Providing a stable physical connection between the nucleus and the cytoskeleton is essential for a wide range of cellular functions and it could also participate in mechanosensing by transmitting intra- and extra-cellular mechanical stimuli via the cytoskeleton to the nucleus. Nesprins and SUN proteins, located at the nuclear envelope, form the LINC (linker of nucleoskeleton and cytoskeleton) complex that connects the nucleus to the cytoskeleton; underlying nuclear lamins contribute to anchoring LINC complex components at the nuclear envelope. Disruption of the LINC complex or loss of lamins can result in disturbed perinuclear actin and intermediate filament networks and causes severe functional defects, including impaired nuclear positioning, cell polarization and cell motility. Recent studies have identified the LINC complex as the major force-transmitting element at the nuclear envelope and suggest that many of the aforementioned defects can be attributed to disturbed force transmission between the nucleus and the cytoskeleton. Thus mutations in nesprins, SUN proteins or lamins, which have been linked to muscular dystrophies and cardiomyopathies, may weaken or completely eliminate LINC complex function at the nuclear envelope and result in impaired intracellular force transmission, thereby disrupting critical cellular functions.
- cell mechanics
- force transmission
- SUN protein
The importance of efficient force transmission between cells and their surroundings has long been recognized and studied in the context of cell adhesion, migration, and cell–cell and cell–matrix interactions. Many of the molecular players, such as integrins, cadherins or members of the dystroglycan complex, are now well characterized. In contrast, even though it is evident that extracellular forces are transmitted across the cytoskeleton to the nucleus , the molecular details involved in coupling the nucleus to the cytoskeleton have only recently begun to emerge. The current model suggests that nesprins, a family of spectrin-repeat proteins located at the nuclear envelope, can simultaneously bind to cytoskeletal filaments and interact across the luminal space with the inner nuclear membrane proteins SUN1 and SUN2. This physical link between the cytoskeleton and the nucleus is referred to as the LINC (linker of nucleoskeleton and cytoskeleton) complex [2,3]. SUN proteins can, in turn, bind to lamins, chromatin and other nuclear envelope proteins , extending further the physical connection to the nuclear interior . Nonetheless, despite the many recent advances, it remains unclear whether or how other nuclear envelope proteins can modulate the interaction of LINC complex proteins or independently contribute to nucleocytoskeletal coupling.
To date, at least four nesprin genes (encoding nesprin-1–4) have been identified. As a result of multiple initiation and splicing sites, nesprins are expressed in multiple isoforms that vary in length, domain composition and expression patterns. Nesprins can be located at the inner or outer nuclear membrane, probably in the form of dimers or other complexes. The largest (>800 kDa) isoforms of nesprin-1 and nesprin-2 contain an N-terminal actin-binding domain that can interact with cytoplasmic actin filaments [6,7], whereas nesprin-3 isoforms (~100 kDa) bind to plectin proteins, which cross-link intermediate filaments . Nesprin-4, which is only expressed in secretory epithelial cells, is a kinesin-binding protein that connects the nucleus to microtubules . The C-terminal domain of all four nesprins consists of a highly conserved KASH (Klarsicht/ANC-1/SYNE homology) domain  that can bind to the C-terminal luminal domain of SUN proteins and is required for the localization of nesprins at the nuclear envelope [2,4,11,12]. SUN1 and SUN2 are retained at the inner nuclear membrane by the interaction of their N-terminal domain with lamins, chromatin and other nuclear membrane proteins, and the loss of lamins A/C results in increased mobility of nesprins and SUN proteins at the nuclear envelope [4,13,14].
Loss of nesprins, SUN proteins and lamins from the nuclear envelope results in defects in nuclear positioning, cell polarization and migration
Many of the initial insights into nucleocytoskeletal coupling have come from studies in Caenorhabditis elegans and Drosophila melanogaster. In C. elegans, the nesprin-1/2 orthologue ANC-1 is critical for actin-dependent nuclear positioning in muscle cells during development , and mutations in the SUN1 orthologue UNC-84 similarly result in defective nuclear migration and anchoring . Recent independent studies in mouse and human fibroblasts expressing dominant-negative LINC complex components or lacking specific nesprin or SUN proteins have reported defects in centrosome attachment to the nucleus ; in scratch-wound assays, the initial cell polarization towards the wound, which consists of rearward nuclear movement and positioning of the centrosome towards the leading edge, is often absent, and the subsequent migration of cells into the wound is impaired [13,17–21]. For example, knockdown of nesprin-1 prevents the reorientation of endothelial cells in response to cyclic strain and causes a decrease in endothelial cell migration in a scratch-wound assay . In fibroblasts, nesprin-2 giant is required for the rearward nuclear movement that precedes polarization and migration . Similar results can be seen when disrupting the LINC complex with dominant-negative constructs and plating cells on micropatterned substrates that induce cell polarization (Figure 1). In addition to nesprin and SUN proteins, impaired nuclear polarization and migration has been demonstrated in lamin A/C-deficient fibroblasts . In genetically engineered mice, mutations in the LINC complex proteins or lamins have been shown to cause abnormal nuclear positioning of synaptic nuclei at the neuromuscular junction in skeletal muscle [23–26].
Nucleocytoskeletal coupling is crucial for intracellular force transmission
How can mutations in nuclear envelope proteins result in such dramatic defects in cellular organization and function? Force transmission from the cytoskeleton to the nuclear interior is essential for many cellular processes (Figure 2). In migrating cells, movement of the nucleus is closely co-ordinated with the dynamic remodelling of the cytoskeleton, resulting in the nucleus appearing to remain stationary relative to the moving cell; during interkinetic nuclear migration, the nucleus periodically moves between the basal and apical side of the neuron; in muscle cells, synaptic nuclei are moved towards the cell periphery and anchored at neuromuscular junctions . To shift the nucleus within the cell or, in the case of migrating cells, along with the cell, cytoskeletal forces must pull or push on the nucleus ; even to retain the nucleus in a fixed position within the cell, the nucleus must be tightly anchored to the surrounding cytoskeleton to prevent random movement. In a seminal study, Luxton et al.  identified the interaction of SUN proteins and nesprins as critical for intracellular force transmission in mammalian cells by demonstrating that the rearward nuclear movement during the initial cell polarization in a scratch-wound assay is driven by the retrograde flow of actin cables that engage the nucleus via nesprin-2 and SUN2, creating structures referred to as TAN (transmembrane actin-associated nuclear) lines, and that knockdown of either nesprin-2 or SUN2 is sufficient to abrogate nuclear movement. In addition to actin-mediated forces, microtubule-associated motors such as dynein or kinesin can also propel the nucleus within the cell. A nuclear envelope-spanning complex of kinesin-1, nesprin-2, SUN proteins and lamins is required for cell polarization in a scratch-wound assay , and ectopic expression of nesprin-4, which interacts with kinesin-1, can result in dramatic cellular rearrangement and separation between the nucleus and the centrosome . Further support for the importance of the LINC complex in intracellular force transmission comes from a recent study in which a precisely controlled strain was applied to adherent fibroblasts either locally by displacing a microneedle inserted into the cytoskeleton in close proximity to the nucleus, or globally by stretching the elastic substrate on which the cells were cultured . Intracellular force transmission can then be inferred from the induced cytoskeletal and nuclear displacements (Figure 3) and compared between cells in which the LINC complex had been disrupted by expression of dominant-negative nesprin (or SUN constructs) and mock controls. In these experiments, LINC complex disruption resulted in impaired propagation of intracellular forces between the cytoskeleton and the nucleus, as is evident by reduced nuclear displacements in cells subjected to internal or external mechanical strain . Taken together, these studies indicate that the LINC complex is critical for efficient force transmission between the nucleus and the cytoskeleton.
Disruption of nucleocytoskeletal coupling alters perinuclear cytoskeletal organization
Cytoskeletal components remodel in response to applied forces, and this dynamic feedback with intra- and extra-cellular forces has an important impact on cellular morphology. For example, in cells subjected to fluid shear stress, actin stress fibres align with the flow direction within 12 h . Since cytoskeletal components, such as actin, vimentin and microtubules, are directly associated with the LINC complex, it is not surprising that disruption of the LINC complex can result in altered cytoskeletal organization by eliminating anchoring sites at the nuclear envelope and reducing force transmission between the cytoskeleton and the nucleus. Cultured fibroblasts expressing dominant-negative forms of nesprin and SUN proteins have discontinuous and fragmented actin stress fibres in the perinuclear area and also disturbed organization of the perinuclear vimentin filament systems, characterized by a looser and more irregular network surrounding the nucleus [13,17,18,20,21,28]. The reduced density of the perinuclear cytoskeleton could, in turn, contribute further to the reduced force transmission between the nucleus and the cytoskeleton. Of note, lamin A/C-deficient cells display similar defects in perinuclear cytoskeletal organization [18,30] and have reduced cytoskeletal stiffness compared with wild-type controls [19,31], suggesting that lamins may play a similar role in nucleocytoskeletal coupling and cytoskeletal organization as that of nesprins and SUN proteins, possibly by anchoring LINC complex proteins at the nuclear envelope.
Defective nucleocytoskeletal coupling may impair activation of mechanosensitive genes
Cells respond to mechanical stimulation with activation of specific signalling pathways and expression of mechanosensitive genes, a process referred to as mechanotransduction. The nucleus itself has often been proposed as a cellular mechanosensor ; for example, nuclear deformations could cause conformational changes in chromatin structure and organization that modulate accessibility of transcription factors or transcriptional processes . Initial support for the existence of nuclear mechanosensing came from findings in lamin A/C-deficient and emerin-deficient mouse embryonic fibroblasts that have altered nuclear mechanics and impaired expression of the mechanosensitive genes Iex-1 and Egr-1 in response to cyclic strain application [31,32]. However, since lamins A and C can also modulate signalling pathways directly by binding to transcriptional regulars such as c-Fos and ERK1/2 (extracellular-signal-regulated kinase 1/2) , it remains unclear whether the observed mechanotransduction defects result from impaired nucleocytoskeletal coupling or from the loss of biochemical interactions between transcriptional regulators and specific nuclear envelope proteins. Recent experiments in which nucleocytoskeletal coupling was disrupted with dominant-negative nesprin constructs without affecting lamin A/C or emerin levels suggest the latter, as the mechanically induced expression of Iex-1 and Egr-1 was indistinguishable from control cells, in spite of dramatically reduced nuclear deformations in the LINC complex-disrupted cells . One likely explanation is that mechanotransduction signals triggered at focal adhesions, the plasma membrane or the peripheral cytoskeleton are sufficient to initiate cellular mechanotransduction pathways . Nonetheless, disturbed nuclear and/or cytoskeletal organization resulting from LINC complex disruption could have additional, possibly modulating, effects on specific transcriptional pathways. In one recent example, C2C12 cells expressing dominant-negative nesprin and SUN protein constructs showed enhanced activation of NF-KB (nuclear factor κB) signalling, regardless of whether the stimulation was mechanical (cyclic strain) or chemical [TNFα (tumour necrosis factor α)] .
Defective nucleocytoskeletal coupling and force transmission in human diseases
Muscular dystrophies and congenital dilated cardiomyopathies are often caused by mutations in cytoskeletal proteins that disrupt cellular force transmission [35,36]. For example, Duchenne muscular dystrophy, the most common form of muscular dystrophy, is caused by mutations in the dystrophin gene, which disturbs connections between the cytoskeleton and the extracellular matrix and renders the plasma membrane more fragile . Recently, nesprin-1 and -2 and SUN proteins joined lamins A/C, emerin and torsin A in the growing group of nuclear envelope proteins that can cause muscular dystrophies and dilated cardiomyopathy when mutated [20,37–40]. Of note, all of these nuclear envelope proteins are either part of the LINC complex or interact directly with LINC complex proteins. For most of these genes, it has already been demonstrated that functional loss causes impaired nucleocytoskeletal coupling and defects in intracellular force transmission, cell polarization and migration. Future experiments should be aimed at investigating whether the disease-causing mutations elicit similar phenotypes to the complete loss of protein or the dominant-negative constructs used in the current studies. This is particularly important in the light of the fact that the nesprin mutations identified in muscular dystrophy patients to date lie outside the KASH domain. For lamins A and C, work by the Gundersen group revealed that lamin mutants associated with muscular dystrophy, but not those linked to lipodystrophy, abolished retrograde nuclear movement in a scratch wound assay , suggesting that loss of nucleocytoskeletal coupling is particularly relevant to muscular laminopathies.
Nonetheless, the consequences of defects in nucleocytoskeletal coupling are not limited to muscle cells. Mutations in the gene encoding lamins A and C are notorious for causing a plethora of human diseases that include Emery–Dreifuss muscular dystrophy and limb-girdle muscular dystrophy, and also Hutchinson–Gilford progeria syndrome, familial partial lipodystrophy and Charcot–Marie–Tooth disease ; mutations in nesprin-1 also cause Meckel–Gruber syndrome and autosomal recessive cerebellar ataxia with neurological defects in the cerebellum [43,44]; duplication of the lamin B1 gene has been linked to autosomal dominant leukodystrophy, a neurological disorder ; and mouse embryonic fibroblasts lacking full-length lamin B1 have defective nucleocytoskeletal coupling, causing the nuclei of these cells to undergo spontaneous sustained rotations . In mice, loss of lamin B2 results in severe defects in neuronal migration in the cerebral cortex , and mice lacking either nesprin-2 or SUN1 and SUN2 have mislocalized photoreceptor nuclei in the retina , illustrating further the importance of sufficient nucleocytoskeletal coupling in neuronal development and intracellular nuclear migration.
Conclusions and perspectives
The LINC complex and LINC complex-associated proteins provide an essential connection between the cytoskeleton and the nucleus that is critical for intracellular force transmission. Functional loss, by mutation, dominant-negative constructs or genetic manipulation, of these proteins can result in defective cytoskeletal organization, impaired nuclear positioning and anchoring and loss of cell polarization and reduced motility. In recent years, a rapidly growing number of mutations in nuclear envelope proteins have been identified as the cause of at least 15 human diseases; impaired nucleocytoskeletal coupling and disturbed intracellular force transmission are likely to contribute to the disease mechanism, particularly in muscle and neuronal cells. Despite many recent advances, we still have an incomplete understanding of the various functions of the diverse nuclear envelope proteins and their complex interactions. For example, just in the case of nesprins, numerous isoforms exist, and yet even the functional differences between nesprin-1 and -2 have not been fully addressed. Similarly, considering how promiscuous the interactions between KASH and SUN domain proteins appear, little is known about the mechanism by which interactions between nesprins, SUN proteins and other nuclear envelope proteins are regulated to control nucleocytoskeletal coupling in cells. Future research, combining clever genetic and experimental approaches to probe nucleocytoskeletal coupling in vitro and in vivo, will be required to address these questions.
This work was supported by the National Institutes of Health [grant numbers R01 NS059348 and R01 HL082792] and the Brigham and Women's Hospital Cardiovascular Leadership Group Award.
We apologize to all authors whose work could not be cited because of space constraints.
Nuclear Envelope Disease and Chromatin Organization: A Biochemical Society Focused Meeting held at Robinson College, Cambridge, U.K., 13–15 July 2011. Organized and Edited by Juliet Ellis (King's College London, U.K.) and Sue Shackleton (Leicester, U.K.).
Abbreviations: KASH, Klarsicht/ANC-1/SYNE homology; LINC, linker of nucleoskeleton and cytoskeleton
- © The Authors Journal compilation © 2011 Biochemical Society