The cell nucleus is the largest and quite often the stiffest organelle in a cell, and is a richly complex and dynamic structure that supports a surprisingly diverse set of functions [1]. Rather than simply housing the genetic material, the nucleus acts as a hub for genome organization, transcriptional regulation, DNA damage repair, and mechanosensing; as a result, many human diseases are genetically linked to the nucleus.
The nucleus is comprised of the nuclear interior, including chromatin, nuclear bodies, and other intranuclear components, and the peripheral region adjacent to the nuclear envelope. The nuclear envelope, a double membrane structure consisting of the inner and outer nuclear membranes (INM and ONM, respectively), is contiguous with the endoplasmic reticulum in the cytosol. The nucleoplasmic face of the INM lies adjacent to the nuclear lamina, a proteinaceous meshwork of type V intermediate filaments consisting of A-type and B-type lamins and their associated proteins. The B-type lamins, Lamin B1 and Lamin B2, are transcribed from the LMNB1 and LMNB2 genes, respectively, while Lamins A and C (often referred to as Lamins A + C) arise from alternative splicing of the LMNA gene.
While A-type lamins are only expressed in differentiated cell types, B-type lamins are present throughout development, including in embryonic stem cells [2-4]. Recent work indicates that the two lamin types form partially overlapping concentric meshworks, with the Lamin B1 network located adjacent to the INM and the Lamin A + C network facing the nucleoplasm [5,6]. The structure of these meshworks is regulated by farnesylation of the lamins during their biosynthesis. While B-type lamins are constitutively farnesylated, mediating their association with the INM, A-type lamins undergo proteolytic cleavage of the farnesyl group [7]. Defective removal of the farnesyl group from Lamin A produces the pathogenic progerin form of the protein, which gives rise to the accelerated aging disorder progeria [8].
Multiple proteins located at the INM reinforce the interaction between the lamina and the INM. LEM (LAP2, Emerin, and MAN1) domain family proteins interact with both lamins and BAF (Barrier-to-Autointegration Factor), providing a link to chromatin. LAP1 (Lamina-Associated Polypeptide 1), LAP2 (Lamina-Associated Polypeptide 2), and LBR (Lamin B Receptor) also interact with lamins and carry out distinct biochemical functions [9-11].
Spanning both INM and ONM are nuclear pore complexes (NPCs), huge multi-protein assemblies of around 120 MDa, in metazoans, that mediate transport between the cytosol and nuclear interior [12]. They are composed of several copies of ~30 different proteins, called nucleoporins (Nups), which assemble into octagonal structures of 50-100 nm diameter. NPCs also feature a selective central pore composed of intrinsically disordered proteins called FG Nups. NPCs are permeable to molecules under 40 kDa, while larger macromolecules are actively transported across the pore by karyopherins and Kaps [13]. Much effort has been devoted to determining the method by which the FG Nups of the central channel mediate NPC barrier function, with several models still under consideration [13].
LINC complexes (Linker of Nucleoskeleton and Cytoskeleton) also span the nuclear envelope, with SUN domain proteins embedded in the INM and KASH domain proteins embedded in the ONM. These structures uniquely mediate connections between the nuclear interior and cytosolic cytoskeleton, including actin, microtubules, and intermediate filaments [14], and thus provide a conduit for force transmission across the nuclear envelope. In addition, they are crucial for positioning of the nucleus in a broad range of contexts [15].
A number of different nuclear bodies, dynamic structures composed of proteins and often RNA molecules, are also found within the nuclear interior. Nuclear bodies are non-membrane bound structures that exhibit rapid compositional exchange with the surrounding nucleoplasm, and include nucleoli, Cajal bodies, PML bodies, and nuclear speckles, among others [16]. These structures are involved in a multitude of activities, including RNA processing, telomere maintenance, the cellular stress response, DNA repair, and protein and RNA sequestration [16].
Recent work has shown that cytoskeletal proteins are also present in the nucleus, with actin being the most well characterized to date. The nuclear actin pool has been shown to interact directly with chromatin remodeling complexes and RNA polymerases, and functions in gene expression, chromosome mobility, DNA damage repair, and differentiation [17]. Compelling evidence suggests that actin is present in both monomeric and polymerized forms in the nucleus [17].
Within the eukaryotic nucleus, DNA is compacted into chromatin through its association with nucleosome proteins and non-histone proteins. Chromatin is broadly classified as euchromatin or heterochromatin based on its compaction state, histone modifications, gene-richness, and replication timing during the cell cycle. While euchromatin is typically gene-rich, replicates early in S-phase of the cell cycle, and occupies the nuclear interior, heterochromatin tends to be gene-poor, includes tissue-specific genes, replicates later in S-phase, and is generally located at the nuclear periphery or nucleoli [18]. Chromosome conformation capture techniques, which reveal the 3D spatial organization of chromatin within the nucleus, have shown that chromatin is organized at the largest scale into two compartments, A and B. These are differentially characterized by their compaction state, replication timing, and gene-richness, and therefore closely align with euchromatic and heterochromatic domains [19]. Within these compartments, chromatin is further organized into distinct megabase-sized topologically associated domains (TADs), the boundaries of which are defined by architectural proteins like CTCF and cohesin, and likely represent differentially-transcribed units of the genome [20].
In addition to TADs, chromatin is further functionally and structurally segregated by its association with the nuclear lamina and nucleolus. 35-40% of the genome interacts with lamina proteins, including LBR and Lamin A + C, in roughly 0.5 megabase-spanning regions called lamina associated domains (LADs) [21]. LADs were first described based in DNA fluorescence in situ hybridization experiments (FISH), which showed that specific genomic loci exhibited close apposition with the nuclear periphery. Further technological advancements have refined our understanding of LADs [22]. One major tool used to map LADs is DamID, which allows binding interactions between a protein of interest and specific chromatin sites to be identified [22].
LADs typically contain genes that are transcriptionally inactive and enriched in heterochromatin-associated histone modifications, including H3K9me2 and H3K9me3 [21]. The nuclear periphery has thus been postulated to drive transcriptional repression. Further, transcriptional activation of genes during cell differentiation is often accompanied by relocation of those genes away from the nuclear periphery to more internal sites [23,24]. However, recent work suggests that transcriptional repression may actually drive peripheral localization of genes, indicating that the role of the nuclear periphery in transcriptional regulation is more complicated than once thought [25].
Despite these multiple levels of organization, chromatin is capable of significant rearrangements within these compartments. Following mitosis, for example, LADs can reposition from the nuclear periphery to nucleoli at nucleolus associated domains (NADs) [26]. In addition, significant changes in chromatin organization occur over the course of cell differentiation, such as chromatin compartment switching and changes in compaction state [27]. Live cell imaging of specific genomic loci using LacO/LacI-GFP technology and single nucleosome imaging strategies has also revealed significant mobility within the nucleus [28,29]. This may increase DNA accessibility for replication, repair, and transcription machinery. For example, DNA double-strand breaks have been shown to be highly mobile, which might facilitate DNA repair processes [30].
In addition to serving as tethers for chromatin association with the nuclear periphery, the nuclear lamina also acts as a platform for proteins that can regulate gene expression, including chromatin remodelers, histone modifiers, and transcription factors. The lamina also interacts with components of biochemical signaling pathways. For example, Lamins A + C have been implicated in regulating YAP/TAZ signaling [3,31], while MAN1 and LINC complexes may differentially modulate TGF-β/SMAD signaling [32,33].
While textbooks typically depict the nucleus as a static body at the center of the cell, nuclear positioning is actually highly dynamic, responsive to external stimuli, and typically dependent on interactions between the nuclear envelope and cytosolic cytoskeleton. Further, regulated nuclear positioning plays an important role in a number of fundamental biological processes. One classic example, first described in the 1930s, occurs during development of the vertebrate neuroepithelium. In a process known as interkinetic nuclear migration, the nuclei of neural progenitor cells in the developing neuroepithelium undergo apical-basal repositioning events that are tied to the cell cycle [34]. This process likely contributes to the fate decisions of these progenitor cells [34]. In the developing murine neuroepithelium, this process is dependent on the recruitment of dynein motors to the nuclear envelope by KASH domain proteins [34].
In addition, proper nuclear positioning is important for muscle development, as the nuclear envelope appears to serve as a nucleation site for cytoskeleton organization during myofibrillogenesis [35]. LINC complex-dependent regulation of nuclear positioning is also implicated in the migration of male and female pronuclei toward one another in newly fertilized zygotes.
Nuclear positioning also plays a crucial role during cell migration, as the nucleus is positioned towards the rear of migratory fibroblasts, neurons, mesenchymal cells, and many cancer cell types. Nuclear polarization during 2D cell migration occurs through the harnessing of the retrograde flow of perinuclear actin cables by transmembrane actin-associated nuclear (TAN) lines, composed of LINC complex components nesprin-2G and SUN2, nuclear envelope proteins Lamin A, Emerin, Samp1, TorsinA, and LAP1, and the formin FHOD1 [36,37]. Disruption of TAN lines prevents nuclear polarization during migration, leading to inefficient directed cell migration [38].
As the size and stiffness of the nucleus presents a barrier to cell migration through confined spaces, as occurs during cancer metastasis or leukocyte transendothelial migration, a number of mechanisms exist to promote nuclear deformation during 3D cell migration. It is not yet clear if TAN lines are involved in 3D cell migration, although Nesprin-2 and the actin regulator Fascin are important for nuclear deformation of cancer cells during cell invasion in 3D microenvironments [39]. In addition, it has been shown that actomyosin-based forces pull the nucleus forward during 3D migration, creating hydraulic pressure in the front of the cell to drive forward movement [40]. Further, cancer cells and leukocytes exhibit transient nuclear envelope ruptures during migration through tight spaces, which are repaired by the same ESCRT III machinery that reseals the nuclear envelope following mitosis [41-43].
Work in the 1990s demonstrated that mechanical forces can be transmitted from the cell surface to the nuclear interior [44]. A large body of work now indicates that nuclear properties are responsive to both extracellular and intracellular mechanical cues [45]. Intracellular cues include forces applied directly to the nucleus during 3D cell migration and nuclear repositioning, while extracellular cues include forces transmitted via inter- and intracellular adhesions, like fluid sheer stress, substrate stiffness, and substrate stretch.
Of the nuclear lamina components, Lamin A + C appears to be uniquely responsive to mechanical inputs, as Lamin A + C protein levels, but not Lamin B levels, scale with tissue stiffness in vivo and substrate stiffness in vitro; for example, Lamin A + C levels are higher in bone and lower in brain [3]. Further, Lamin A + C undergoes substrate stiffness-dependent phosphorylation on serine 22, potentially in response to unfolding of its immunoglobulin-like domain, which prompts disassembly of the lamin network [46,47]. Mesenchymal stem cells exposed to stiff substrates, and therefore high intracellular cytoskeletal tension, exhibit decreased phosphorylation of serine 22, while cells exposed to soft substrates exhibit increased phosphorylation [46]. Compressive forces on cells can also modulate the accessibility of mechanically-sensitive epitopes in Lamin A + C in a LINC complex-dependent manner [48].
Other components of the nuclear periphery are also mechanosensitive. Direct force application to the KASH domain protein Nesprin-1 on isolated nuclei using magnetic tweezers induces nuclear stiffening, Emerin phosphorylation, and increased association of LINC complexes with Lamin A + C [49]. Shear stress can also induce nuclear stiffening in endothelial cells [50] and peripheral recruitment of Lamin A + C in HeLa cells [51]. Uniaxial stretch has been shown to drive the redistribution of Emerin from the INM to the ONM, accompanied by H2K27me3 and PRC2-dependent transcriptional repression of genes associated with epidermal differentiation [52].
In addition to these changes in nuclear peripheral proteins, extrinsic force application to cells has been shown to influence nuclear shape, chromosome organization, the position of subnuclear bodies, and even gene expression [53-58]. Further work will be required to directly associate force transmission through the LINC complex with the regulation of mechanosensitive gene expression.
Changes in nuclear properties can likewise influence cytosolic and plasma membrane properties. Emerging evidence suggests that the LINC complex may exist in a feedback circuit with RhoA to modulate actomyosin contractility [59,60] and actin dynamics [61,62]. LINC complexes also regulate microtubule organization in many contexts [63,64]. Nuclear lamina components can also mediate changes in cell-cell and cell-extracellular matrix adhesions, potentially downstream of their influence on the cytoskeleton. Disruption of LINC complexes and Lamin A + C, for example, result in altered focal adhesion size, number, and function in cultured cells [65,66]. Similar disruptions appear to cause defects in intercellular adhesion, as well [67-69].
As discussed above, the nucleus, and the nuclear periphery in particular, serves many vital biological functions. The biological necessity of these components is demonstrated by the fact that mice deficient for SUN domain proteins exhibit perinatal lethality [70], while mice that lack functional Lamin A + C exhibit early death at 2-8 weeks of age [71]. Consequently, there are a number of human diseases linked to mutations in genes encoding proteins that reside at the nuclear envelope, which are collectively referred to as “laminopathies” or “nuclear envelopathies”. These diseases appear to predominantly affect mechanically active tissues, including the myocardium, skeletal muscle, and the skin. LINC complexes, Emerin, and Lamins A + C are linked to various human myopathies, including Emery-Dreifuss muscular dystrophy [72,73]. Further, many individuals with dilated cardiomyopathies exhibit mutations in Lamins A + C [74,75]. Mutations in lamins are also known to drive the premature aging disorder Hutchinson-Gilford progeria syndrome, implicating the nuclear periphery in cellular aging.
While the exact etiology of these diseases remains unclear, it is currently believed that a combination of altered mechanotransduction and biochemical signaling, altered gene expression, increased DNA damage, and increased nuclear fragility may all contribute. For example, increased MAP kinase and AKT/mTOR signaling appears to be a major driver of cardiac defects in laminopathy mouse models, which can be ameliorated with compounds that suppress these pathways [76-79].
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