of many nuclear functions. The results of this study demonstrate that LB1 expression in WI-38 cells decreases during cellular senescence. Premature senescence induced by oncogenic Ras also decreases LB1 expression through a retinoblastoma protein (pRb)-dependent mechanism. Silencing the expression of LB1 slows cell proliferation and induces premature senescence in WI-38 cells. The effects of LB1 silencing on proliferation require the activation of p53, but not pRb. However, the induction of premature senescence requires both p53 and pRb. The proliferation defects induced by silencing LB1 are accompanied by a p53-dependent reduction in mitochondrial reactive oxygen species (ROS), which can be rescued by growth under hypoxic conditions. In contrast to the effects of LB1 silencing, overexpression of LB1 increases the proliferation rate and delays the onset of senescence of WI-38 cells. This overexpression eventually leads to cell cycle arrest at the G1/S boundary. These results demonstrate the importance of LB1 in regulating the proliferation and senescence of human diploid cells through a ROS signaling pathway.

Deleterious assembly of the lamin A/C mutant p.S143P causes ER stress in familial dilated cardiomyopathy.

Mutation of the LMNA gene, encoding nuclear lamin A and lamin C (hereafter lamin A/C), is a common cause of familial dilated cardiomyopathy (DCM). Among Finnish DCM patients, the founder mutation c.427T>C (p.S143P) is the most frequently reported genetic variant. Here, we show that p.S143P lamin A/C is more nucleoplasmic and soluble than wild-type lamin A/C and accumulates into large intranuclear aggregates in a fraction of cultured patient fibroblasts as well as in cells ectopically expressing either FLAG- or GFP-tagged p.S143P lamin A. In fluorescence loss in photobleaching (FLIP) experiments, non-aggregated EGFP-tagged p.S143P lamin A was significantly more dynamic. In in vitro association studies, p.S143P lamin A failed to form appropriate filament structures but instead assembled into disorganized aggregates similar to those observed in patient cell nuclei. A whole-genome expression analysis revealed an elevated unfolded protein response (UPR) in cells expressing p.S143P lamin A/C. Additional endoplasmic reticulum (ER) stress induced by tunicamycin reduced the viability of cells expressing mutant lamin further. In summary, p.S143P lamin A/C affects normal lamina structure and influences the cellular stress response, homeostasis and viability.

Interphase phosphorylation of lamin A.

Nuclear lamins form the major structural elements that comprise the nuclear lamina. Loss of nuclear structural integrity has been implicated as a key factor in the lamin A/C gene mutations that cause laminopathies, whereas the normal regulation of lamin A assembly and organization in interphase cells is still undefined. We assumed phosphorylation to be a major determinant, identifying 20 prime interphase phosphorylation sites, of which eight were high-turnover sites. We examined the roles of these latter sites by site-directed mutagenesis, followed by detailed microscopic analysis - including fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and nuclear extraction techniques. The results reveal three phosphorylation regions, each with dominant sites, together controlling lamin A structure and dynamics. Interestingly, two of these interphase sites are hyper-phosphorylated in mitotic cells and one of these sites is within the sequence that is missing in progerin of the Hutchinson-Gilford progeria syndrome. We present a model where different phosphorylation combinations yield markedly different effects on the assembly, subunit turnover and the mobility of lamin A between, and within, the lamina, the nucleoplasm and the cytoplasm of interphase cells.

Mechanical model of blebbing in nuclear lamin meshworks.

Much of the structural stability of the nucleus comes from meshworks of intermediate filament proteins known as lamins forming the inner layer of the nuclear envelope called the nuclear lamina. These lamin meshworks additionally play a role in gene expression. Abnormalities in nuclear shape are associated with a variety of pathologies, including some forms of cancer and Hutchinson-Gilford Progeria Syndrome, and often include protruding structures termed nuclear blebs. These nuclear blebs are thought to be related to pathological gene expression; however, little is known about how and why blebs form. We have developed a minimal continuum elastic model of a lamin meshwork that we use to investigate which aspects of the meshwork could be responsible for bleb formation. Mammalian lamin meshworks consist of two types of lamin proteins, A type and B type, and it has been reported that nuclear blebs are enriched in A-type lamins. Our model treats each lamin type separately and thus, can assign them different properties. Nuclear blebs have been reported to be located in regions where the fibers in the lamin meshwork have a greater separation, and we find that this greater separation of fibers is an essential characteristic for generating nuclear blebs. The model produces structures with comparable morphologies and distributions of lamin types as real pathological nuclei. Thus, preventing this opening of the meshwork could be a route to prevent bleb formation, which could be used as a potential therapy for the pathologies associated with nuclear blebs.

Meeting report: mitosis and nuclear structure.

The Company of Biologists Workshop entitled 'Mitosis and Nuclear Structure' was held at Wiston House, West Sussex in June 2013. It provided a unique and timely opportunity for leading experts from different fields to discuss not only their own work but also its broader context. Here we present the proceedings of this meeting and several major themes that emerged from the crosstalk between the two, as it turns out, not so disparate fields of mitosis and nuclear structure. Co-chaired by Katherine Wilson (Johns Hopkins School of Medicine, Baltimore, MD), Timothy Mitchison (Harvard University, Cambridge, MA) and Michael Rout (Rockefeller University, New York, NY), this workshop brought together a small group of scientists from a range of disciplines to discuss recent advances and connections between the areas of mitosis and nuclear structure research. Several early-career researchers (students, postdoctoral researchers, junior faculty) participated along with 20 senior scientists, including the venerable and affable Nobel Laureate Tim Hunt. Participants were encouraged to embrace unconventional thinking in the 'scientific sandbox' created by this unusual combination of researchers in the inspiring, isolated setting of the 16th-century Wiston House.

Nuclear lamins and oxidative stress in cell proliferation and longevity.

In mammalian cells, the nuclear lamina is composed of a complex fibrillar network associated with the inner membrane of the nuclear envelope. The lamina provides mechanical support for the nucleus and functions as the major determinant of its size and shape. At its innermost aspect it associates with peripheral components of chromatin and thereby contributes to the organization of interphase chromosomes. The A- and B-type lamins are the major structural components of the lamina, and numerous mutations in the A-type lamin gene have been shown to cause many types of human diseases collectively known as the laminopathies. These mutations have also been shown to cause a disruption in the normal interactions between the A and B lamin networks. The impact of these mutations on nuclear functions is related to the roles of lamins in regulating various essential processes including DNA synthesis and damage repair, transcription and the regulation of genes involved in the response to oxidative stress. The major cause of oxidative stress is the production of reactive oxygen species (ROS), which is critically important for cell proliferation and longevity. Moderate increases in ROS act to initiate signaling pathways involved in cell proliferation and differentiation, whereas excessive increases in ROS cause oxidative stress, which in turn induces cell death and/or senescence. In this review, we cover current findings about the role of lamins in regulating cell proliferation and longevity through oxidative stress responses and ROS signaling pathways. We also speculate on the involvement of lamins in tumor cell proliferation through the control of ROS metabolism.

Crosstalk between mitotic reassembly and repair of the nuclear envelope.

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.

Chromosomal regions associated with prostate cancer risk localize to lamin B-deficient microdomains and exhibit reduced gene transcription.

The lamins are major determinants of nuclear shape and chromatin organization and these features are frequently altered in prostate cancer (CaP). Human CaP cell lines frequently have nuclear lobulations, which are enriched in A-type lamins but lack B-type lamins and have been defined as lamin B-deficient microdomains (LDMDs). LDMD frequency is correlated with CaP cell line aggressiveness and increased cell motility. In addition, LNCaP cells grown in the presence of dihydrotestosterone (DHT) show an increased frequency of LDMDs. The LDMDs are enriched in activated RNA polymerase II (Pol IIo) and androgen receptor (AR) and A-type lamins form an enlarged meshwork that appears to co-align with chromatin fibres and AR. Furthermore, fluorescence in situ hybridization and comparative genomic hybridization demonstrated that chromosomal regions associated with CaP susceptibility are preferentially localized to LDMDs. Surprisingly, these regions lack histone marks for transcript elongation and exhibit reduced BrU incorporation, suggesting that Pol II is stalled within LDMDs. Real-time PCR of genes near androgen response elements (AREs) was used to compare transcription between cells containing LDMDs and controls. Genes preferentially localized to LDMDs showed significantly decreased expression, while genes in the main nuclear body were largely unaffected. Furthermore, LDMDs were observed in human CaP tissue and the frequency was correlated with increased Gleason grade. These results imply that lamins are involved in chromatin organization and Pol II transcription, and provide insights into the development and progression of CaP.

A progeria mutation reveals functions for lamin A in nuclear assembly, architecture, and chromosome organization.

Numerous mutations in the human A-type lamin gene (LMNA) cause the premature aging disease, progeria. Some of these are located in the alpha-helical central rod domain required for the polymerization of the nuclear lamins into higher order structures. Patient cells with a mutation in this domain, 433G>A (E145K) show severely lobulated nuclei, a separation of the A- and B-type lamins, alterations in pericentric heterochromatin, abnormally clustered centromeres, and mislocalized telomeres. The induction of lobulations and the clustering of centromeres originate during postmitotic nuclear assembly in daughter cells and this early G1 configuration of chromosomes is retained throughout interphase. In vitro analyses of E145K-lamin A show severe defects in the assembly of protofilaments into higher order lamin structures. The results show that this central rod domain mutation affects nuclear architecture in a fashion distinctly different from the changes found in the most common form of progeria caused by the expression of LADelta50/progerin. The study also emphasizes the importance of lamins in nuclear assembly and chromatin organization.

Superfluid helium nanoscope insert with millimeter working range.

A superfluid helium insert was developed for cryogenic microscopy of millimeter-sized specimens. An optical-interferometric position sensor, cryogenic objective mirror, and piezo-driven cryogenic stage were fixed to an insert holder that was immersed in superfluid helium. The single-component design stabilized the three-dimensional position of the sample, with root-mean-square deviations of (x, lateral) 0.33 nm, (y, lateral) 0.29 nm, and (z, axial) 0.25 nm. Because of the millimeter working range of the optical sensor, the working range of the sample under the active stabilization was (x, y) 5 mm and (z) 3 mm in superfluid helium at 1.8 K. The insert was used to obtain the millimeter-sized fluorescence image of cell nuclei at 1.8 K without a sample exchange.

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 the nucleoplasmic pool of lamin C rapidly accumulated at sites of NE rupture induced by laser microirradiation in mouse embryonic fibroblasts. The accumulation of lamin C at the rupture sites required both the immunoglobulin-like fold domain that binds to barrier-to-autointegration factor (BAF) and a nuclear localization signal. The accumulation of nuclear BAF and cytoplasmic cyclic GMP-AMP 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 rapid repair.

Computational analyses reveal spatial relationships between nuclear pore complexes and specific lamins.

Nuclear lamin isoforms form fibrous meshworks associated with nuclear pore complexes (NPCs). Using datasets prepared from subpixel and segmentation analyses of 3D-structured illumination microscopy images of WT and lamin isoform knockout mouse embryo fibroblasts, we determined with high precision the spatial association of NPCs with specific lamin isoform fibers. These relationships are retained in the enlarged lamin meshworks of Lmna-/- and Lmnb1-/- fibroblast nuclei. Cryo-ET observations reveal that the lamin filaments composing the fibers contact the nucleoplasmic ring of NPCs. Knockdown of the ring-associated nucleoporin ELYS induces NPC clusters that exclude lamin A/C fibers but include LB1 and LB2 fibers. Knockdown of the nucleoporin TPR or NUP153 alters the arrangement of lamin fibers and NPCs. Evidence that the number of NPCs is regulated by specific lamin isoforms is presented. Overall the results demonstrate that lamin isoforms and nucleoporins act together to maintain the normal organization of lamin meshworks and NPCs within the nuclear envelope.

A mosaic of old and young nucleoporins.

Some nucleoporins, the nuclear pore complex (NPC) components, have exceptionally long lifetimes. In this issue, Toyama et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201809123) report that NPCs are maintained by a slow piecemeal replacement of NPC components in dividing and terminally differentiated cells and by whole-pore exchange in quiescent cells.