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The nuclear envelope plays an essential role in organizing the genome inside of the nucleus. The inner nuclear membrane is coated with a meshwork of filamentous lamin proteins that provide a surface to organize a variety of cellular processes. A subset of nuclear lamina- and membrane-associated proteins functions as anchors to hold transcriptionally silent heterochromatin at the nuclear periphery. While most chromatin tethers are integral membrane proteins, a limited number are lamina-bound. One example is the mammalian proline-rich 14 (PRR14) protein. PRR14 is a recently characterized protein with unique function that is different from other known chromatin tethers. Here, we review our current understanding of PRR14 structure and function in organizing heterochromatin at the nuclear periphery., (© 2023 Federation of European Biochemical Societies.)

Background: Lamina-associated domains (LADs) are large genomic regions that are positioned at the nuclear lamina. It has remained largely unclear what drives the positioning and demarcation of LADs. Because the insulator protein CTCF is enriched at LAD borders, it was postulated that CTCF binding could position some LAD boundaries, possibly through its function in stalling cohesin and hence preventing cohesin invading into the LAD. To test this, we mapped genome-nuclear lamina interactions in mouse embryonic stem cells after rapid depletion of CTCF and other perturbations of cohesin dynamics., Results: CTCF and cohesin contribute to a sharp transition in lamina interactions at LAD borders, while LADs are maintained after depletion of these proteins, also at borders marked by CTCF. CTCF and cohesin may thus reinforce LAD borders, but do not position these. CTCF binding sites within LADs are locally detached from the lamina and enriched for accessible DNA and active histone modifications. Remarkably, despite lamina positioning being strongly correlated with genome inactivity, this DNA remains accessible after the local detachment is lost following CTCF depletion. At a chromosomal scale, cohesin depletion and cohesin stabilization by depletion of the unloading factor WAPL quantitatively affect lamina interactions, indicative of perturbed chromosomal positioning in the nucleus. Finally, while H3K27me3 is locally enriched at CTCF-marked LAD borders, we find no evidence for an interplay between CTCF and H3K27me3 on lamina interactions., Conclusions: These findings illustrate that CTCF and cohesin are not primary determinants of LAD patterns. Rather, these proteins locally modulate NL interactions., (© 2022. The Author(s).)

The link between genomic structure and biological function is yet to be consolidated, it is, however, clear that physical manipulation of the genome, driven by the activity of a variety of proteins, is a crucial step. To understand the consequences of the physical forces underlying genome organization, we build a coarse-grained polymer model of the genome, featuring three fundamentally distinct classes of interactions: lengthwise compaction, i.e., compaction of chromosomes along its contour, self-adhesion among epigenetically similar genomic segments, and adhesion of chromosome segments to the nuclear envelope or lamina. We postulate that these three types of interactions sufficiently represent the concerted action of the different proteins organizing the genome architecture and show that an interplay among these interactions can recapitulate the architectural variants observed across the tree of life. The model elucidates how an interplay of forces arising from the three classes of genomic interactions can drive drastic, yet predictable, changes in the global genome architecture, and makes testable predictions. We posit that precise control over these interactions in vivo is key to the regulation of genome architecture., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

More than one third of the mammalian genome is in a close association with the nuclear lamina, thus these genomic regions were termed lamina-associated domains (LADs). This association is fundamental for many aspects of chromatin biology including transcription, replication, and DNA damage repair. LADs association with the nuclear envelope is thought to be dependent on two major mechanisms: The first mechanism is the interaction between nuclear membrane proteins such as LBR with heterochromatin modifications that are enriched in LADs chromatin. The second mechanism is based on proteins that bind the borders of the LADs and support the association of the LADs with the nuclear envelope. Two factors were suggested to support the second mechanism: CCCTC-binding factor (CTCF) and YY1 based on their enriched binding to LADs borders. However, this mechanism has not been proven yet at a whole genome level. Here, to test if CTCF supports the LADs landscape, we generated melanoma cells with a partial loss of function (pLoF) of CTCF by the CRISPR-Cas9 system and determined the LADs landscape by lamin B ChIP-seq analysis. We found that under regular growth conditions, CTCF pLoF led to modest changes in the LADs landscape that included an increase in the signal of 2% of the LADs and a decrease in the signal of 8% of the LADs. However, CTCF importance for the LADs landscape was much higher upon induction of a chromatin stress. We induced chromatin stress by inhibiting RNA polymerase II, an intervention that is known to alter chromatin compaction and supercoiling. Notably, only in CTCF pLoF cells, the chromatin stress led to the dissociation of 7% of the LADs from the lamina. The CTCF-dependent LADs had almost three times shorter median length than the non-affected LADs, were enriched in CTCF binding at their borders, and were higher in their facultative-status (cell-type specific). Thus, it appears that CTCF is a key factor in facilitating the association of short facultative LADs with the nuclear lamina upon chromatin stress., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The organization of DNA within the eukaryotic nucleus is important for cellular processes such as regulation of gene expression and repair of DNA damage. To comprehend cell-to-cell variation within a complex system, systematic analysis of individual cells is necessary. While many tools exist to capture DNA conformation and chromatin context, these methods generally require large populations of cells for sufficient output. Here we describe single-cell DamID, a technique to capture contacts between DNA and a given protein of interest. By fusing the bacterial methyltransferase Dam to nuclear lamina protein lamin B1, genomic regions in contact with the nuclear periphery can be mapped. Single-cell DamID generates contact maps with sufficient throughput and resolution to reliably identify patterns of similarity as well as variation in nuclear organization of interphase chromosomes.

At the nuclear periphery, associations of chromatin with the nuclear lamina through lamina-associated domains (LADs) aid functional organization of the genome. We review the organization of LADs and provide evidence of LAD heterogeneity from cell ensemble and single-cell data. LADs are typically repressive environments in the genome; nonetheless, we discuss findings of lamin interactions with regulatory elements of active genes, and the role lamins may play in genome regulation. We address the relationship between LADs and other genome organizers, and the involvement of LADs in laminopathies. The current data lay the basis for future studies on the significance of lamin-chromatin interactions in health and disease.

The nucleus of oocytes (germinal vesicle) is unusually large and its nuclear envelope (NE) is densely packed with nuclear pore complexes (NPCs) that are stockpiled for embryonic development. We showed that breakdown of this specialized NE is mediated by an Arp2/3-nucleated F-actin 'shell' in starfish oocytes, in contrast to microtubule-driven tearing in mammalian fibroblasts. Here, we address the mechanism of F-actin-driven NE rupture by correlated live-cell, super-resolution and electron microscopy. We show that actin is nucleated within the lamina, sprouting filopodia-like spikes towards the nuclear membranes. These F-actin spikes protrude pore-free nuclear membranes, whereas the adjoining stretches of membrane accumulate NPCs that are associated with the still-intact lamina. Packed NPCs sort into a distinct membrane network, while breaks appear in ER-like, pore-free regions. We reveal a new function for actin-mediated membrane shaping in nuclear rupture that is likely to have implications in other contexts, such as nuclear rupture observed in cancer cells., Competing Interests: NW, IA, PM, CG, HK, MM, PL No competing interests declared, (© 2020, Wesolowska et al.)

Phasor-assisted Metal Induced Energy Transfer-Fluorescence Lifetime Imaging Microscopy (MIET-FLIM) nanoscopy is introduced as a powerful tool for functional cell biology research. Thin metal substrates can be used to obtain axial super-resolution via nanoscale distance-dependent MIET from fluorescent dyes towards a nearby metal layer, thereby creating fluorescence lifetime contrast between dyes located at different nanoscale distance from the metal. Such data can be used to achieve axially super-resolved microscopy images, a process known as MIET-FLIM nanoscopy. Suitability of the phasor approach in MIET-FLIM nanoscopy is first demonstrated using nanopatterned substrates, and furthermore applied to characterize the distance distribution of the epithelial basal membrane of a biological cell from the gold substrate. The phasor plot of an entire cell can be used to characterize the full Förster resonance energy transfer (FRET) trajectory as a large distance heterogeneity within the sensing range of about 100 nm from the metal surface is present due to the extended shape of cell with curvatures. In contrast, the different proteins of nuclear lamina show strong confinement close to the nuclear envelope in nanoscale. We find the lamin B layer resides in average at shorter distances from the gold surface compared to the lamin A/C layer located in more extended ranges. This and the observed heterogeneity of the protein layer thicknesses suggests that A- and B-type lamins form distinct networks in the nuclear lamina. Our results provide detailed insights for the study of the different roles of lamin proteins in chromatin tethering and nuclear mechanics., (Copyright © 2019. Published by Elsevier B.V.)

The nuclear lamina consists of a dense fibrous meshwork of nuclear lamins, Type V intermediate filaments, and is ~14 nm thick according to recent cryo-electron tomography studies. Recent advances in light microscopy have extended the resolution to a scale allowing for the fine structure of the lamina to be imaged in the context of the whole nucleus. We review quantitative approaches to analyze the imaging data of the nuclear lamina as acquired by structured illumination microscopy (SIM) and single molecule localization microscopy (SMLM), as well as the requisite cell preparation techniques. In particular, we discuss the application of steerable filters and graph-based methods to segment the structure of the four mammalian lamin isoforms (A, C, B1, and B2) and extract quantitative information.

Mammalian nuclei are equipped with a framework of intermediate filaments that function as a karyoskeleton. This nuclear scaffold, formed primarily by lamins (A-type and B-type), maintains the spatial and functional organization of the genome and of sub-nuclear compartments. Over the past decade, a body of evidence has highlighted the significance of these structural nuclear proteins in the maintenance of nuclear architecture and mechanical stability, as well as genome function and integrity. The importance of these structures is now unquestioned given the wide range of degenerative diseases that stem from LMNA gene mutations, including muscular dystrophy disorders, peripheral neuropathies, lipodystrophies, and premature aging syndromes. Here, we review our knowledge about how alterations in nuclear lamins, either by mutation or reduced expression, impact cellular mechanisms that maintain genome integrity. Despite the fact that DNA replication is the major source of DNA damage and genomic instability in dividing cells, how alterations in lamins function impact replication remains minimally explored. We summarize recent studies showing that lamins play a role in DNA replication, and that the DNA damage that accumulates upon lamins dysfunction is elicited in part by deprotection of replication forks. We also discuss the emerging model that DNA damage and replication stress are "sensed" at the cytoplasm by proteins that normally survey this space in search of foreign nucleic acids. In turn, these cytosolic sensors activate innate immune responses, which are materializing as important players in aging and cancer, as well as in the response to cancer immunotherapy.

The nuclear lamina contributes to the regulation of gene expression and to chromatin organization. Mutations in A-type nuclear lamins cause laminopathies, some of which are associated with a loss of heterochromatin at the nuclear periphery. Until recently however, little if any information has been provided on where and how lamin A interacts with the genome and on how disease-causing lamin A mutations may rearrange genome conformation. Here, we review aspects of nuclear lamin association with the genome. We highlight recent evidence of reorganization of lamin A-chromatin interactions in cellular models of laminopathies, and implications on the 3-dimensional rearrangement of chromatin in these models, including patient cells. We discuss how a hot-spot lipodystrophic lamin A mutation alters chromatin conformation and epigenetic patterns at an anti-adipogenic locus, and conclude with remarks on links between lamin A, Polycomb and the pathophysiology of laminopathies. The recent findings presented here collectively argue towards a deregulation of large-scale and local spatial genome organization by a subset of lamin A mutations causing laminopathies.

The nucleus is a mechanosensitive and load-bearing structure. Structural components of the nucleus interact to maintain nuclear integrity and have become subjects of exciting research that is relevant to cell and developmental biology. Here we outline the boundaries of what is known about key architectural elements within the nucleus and highlight their potential structural and transcriptional regulatory functions., (Copyright © 2017. Published by Elsevier Ltd.)

In metazoan cell nuclei, hundreds of large chromatin domains are in close contact with the nuclear lamina. Such lamina-associated domains (LADs) are thought to help organize chromosomes inside the nucleus and have been associated with gene repression. Here, we discuss the properties of LADs, the molecular mechanisms that determine their association with the nuclear lamina, their dynamic links with other nuclear compartments, and their proposed roles in gene regulation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.

Intermediate filament (IF) proteins are known mainly by their propensity to form viscoelastic filamentous networks within cells. In addition, IF-proteins are essential parts of various biological materials, such as horn and hagfish slime threads, which exhibit a range of mechanical properties from hard to elastic. These properties and their self-assembly nature made IF-proteins attractive building blocks for biomimetic and biological materials in diverse applications. Here we show that a type V IF-protein, the Caenorhabditis elegans nuclear lamin (Ce-lamin), is a promising building block for protein-based fibers. Electron cryo-tomography of vitrified sections enabled us to depict the higher ordered assembly of the Ce-lamin into macroscopic fibers through the creation of paracrystalline fibers, which are prominent in vitro structures of lamins. The lamin fibers respond to tensile force as other IF-protein-based fibers, i.e., hagfish slime threads, and possess unique mechanical properties that may potentially be used in certain applications. The self-assembly nature of lamin proteins into a filamentous structure, which is further assembled into a complex network, can be easily modulated. This knowledge may lead to a better understanding of the relationship in IF-proteins-based fibers and materials, between their hierarchical structures and their mechanical properties., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The nuclear lamina represents a multifunctional platform involved in such diverse yet interconnected processes as spatial organization of the genome, maintenance of mechanical stability of the nucleus, regulation of transcription and replication. Most of lamina activities are exerted through tethering of lamina-associated chromatin domains (LADs) to the nuclear periphery. Yet, the lamina is a dynamic structure demonstrating considerable expansion during the cell cycle to accommodate increased number of LADs formed during DNA replication. We analyzed dynamics of nuclear growth during interphase and changes in lamina structure as a function of cell cycle progression. The nuclear lamina demonstrates steady growth from G1 till G2, while quantitative analysis of lamina meshwork by super-resolution microscopy revealed that microdomain organization of the lamina is maintained, with lamin A and lamin B microdomain periodicity and interdomain gap sizes unchanged. FRAP analysis, in contrast, demonstrated differences in lamin A and B1 exchange rates; the latter showing higher recovery rate in S-phase cells. In order to further analyze the mechanism of lamina growth in interphase, we generated a lamina-free nuclear envelope in living interphase cells by reversible hypotonic shock. The nuclear envelope in nuclear buds formed after such a treatment initially lacked lamins, and analysis of lamina formation revealed striking difference in lamin A and B1 assembly: lamin A reassembled within 30 min post-treatment, whereas lamin B1 did not incorporate into the newly formed lamina at all. We suggest that in somatic cells lamin B1 meshwork growth is coordinated with replication of LADs, and lamin A meshwork assembly seems to be chromatin-independent process.

Nuclear lamins play central roles at the intersection between cytoplasmic signalling and nuclear events. Here, we show that at least two N- and C-terminal lamin epitopes are not accessible at the basal side of the nuclear envelope under environmental conditions known to upregulate cell contractility. The conformational epitope on the Ig-domain of A-type lamins is more buried in the basal than apical nuclear envelope of human mesenchymal stem cells undergoing osteogenesis (but not adipogenesis), and in fibroblasts adhering to rigid (but not soft) polyacrylamide hydrogels. This structural polarization of the lamina is promoted by compressive forces, emerges during cell spreading, and requires lamin A/C multimerization, intact nucleoskeleton-cytoskeleton linkages (LINC), and apical-actin stress-fibre assembly. Notably, the identified Ig-epitope overlaps with emerin, DNA and histone binding sites, and comprises various laminopathy mutation sites. Our findings should help decipher how the physical properties of cellular microenvironments regulate nuclear events.

In the interphase nucleus, chromatin is organized into three-dimensional conformation to coordinate genome functions. The lamina-chromatin association is important to facilitate higher-order chromatin in mammalian cells, but its biological significances and molecular mechanisms remain poorly understood. One obstacle is that the list of lamina-associated proteins remains limited, presumably due to the inherent insolubility of lamina proteins. In this report, we identified 182 proteins associated with lamin B1 (a constitutive component of lamina) in mouse hepatocytes, by adopting virus-based proximity-dependent biotin identification. These proteins are functionally related to biological processes such as chromatin organization. As an example, we validated the association between lamin B1 and core histone macroH2A1, a histone associated with repressive chromatin. Furthermore, we mapped Lamina-associated domains (LADs) in mouse liver cells and found that boundaries of LADs are enriched for macroH2A. More interestingly, knocking-down of macroH2A1 resulted in the release of heterochromatin foci marked by histone lysine 9 trimethylation (H3K9me3) and the decondensation of global chromatin structure. However, down-regulation of lamin B1 led to redistribution of macroH2A1. Taken together, our data indicated that macroH2A1 is associated with lamina and is required to maintain chromatin architecture in mouse liver cells.

The nuclear lamina is a key structural element of the metazoan nucleus. However, the structural organization of the major proteins composing the lamina is poorly defined. Using three-dimensional structured illumination microscopy and computational image analysis, we characterized the supramolecular structures of lamin A, C, B1, and B2 in mouse embryo fibroblast nuclei. Each isoform forms a distinct fiber meshwork, with comparable physical characteristics with respect to mesh edge length, mesh face area and shape, and edge connectivity to form faces. Some differences were found in face areas among isoforms due to variation in the edge lengths and number of edges per face, suggesting that each meshwork has somewhat unique assembly characteristics. In fibroblasts null for the expression of either lamins A/C or lamin B1, the remaining lamin meshworks are altered compared with the lamin meshworks in wild-type nuclei or nuclei lacking lamin B2. Nuclei lacking LA/C exhibit slightly enlarged meshwork faces and some shape changes, whereas LB1-deficient nuclei exhibit primarily a substantial increase in face area. These studies demonstrate that individual lamin isoforms assemble into complex networks within the nuclear lamina and that A- and B-type lamins have distinct roles in maintaining the organization of the nuclear lamina., (© 2015 Shimi, Kittisopikul, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

This article discusses three reviews on the theme of nuclear organization.

Gradual loss of tissue function (or homeostasis) is a natural process of aging and is believed to cause many age-associated diseases. In human epidemiology studies, the low-grade and chronic systemic inflammation in elderly has been correlated with the development of aging related pathologies. Although it is suspected that tissue decline is related to systemic inflammation, the cause and consequence of these aging phenomena are poorly understood. By studying the Drosophila fat body and gut, we have uncovered a mechanism by which lamin-B loss in the fat body upon aging induces age-associated systemic inflammation. This chronic inflammation results in the repression of gut local immune response, which in turn leads to the over-proliferation and mis-differentiation of the intestinal stem cells, thereby resulting in gut hyperplasia. Here we discuss the implications and remaining questions in light of our published findings and new observations.

Lamin A/C is part of the nuclear lamina, a meshwork of intermediate filaments underlying the inner nuclear membrane. The lamin network is anchoring a complex set of structural and linker proteins and is either directly or through partner proteins also associated or interacting with a number of signaling protein and transcription factors. During mitosis the nuclear lamina is dissociated by well established phosphorylation- dependent mechanisms. A-type lamins are, however, also phosphorylated during interphase. A recent study identified 20 interphase phosphorylation sites on lamin A/C and explored their functions related to lamin dynamics; movements, localization and solubility. Here we discuss these findings in the light of lamin functions in health and disease.

The cell nucleus is structurally and functionally organized by lamins, intermediate filament proteins that form the nuclear lamina. Point mutations in genes that encode a specific subset of lamins, the A-type lamins, cause a spectrum of diseases termed laminopathies. Recent evidence points to a role for A-type lamins in intracellular redox homeostasis. To determine whether lamin A/C depletion and prelamin A accumulation differentially induce oxidative stress, we have performed a quantitative microscopy-based analysis of reactive oxygen species (ROS) levels and mitochondrial membrane potential (Δψm) in human fibroblasts subjected to sustained siRNA-mediated knockdown of LMNA and ZMPSTE24, respectively. We measured a highly significant increase in basal ROS levels and an even more prominent rise of induced ROS levels in lamin A/C depleted cells, eventually resulting in Δψm hyperpolarization and apoptosis. Depletion of ZMPSTE24 on the other hand, triggered a senescence pathway that was associated with moderately increased ROS levels and a transient Δψm depolarization. Both knockdowns were accompanied by an upregulation of several ROS detoxifying enzymes. Taken together, our data suggest that both persistent prelamin A accumulation and lamin A/C depletion elevate ROS levels, but to a different extent and with different effects on cell fate. This may contribute to the variety of disease phenotypes witnessed in laminopathies.

Emerin is a conserved membrane component of nuclear lamina structure. Here, we report an advance in understanding the molecular basis of emerin function: intermolecular emerin-emerin association. There were two modes: one mediated by association of residues 170-220 in one emerin molecule to residues 170-220 in another, and the second involving residues 170-220 and 1-132. Deletion analysis showed residues 187-220 contain a positive element essential for intermolecular association in cells. By contrast, deletion of residues 168-186 inactivated a proposed negative element, required to limit or control association. Association of GFP-emerin with nuclear BAF in cells required the LEM domain (residues 1-47) and the positive element. Emerin peptide arrays revealed direct binding of residues 170-220 to residues 206-225 (the proposed positive element), residues 147-174 (particularly P(153)MYGRDSAYQSITHYRP(169)) and the LEM domain. Emerin residues 1-132 and 159-220 were each sufficient to bind lamin A or B1 tails in vitro, identifying two independent regions of molecular contact with lamins. These results, and predicted emerin intrinsic disorder, support the hypothesis that there are multiple 'backbone' and LEM-domain configurations in a proposed intermolecular emerin network at the nuclear envelope., (© 2014. Published by The Company of Biologists Ltd.)

The nuclear periphery has been implicated in gene regulation and it has been proposed that proximity to the nuclear lamina and inner nuclear membrane (INM) leads to gene repression. More recently, it appears that there is a correlation and interdependence between lamina associated domains (LADs), the epigenome and overall three-dimensional architecture of the genome. However, the mechanisms of such organization at the 'peripheral zone' and the functional significance of these associations are poorly understood. The role these domains play in development and disease is an active and exciting area of research, expanding our knowledge of how the three-dimensional (3D) genome is regulated., (Copyright © 2014. Published by Elsevier Ltd.)

The nuclear lamina is a protein meshwork that lies under the inner nuclear membrane of metazoan cells. One function of the nuclear lamina is to organize heterochromatin at the inner nuclear periphery. However, very little is known about how heterochromatin attaches to the nuclear lamina and how such attachments are restored at mitotic exit. Here, we show that a previously unstudied human protein, PRR14, functions to tether heterochromatin to the nuclear periphery during interphase, through associations with heterochromatin protein 1 (HP1) and the nuclear lamina. During early mitosis, PRR14 is released from the nuclear lamina and chromatin and remains soluble. Strikingly, at the onset of anaphase, PRR14 is incorporated rapidly into chromatin through HP1 binding. Finally, in telophase, PRR14 relocalizes to the reforming nuclear lamina. This stepwise reassembly of PRR14 suggests a function in the selection of HP1-bound heterochromatin for reattachment to the nuclear lamina as cells exit mitosis.

Lamin proteins are the major constituents of the nuclear lamina, a proteinaceous network that lines the inner nuclear membrane. Primarily, the nuclear lamina provides structural support for the nucleus and the nuclear envelope; however, lamins and their associated proteins are also involved in most of the nuclear processes, including DNA replication and repair, regulation of gene expression, and signaling. Mutations in human lamin A and associated proteins were found to cause a large number of diseases, termed 'laminopathies.' These diseases include muscular dystrophies, lipodystrophies, neuropathies, and premature aging syndromes. Despite the growing number of studies on lamins and their associated proteins, the molecular organization of lamins in health and disease is still elusive. Likewise, there is no comprehensive view how mutations in lamins result in a plethora of diseases, selectively affecting different tissues. Here, we discuss some of the structural aspects of lamins and the nuclear lamina organization, in light of recent results.

Lamins are intermediate filament proteins that assemble into a meshwork underneath the inner nuclear membrane, the nuclear lamina. Mutations in the LMNA gene, encoding lamins A and C, cause a variety of diseases collectively called laminopathies. The disease mechanism for these diverse conditions is not well understood. Since lamins A and C are fundamental determinants of nuclear structure and stability, we tested whether defects in nuclear mechanics could contribute to the disease development, especially in laminopathies affecting mechanically stressed tissue such as muscle. Using skin fibroblasts from laminopathy patients and lamin A/C-deficient mouse embryonic fibroblasts stably expressing a broad panel of laminopathic lamin A mutations, we found that several mutations associated with muscular dystrophy and dilated cardiomyopathy resulted in more deformable nuclei; in contrast, lamin mutants responsible for diseases without muscular phenotypes did not alter nuclear deformability. We confirmed our results in intact muscle tissue, demonstrating that nuclei of transgenic Drosophila melanogaster muscle expressing myopathic lamin mutations deformed more under applied strain than controls. In vivo and in vitro studies indicated that the loss of nuclear stiffness resulted from impaired assembly of mutant lamins into the nuclear lamina. Although only a subset of lamin mutations associated with muscular diseases caused increased nuclear deformability, almost all mutations tested had defects in force transmission between the nucleus and cytoskeleton. In conclusion, our results indicate that although defective nuclear stability may play a role in the development of muscle diseases, other factors, such as impaired nucleo-cytoskeletal coupling, likely contribute to the muscle phenotype.

Human cytomegalovirus (HCMV) encodes one conventional protein kinase, UL97. During infection, UL97 phosphorylates the retinoblastoma tumor suppressor protein (pRb) on sites ordinarily phosphorylated by cyclin-dependent kinases (CDK), inactivating the ability of pRb to repress host genes required for cell cycle progression to S phase. UL97 is important for viral DNA synthesis in quiescent cells, but this function can be replaced by human papillomavirus type 16 E7, which targets pRb for degradation. However, viruses in which E7 replaces UL97 are still defective for virus production. UL97 is also required for efficient nuclear egress of viral nucleocapsids, which is associated with disruption of the nuclear lamina during infection, and phosphorylation of lamin A/C on serine 22, which antagonizes lamin polymerization. We investigated whether inactivation of pRb might overcome the requirement of UL97 for these roles, as pRb inactivation induces CDK1, and CDK1 phosphorylates lamin A/C on serine 22. We found that lamin A/C serine 22 phosphorylation during HCMV infection correlated with expression of UL97 and was considerably delayed in UL97-null mutants, even when E7 was expressed. E7 failed to restore gaps in the nuclear lamina seen in wild-type but not UL97-null virus infections. In electron microscopy analyses, a UL97-null virus expressing E7 was as impaired as a UL97-null mutant in cytoplasmic accumulation of viral nucleocapsids. Our results demonstrate that pRb inactivation is insufficient to restore efficient viral nuclear egress of HCMV in the absence of UL97 and instead argue further for a direct role of UL97 in this stage of the infectious cycle.

The nuclear lamina (NL) interacts with hundreds of large genomic regions termed lamina associated domains (LADs). The dynamics of these interactions and the relation to epigenetic modifications are poorly understood. We visualized the fate of LADs in single cells using a "molecular contact memory" approach. In each nucleus, only ~30% of LADs are positioned at the periphery; these LADs are in intermittent molecular contact with the NL but remain constrained to the periphery. Upon mitosis, LAD positioning is not detectably inherited but instead is stochastically reshuffled. Contact of individual LADs with the NL is linked to transcriptional repression and H3K9 dimethylation in single cells. Furthermore, we identify the H3K9 methyltransferase G9a as a regulator of NL contacts. Collectively, these results highlight principles of the dynamic spatial architecture of chromosomes in relation to gene regulation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Recent studies have shown that premature cellular senescence and normal organ development and function depend on the type V intermediate filament proteins, the lamins, which are major structural proteins of the nucleus. This review presents an up-to-date summary of the literature describing new findings on lamin functions in various cellular processes and emphasizes the relationship between the lamins and devastating diseases ranging from premature aging to cancer. Recent insights into the structure and function of the A- and B- type lamins in normal cells and their dysfunctions in diseased cells are providing novel targets for the development of new diagnostic procedures and disease intervention. We summarize these recent findings, focusing on data from mice and humans, and highlight the expanding knowledge of these proteins in both healthy and diseased cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Lamins are the major components of the nuclear lamina, a filamentous layer underlying the inner nuclear membrane and attached to the peripheral chromatin. Lamins are required for maintaining nuclear shape and are involved in most nuclear activities. Here, we studied the 3D organization of the nuclear lamina formed upon the expression of Caenorhabditis elegans lamin (Ce-lamin) within the nucleus of a Xenopus laevis oocyte. We show that Ce-lamin forms an intricate 3D meshwork of 5-6 nm lamin protofilaments. The diverse protofilament interactions and organization may shed light upon the unique mechano-elastic properties of the nuclear lamina scaffold supporting the nuclear envelope. The Q159K Hutchinson-Gilford Progeria Syndrome-linked mutation alters interactions between protofilaments within the lamina, leading to the formation of more bundled arrays of less isotropically-oriented protofilaments. Using this system, we show for the first time the organization of lamin proteins that were translated and assembled within the environment of a living cell., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The nuclear lamina is a major structural element of the nucleus and is predominately composed of the intermediate filament lamin proteins. Missense mutations in the human lamins A/C cause a family of laminopathic diseases, with no known mechanistic link between the position of the mutation and the resulting disease phenotypes. The Caenorhabditis elegans lamin (Ce-lamin) is structurally and functionally homologous to human lamins, and recent advances have allowed detailed structural analysis of Ce-lamin filaments both in vitro and in vivo. Here, we studied the effect of laminopathic mutations on Ce-lamin filament assembly in vitro and the corresponding physiological phenotypes in animals. We focused on three disease-linked mutations, Q159K, T164P, and L535P, which have previously been shown to affect lamin structure and nuclear localization. Mutations prevented the proper assembly of Ce-lamin into filament and/or paracrystalline arrays. Disease-like phenotypes were observed in strains expressing low levels of these mutant lamins, including decreased fertility and motility coincident with muscle lesions. In addition, the Q159K- and T164P-expressing strains showed a reduced lifespan. Thus, different disease-linked mutations in Ce-lamin exhibit major effects in vivo and in vitro. Using C. elegans as a model system, a comprehensive analysis of the effects of specific lamin mutations from the level of in vitro filament assembly to the physiology of the organism will help uncover the mechanistic differences between these different lamin mutations., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Beneath the inner nuclear membrane lies the dense meshwork of the nuclear lamina, which provides structural support for the nuclear envelope and serves as an important organizing center for a number of nuclear and cytoplasmic constituents and processes. Herpesviruses have a significant and wide-ranging impact on human health, and their capacity to replicate and cause disease includes events that occur in the host cell nucleus. Herpesviruses begin assembly of progeny virus in the nuclei of infected cells and their capsids must escape the confines of the nucleus by budding through the inner nuclear membrane (INM) to proceed with later stages of virion assembly and egress. Access of viral capsids to the INM thus necessitates disruption of the dense nuclear lamina layer. We review herpesvirus effects on the nuclear lamina and in particular the roles of the herpes simplex virus-encoded nuclear envelope complex and viral kinases on lamin phosphorylation, dissociation, and nucleocapsid envelopment at the INM.

The products of numerous open reading frames (ORFs) present in the genome of human cytomegalovirus (CMV) have not been characterized. Here, we describe the identification of a new CMV protein localizing to the nuclear envelope and in cytoplasmic vesicles at late times postinfection. Based on this distinctive localization pattern, we called this new protein nuclear rim-associated cytomegaloviral protein, or RASCAL. Two RASCAL isoforms exist, a short version of 97 amino acids encoded by the majority of CMV strains and a longer version of 176 amino acids encoded by the Towne, Toledo, HAN20, and HAN38 strains. Both isoforms colocalize with lamin B in deep intranuclear invaginations of the inner nuclear membrane (INM) and in novel cytoplasmic vesicular structures possibly derived from the nuclear envelope. INM infoldings have been previously described as sites of nucleocapsid egress, which is mediated by the localized disruption of the nuclear lamina, promoted by the activities of viral and cellular kinases recruited by the lamina-associated proteins UL50 and UL53. RASCAL accumulation at the nuclear membrane required the presence of UL50 but not of UL53. RASCAL and UL50 also appeared to specifically interact, suggesting that RASCAL is a new component of the nuclear egress complex (NEC) and possibly involved in mediating nucleocapsid egress from the nucleus. Finally, the presence of RASCAL within cytoplasmic vesicles raises the intriguing possibility that this protein might participate in additional steps of virion maturation occurring after capsid release from the nucleus.

Nuclear lamins are involved in most nuclear activities and are essential for retaining the mechano-elastic properties of the nucleus. They are nuclear intermediate filament (IF) proteins forming a distinct meshwork-like layer adhering to the inner nuclear membrane, called the nuclear lamina. Here, we present for the first time, the three-dimensional supramolecular organization of lamin 10 nm filaments and paracrystalline fibres. We show that Caenorhabditis elegans nuclear lamin forms 10 nm IF-like filaments, which are distinct from their cytoplasmic counterparts. The IF-like lamin filaments are composed of three and four tetrameric protofilaments, each of which contains two partially staggered anti-parallel head-to-tail polymers. The beaded appearance of the lamin filaments stems from paired globular tail domains, which are spaced regularly, alternating between 21 nm and 27 nm. A mutation in an evolutionarily conserved residue that causes Hutchison-Gilford progeria syndrome in humans alters the supramolecular structure of the lamin filaments. On the basis of our structural analysis, we propose an assembly pathway that yields the observed 10 nm IF-like lamin filaments and paracrystalline fibres. These results serve also as a platform for understanding the effect of laminopathic mutations on lamin supramolecular organization.

The architecture of human chromosomes in interphase nuclei is still largely unknown. Microscopy studies have indicated that specific regions of chromosomes are located in close proximity to the nuclear lamina (NL). This has led to the idea that certain genomic elements may be attached to the NL, which may contribute to the spatial organization of chromosomes inside the nucleus. However, sequences in the human genome that interact with the NL in vivo have not been identified. Here we construct a high-resolution map of the interaction sites of the entire genome with NL components in human fibroblasts. This map shows that genome-lamina interactions occur through more than 1,300 sharply defined large domains 0.1-10 megabases in size. These lamina-associated domains (LADs) are typified by low gene-expression levels, indicating that LADs represent a repressive chromatin environment. The borders of LADs are demarcated by the insulator protein CTCF, by promoters that are oriented away from LADs, or by CpG islands, suggesting possible mechanisms of LAD confinement. Taken together, these results demonstrate that the human genome is divided into large, discrete domains that are units of chromosome organization within the nucleus.

A surprising realization in recent years was that proteins of the nuclear lamina directly regulate gene expression and cell differentiation. Presented here are examples that highlight the diverse roles these proteins can inhabit. Accompanying the primer is a discussion of current topics in the field with nuclear lamina experts Colin Stewart, Ph.D., and Yosef Gruenbaum, Ph.D.

The evolutionary-conserved interactions between KASH and SUN domain-containing proteins within the perinuclear space establish physical connections, called LINC complexes, between the nucleus and the cytoskeleton. Here, we show that the KASH domains of Nesprins 1, 2 and 3 interact promiscuously with luminal domains of Sun1 and Sun2. These constructs disrupt endogenous LINC complexes as indicated by the displacement of endogenous Nesprins from the nuclear envelope. We also provide evidence that KASH domains most probably fit a pocket provided by SUN domains and that post-translational modifications are dispensable for that interaction. We demonstrate that the disruption of endogenous LINC complexes affect cellular mechanical stiffness to an extent that compares to the loss of mechanical stiffness previously reported in embryonic fibroblasts derived from mouse lacking A-type lamins, a mouse model of muscular dystrophies and cardiomyopathies. These findings support a model whereby physical connections between the nucleus and the cytoskeleton are mediated by interactions between diverse combinations of Sun proteins and Nesprins through their respective evolutionary-conserved domains. Furthermore, they emphasize, for the first time, the relevance of LINC complexes in cellular mechanical stiffness suggesting a possible involvement of their disruption in various laminopathies, a group of human diseases linked to mutations of A-type lamins.

We report stimulated emission depletion (STED) fluorescence microscopy with continuous wave (CW) laser beams. Lateral fluorescence confinement from the scanning focal spot delivered a resolution of 29-60 nm in the focal plane, corresponding to a 5-8-fold improvement over the diffraction barrier. Axial spot confinement increased the axial resolution by 3.5-fold. We observed three-dimensional (3D) subdiffraction resolution in 3D image stacks. Viable for fluorophores with low triplet yield, the use of CW light sources greatly simplifies the implementation of this concept of far-field fluorescence nanoscopy.

Cells infected with wild-type herpes simplex virus type 1 (HSV-1) show disruption of the organization of the nuclear lamina that underlies the nuclear envelope. This disruption is reflected in changes in the localization and phosphorylation of lamin proteins. Here, we show that HSV-1 infection causes relocalization of the LEM domain protein emerin. In cells infected with wild-type virus, emerin becomes more mobile in the nuclear membrane, and in cells infected with viruses that fail to express UL34 protein (pUL34) and US3 protein (pUS3), emerin no longer colocalizes with lamins, suggesting that infection causes a loss of connection between emerin and the lamina. Infection causes hyperphosphorylation of emerin in a manner dependent upon both pUL34 and pUS3. Some emerin hyperphosphorylation can be inhibited by the protein kinase Cdelta (PKCdelta) inhibitor rottlerin. Emerin and pUL34 interact physically, as shown by pull-down and coimmunoprecipitation assays. Emerin expression is not, however, necessary for infection, since virus growth is not impaired in cells derived from emerin-null transgenic mice. The results suggest a model in which pUS3 and PKCdelta that has been recruited by pUL34 hyperphosphorylate emerin, leading to disruption of its connections with lamin proteins and contributing to the disruption of the nuclear lamina. Changes in emerin localization, nuclear shape, and lamin organization characteristic of cells infected with wild-type HSV-1 also occur in cells infected with recombinant virus that does not make viral capsids, suggesting that these changes occur independently of capsid envelopment.

Lamina-associated polypeptides (LAPs) are important components of the nuclear lamina, the dense network of filaments that supports the nuclear envelope and also extends into the nucleoplasm. The main protein constituents of the nuclear lamina are the constitutively expressed B-type lamins and the developmentally regulated A- and C-type lamins. LAP2alpha is the only non-membrane-associated member of the LAP family. It preferentially binds lamin A/C, has been implicated in cell-cycle regulation and chromatin organization, and has also been found to be a component of retroviral preintegration complexes. As an approach to understanding the role of LAP2alpha in cellular pathways, we have determined the crystal structure of the C-terminal domain of LAP2alpha, residues 459-693. The C-terminal domain is dimeric and possesses an extensive four-stranded, antiparallel coiled coil. The surface involved in binding lamin A/C is proposed based on results from alanine-scanning mutagenesis and a solid-phase overlay binding assay.

The nuclear lamina is a network of structural filaments, the A and B type lamins, located at the nuclear envelope and throughout the nucleus. Lamin filaments provide the nucleus with mechanical stability and support many basic activities, including gene regulation. Mutations in LMNA, the gene encoding A type lamins, cause numerous human diseases, including the segmental premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). Here we show that structural and mechanical properties of the lamina are altered in HGPS cells. We demonstrate by live-cell imaging and biochemical analysis that lamins A and C become trapped at the nuclear periphery in HGPS patient cells. Using micropipette aspiration, we show that the lamina in HGPS cells has a significantly reduced ability to rearrange under mechanical stress. Based on polarization microscopy results, we suggest that the lamins are disordered in the healthy nuclei, whereas the lamins in HGPS nuclei form orientationally ordered microdomains. The reduced deformability of the HGPS nuclear lamina possibly could be due to the inability of these orientationally ordered microdomains to dissipate mechanical stress. Surprisingly, intact HGPS cells exhibited a degree of resistance to acute mechanical stress similar to that of cells from healthy individuals. Thus, in contrast to the nuclear fragility seen in lmna null cells, the lamina network in HGPS cells has unique mechanical properties that might contribute to disease phenotypes by affecting responses to mechanical force and misregulation of mechanosensitive gene expression.

UNC-84 is required to localize UNC-83 to the nuclear envelope where it functions during nuclear migration. A KASH domain in UNC-83 was identified. KASH domains are conserved in the nuclear envelope proteins Syne/nesprins, Klarsicht, MSP-300, and ANC-1. Caenorhabditis elegans UNC-83 was shown to localize to the outer nuclear membrane and UNC-84 to the inner nuclear membrane in transfected mammalian cells, suggesting the KASH and SUN protein targeting mechanisms are conserved. Deletion of the KASH domain of UNC-83 blocked nuclear migration and localization to the C. elegans nuclear envelope. Some point mutations in the UNC-83 KASH domain disrupted nuclear migration, even if they localized normally. At least two separable portions of the C-terminal half of UNC-84 were found to interact with the UNC-83 KASH domain in a membrane-bound, split-ubiquitin yeast two-hybrid system. However, the SUN domain was essential for UNC-84 function and UNC-83 localization in vivo. These data support the model that KASH and SUN proteins bridge the nuclear envelope, connecting the nuclear lamina to cytoskeletal components. This mechanism seems conserved across eukaryotes and is the first proposed mechanism to target proteins specifically to the outer nuclear membrane.

The nuclear lamina is a protein meshwork lining the nucleoplasmic face of the nuclear envelope. Association of lamins with the inner nuclear membrane is mediated by specific modifications in the CaaX motif at their C-termini. B-type lamins are permanently isoprenylated whereas lamin A loses its modification by a lamin A-specific processing step after incorporation into the lamina. Lamins are differentially expressed during development and tissue differentiation. Here we show that an increased synthesis of lamins B1 and B2 in amphibian oocytes induces the formation of intranuclear membrane structures that form extensive arrays of stacked cisternae. These 'lamin membrane arrays' are attached to the inner nuclear membrane but are not continuous with it. Induction of this membrane proliferation depends on CaaX-specific posttranslational modification. Moreover, in transfected HeLa cells, chimeric GFP containing a nuclear localization signal and a C-terminal CaaX motif of N-Ras induces intranuclear membrane stacks that resemble those induced by lamins and ER-like cisternae that are induced in the cytoplasm upon increased synthesis of integral ER membrane proteins. Implications for the synthesis of CaaX-containing proteins are discussed and the difference from intranuclear fibrous lamina annulate lamellae formations is emphasized.

Mechanical properties of the nuclear envelope have implications for cell and nuclear architecture as well as gene regulation. Using isolated Xenopus oocyte nuclei, we have established swelling conditions that separate the intact nuclear envelope (membranes, pore complexes and underlying lamin filament network) from nucleoplasm and the majority of chromatin. Swelling proves reversible with addition of high molecular mass dextrans. Micropipette aspiration of swollen and unswollen nuclear envelopes is also reversible and yields a network elastic modulus, unaffected by nucleoplasm, that averages 25 mN/m. Compared to plasma membranes of cells, the nuclear envelope is much stiffer and more resilient. Our results suggest that the nuclear lamina forms a compressed network shell of interconnected rods that is extensible but limited in compressibility from the native state, thus acting as a 'molecular shock absorber'. In light of the conservation of B-type lamins in metazoan evolution, the mechanical properties determined in this investigation suggest physical mechanisms by which mutated lamins can either destabilize nuclear architecture or influence nuclear responses to mechanical signals in Emery-Dreifuss muscular dystrophy, cardiomyopathy, progeria syndromes (premature 'aging') and other laminopathies.

The herpes simplex virus type 1 (HSV-1) U(L)31 and U(L)34 proteins are dependent on each other for proper targeting to the nuclear membrane and are required for efficient envelopment of nucleocapsids at the inner nuclear membrane. In this work, we show that whereas the solubility of lamins A and C (lamin A/C) was not markedly increased, HSV induced conformational changes in the nuclear lamina of infected cells, as viewed after staining with three different lamin A/C-specific antibodies. In one case, reactivity with a monoclonal antibody that recognizes an epitope in the lamin tail domain was greatly reduced in HSV-infected cells. This apparent HSV-induced epitope masking required both U(L)31 and U(L)34, but these proteins were not sufficient to mask the epitope in uninfected cells, indicating that other HSV proteins are also required. In the second case, staining with a rabbit polyclonal antibody that primarily recognizes epitopes in the lamin A/C rod domain revealed that U(L)34 is required for HSV-induced decreased availability of epitopes for reaction with the antibody, whereas U(L)31 protein was dispensable for this effect. Still another polyclonal antibody indicated virtually no difference in lamin A/C staining in infected versus uninfected cells, indicating that the HSV-induced changes are more conformational than the result of lamin depletion at the nuclear rim. Further evidence supporting an interaction between the nuclear lamina and the U(L)31/U(L)34 protein complex includes the observations that (i) overexpression of the U(L)31 protein in uninfected cells was sufficient to relocalize lamin A/C from the nuclear rim into nucleoplasmic aggregates, (ii) overexpression of U(L)34 was sufficient to relocalize some lamin A/C into the cytoplasm, and (iii) both U(L)31 and U(L)34 could directly bind lamin A/C in vitro. These studies suggest that the U(L)31 and U(L)34 proteins modify the conformation of the nuclear lamina in infected cells, possibly by direct interaction with lamin A/C, and that other proteins are also likely involved. Given that the nuclear lamina potentially excludes nucleocapsids from envelopment sites at the inner nuclear membrane, the lamina alteration may reflect a role of the U(L)31/U(L)34 protein complex in perturbing the lamina to promote nucleocapsid egress from the nucleus. Alternatively, the data are compatible with a role of the lamina in targeting the U(L)31/U(L)34 protein complex to the nuclear membrane.

The lamins of the tunicate Ciona intestinalis and the nematode Caenorhabditis elegans show unusual sequence features when compared to the more than 35 metazoan lamin sequences currently known. We therefore analyzed the in vitro assembly of these two lamins by electron microscopy using chicken lamin B2 as a control. While lamin dimers usually appear as a rod carrying two globules at one end, these globules are absent from Ciona lamin, which lacks the central 105-residue region of the tail domain. The deletion of 14 residues or two heptads from the coiled coil rod domain of the single C.elegans lamin results in a 1.5-nm shortening of the dimer rod. Similarly, the paracrystals assembled from the C.elegans lamin exhibit a 3.1-nm reduction of the true axial repeat compared to that of chicken lamin B2 paracrystals. We speculate that the banding pattern in the C.elegans lamin paracrystals arises from a relative stagger between dimers and/or a positioning of the globular tail domain relative to the central rod that is distinct from that observed in chicken lamin B2 paracrystals. Here we show that a nuclear lamin can assemble in vitro into 10-nm intermediate filaments (IFs). C.elegans lamin in low ionic strength Tris-buffers at a pH of 7.2-7.4 provides a stable population of lamin IFs. Some implications of this filament formation are discussed.

The nuclear matrix (NM) has been identified as a potential target for heat-induced cell killing. Previous studies have shown that heat-shock may significantly modulate lamin B content. Since changes in NM structure have often been accompanied by changes in protein composition, we investigated whether hyperthermia induced changes in nuclear lamina (NL) structure in non-tolerant and thermotolerant cells, and the implications of these changes on cell survival. Using indirect immunofluorescence techniques and confocal microscopy, we found that heating cells at 42 or 45.5 degrees C caused invaginations and other distortions of the peripheral NL. While hyperthermia did not alter the number or structure of internal lamin B foci, heat-induced alterations to the peripheral NL were dose-dependent. Interestingly, NL structure recovered with time after heating in cells that were destined to live or die. Thermotolerant cells heated at 45.5 degrees C showed similar initial changes in the NL compared to non-tolerant cells, but recovery occurred much faster. Taken together, these results suggest that the amount of initial damage to the peripheral NL is not correlated with heat-induced cell killing. However, the possibility that an increased rate of recovery might confer a survival advantage cannot be discounted., (Copyright 2002 Wiley-Liss, Inc.)