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Abstract Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that primarily affects motor neurons, leading to progressive muscle weakness and loss of voluntary muscle control. While the exact cause of ALS is not fully understood, emerging research suggests that dysfunction of the nuclear envelope (NE) may contribute to disease pathogenesis and progression. The NE plays a role in ALS through several mechanisms, including nuclear pore defects, nucleocytoplasmic transport impairment, accumulation of mislocalized proteins, and nuclear morphology abnormalities. The LINC complex is the second biggest multi-protein complex in the NE and consists of the SUN1/2 proteins spanning the inner nuclear membrane and Nesprin proteins embedded in the outer membrane. The LINC complex, by interacting with both the nuclear lamina and the cytoskeleton, transmits mechanical forces to the nucleus regulating its morphology and functional homeostasis. In this study we show extensive alterations to the LINC complex in motor and cortical iPSC-derived neurons and spinal cord organoids carrying the ALS causative mutation in the C9ORF72 gene (C9). Importantly, we show that such alterations are present in vivo in a cohort of sporadic ALS and C9-ALS postmortem spinal cord and motor cortex specimens. We also found that LINC complex disruption strongly correlated with nuclear morphological alterations occurring in ALS neurons, independently of TDP43 mislocalization. Altogether, our data establish morphological and functional alterations to the LINC complex as important events in ALS pathogenic cascade, making this pathway a possible target for both biomarker and therapy development.

Abstract This review presents a comprehensive exploration of the pivotal role played by the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, with a particular focus on Nesprin proteins, in cellular mechanics and the pathogenesis of muscular diseases. Distinguishing itself from prior works, the analysis delves deeply into the intricate interplay of the LINC complex, emphasizing its indispensable contribution to maintaining cellular structural integrity, especially in mechanically sensitive tissues such as cardiac and striated muscles. Additionally, the significant association between mutations in Nesprin proteins and the onset of Dilated Cardiomyopathy (DCM) and Emery-Dreifuss Muscular Dystrophy (EDMD) is highlighted, underscoring their pivotal role in disease pathogenesis. Through a comprehensive examination of DCM and EDMD cases, the review elucidates the disruptions in the LINC complex, nuclear morphology alterations, and muscular developmental disorders, thus emphasizing the essential function of an intact LINC complex in preserving muscle physiological functions. Moreover, the review provides novel insights into the implications of Nesprin mutations for cellular dynamics in the pathogenesis of muscular diseases, particularly in maintaining cardiac structural and functional integrity. Furthermore, advanced therapeutic strategies, including rectifying Nesprin gene mutations, controlling Nesprin protein expression, enhancing LINC complex functionality, and augmenting cardiac muscle cell function are proposed. By shedding light on the intricate molecular mechanisms underlying nuclear-cytoskeletal interactions, the review lays the groundwork for future research and therapeutic interventions aimed at addressing genetic muscle disorders.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that primarily affects motor neurons, leading to progressive muscle weakness and loss of voluntary muscle control. While the exact cause of ALS is not fully understood, emerging research suggests that dysfunction of the nuclear envelope (NE) may contribute to disease pathogenesis and progression. The NE plays a role in ALS through several mechanisms, including nuclear pore defects, nucleocytoplasmic transport impairment, accumulation of mislocalized proteins, and nuclear morphology abnormalities. The LINC complex is the second biggest multi-protein complex in the NE and consists of the SUN1/2 proteins spanning the inner nuclear membrane and Nesprin proteins embedded in the outer membrane. The LINC complex, by interacting with both the nuclear lamina and the cytoskeleton, transmits mechanical forces to the nucleus regulating its morphology and functional homeostasis. In this study we show extensive alterations to the LINC complex in motor and cortical iPSC-derived neurons and spinal cord organoids carrying the ALS causative mutation in the C9ORF72 gene (C9). Importantly, we show that such alterations are present in vivo in a cohort of sporadic ALS and C9-ALS postmortem spinal cord and motor cortex specimens. We also found that LINC complex disruption strongly correlated with nuclear morphological alterations occurring in ALS neurons, independently of TDP43 mislocalization. Altogether, our data establish morphological and functional alterations to the LINC complex as important events in ALS pathogenic cascade, making this pathway a possible target for both biomarker and therapy development. [ABSTRACT FROM AUTHOR]

This review presents a comprehensive exploration of the pivotal role played by the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, with a particular focus on Nesprin proteins, in cellular mechanics and the pathogenesis of muscular diseases. Distinguishing itself from prior works, the analysis delves deeply into the intricate interplay of the LINC complex, emphasizing its indispensable contribution to maintaining cellular structural integrity, especially in mechanically sensitive tissues such as cardiac and striated muscles. Additionally, the significant association between mutations in Nesprin proteins and the onset of Dilated Cardiomyopathy (DCM) and Emery-Dreifuss Muscular Dystrophy (EDMD) is highlighted, underscoring their pivotal role in disease pathogenesis. Through a comprehensive examination of DCM and EDMD cases, the review elucidates the disruptions in the LINC complex, nuclear morphology alterations, and muscular developmental disorders, thus emphasizing the essential function of an intact LINC complex in preserving muscle physiological functions. Moreover, the review provides novel insights into the implications of Nesprin mutations for cellular dynamics in the pathogenesis of muscular diseases, particularly in maintaining cardiac structural and functional integrity. Furthermore, advanced therapeutic strategies, including rectifying Nesprin gene mutations, controlling Nesprin protein expression, enhancing LINC complex functionality, and augmenting cardiac muscle cell function are proposed. By shedding light on the intricate molecular mechanisms underlying nuclear-cytoskeletal interactions, the review lays the groundwork for future research and therapeutic interventions aimed at addressing genetic muscle disorders. [ABSTRACT FROM AUTHOR]

SYNE2 mutations have been associated with skeletal and cardiac muscle diseases, including Emery-Dreifuss muscular dystrophy (EDMD). Here, we present a 70-year-old male patient with muscle pain and elevated serum creatine kinase levels in whom whole-exome sequencing revealed a novel heterozygous SYNE2 splice site mutation (NM_182914.3:c.15306+2T>G). This mutation is likely to result in the loss of the donor splice site in intron 82. While a diagnostic muscle biopsy showed unspecific myopathological findings, immunofluorescence analyses of skeletal muscle and dermal cells derived from the patient showed nuclear shape alterations when compared to control cells. In addition, a significantly reduced nesprin-2 giant protein localisation to the nuclear envelope was observed in patient-derived dermal fibroblasts. Our findings imply that the novel heterozygous SYNE2 mutation results in a monoallelic splicing defect of nesprin-2, thereby leading to a rare cause of myalgia and hyperCKemia.

SYNE2 mutations have been associated with skeletal and cardiac muscle diseases, including Emery-Dreifuss muscular dystrophy (EDMD). Here, we present a 70-year-old male patient with muscle pain and elevated serum creatine kinase levels in whom whole-exome sequencing revealed a novel heterozygous SYNE2 splice site mutation (NM_182914.3:c.15306+2T>G). This mutation is likely to result in the loss of the donor splice site in intron 82. While a diagnostic muscle biopsy showed unspecific myopathological findings, immunofluorescence analyses of skeletal muscle and dermal cells derived from the patient showed nuclear shape alterations when compared to control cells. In addition, a significantly reduced nesprin-2 giant protein localisation to the nuclear envelope was observed in patient-derived dermal fibroblasts. Our findings imply that the novel heterozygous SYNE2 mutation results in a monoallelic splicing defect of nesprin-2, thereby leading to a rare cause of myalgia and hyperCKemia. [ABSTRACT FROM AUTHOR]

Nuclear positioning is important for the functionality of many cell types and is mediated by interactions of cytoskeletal elements and nucleoskeleton proteins. Nesprin proteins, part of the linker of nucleoskeleton and cytoskeleton (LINC) complex, have been shown to participate in nuclear positioning in multiple cell types. Outer hair cells (OHCs) in the inner ear are specialized sensory epithelial cells that utilize somatic electromotility to amplify auditory signals in the cochlea. Recently, Nesprin-4 (encoded by Syne4) was shown to play a crucial role in nuclear positioning in OHCs. Syne4 deficiency in humans and mice leads to mislocalization of the OHC nuclei and cell death resulting in deafness. However, it is unknown how Nesprin-4 mediates the position of the nucleus, and which other molecular components are involved in this process. Here, we show that the interaction of Nesprin-4 and the microtubule motor kinesin-1 is mediated by a conserved 4 amino-acid motif. Using in vivo AAV gene delivery, we show that this interaction is critical for nuclear positioning and hearing in mice. Nuclear mislocalization and cell death of OHCs coincide with the onset of hearing and electromotility and are solely restricted to outer, but not inner, hair cells. Likewise, the C. elegans functional homolog of Nesprin-4, UNC-83, uses a similar motif to mediate interactions between migrating nuclei and kinesin-1. Overall, our results suggest that OHCs require unique cellular machinery for proper nuclear positioning at the onset of electromotility. This machinery relies on the interaction between Nesprin-4 and kinesin-1 motors supporting a microtubule cargo model for nuclear positioning.

KASH proteins in the outer nuclear membrane comprise the cytoplasmic half of linker of nucleoskeleton and cytoskeleton (LINC) complexes that connect nuclei to the cytoskeleton. Caenorhabditis elegans ANC-1, an ortholog of Nesprin-1/2, contains actin-binding and KASH domains at opposite ends of a long spectrin-like region. Deletion of either the KASH or calponin homology (CH) domains does not completely disrupt nuclear positioning, suggesting neither KASH nor CH domains are essential. Deletions in the spectrin-like region of ANC-1 led to significant defects, but only recapitulated the null phenotype in combination with mutations in the transmembrane (TM) span. In anc-1 mutants, the endoplasmic reticulum ER, mitochondria, and lipid droplets were unanchored, moving throughout the cytoplasm. The data presented here support a cytoplasmic integrity model where ANC-1 localizes to the ER membrane and extends into the cytoplasm to position nuclei, ER, mitochondria, and other organelles in place.

Many nuclear positioning events involve linker of nucleoskeleton and cytoskeleton (LINC) complexes, which transmit forces generated by the cytoskeleton across the nuclear envelope. LINC complexes are formed by trans-luminal interactions between inner nuclear membrane SUN proteins and outer nuclear membrane KASH proteins, but how these interactions are regulated is poorly understood. We combine in vivo C. elegans genetics, in vitro wounded fibroblast polarization, and in silico molecular dynamics simulations to elucidate mechanisms of LINC complexes. The extension of the KASH domain by a single alanine residue or the mutation of the conserved tyrosine at -7 completely blocked the nuclear migration function of C. elegans UNC-83. Analogous mutations at -7 of mouse nesprin-2 disrupted rearward nuclear movements in NIH 3T3 cells, but did not disrupt ANC-1 in nuclear anchorage. Furthermore, conserved cysteines predicted to form a disulfide bond between SUN and KASH proteins are important for the function of certain LINC complexes, and might promote a developmental switch between nuclear migration and nuclear anchorage. Mutations of conserved cysteines in SUN or KASH disrupted ANC-1-dependent nuclear anchorage in C. elegans and Nesprin-2G-dependent nuclear movements in polarizing fibroblasts. However, the SUN cysteine mutation did not disrupt nuclear migration. Moreover, molecular dynamics simulations showed that a disulfide bond is necessary for the maximal transmission of cytoskeleton-generated forces by LINC complexes in silico. Thus, we have demonstrated functions for SUN-KASH binding interfaces, including a predicted intermolecular disulfide bond, as mechanistic determinants of nuclear positioning that may represent targets for regulation.

Mutations in genes encoding proteins associated with the linker of nucleoskeleton and cytoskeleton (LINC) complex within the nuclear envelope cause different diseases with varying phenotypes including skeletal muscle, cardiac, metabolic, or nervous system pathologies. There is some understanding of the structure of LINC complex-associated proteins and how they interact, but it is unclear how mutations in genes encoding them can cause the same disease, and different diseases with different phenotypes. Here, published mutations in LINC complex-associated proteins were systematically reviewed and analyzed to ascertain whether patterns exist between the genetic sequence variants and clinical phenotypes. This revealed LMNA is the only LINC complex-associated gene in which mutations commonly cause distinct conditions, and there are no clear genotype-phenotype correlations. Clusters of LMNA variants causing striated muscle disease are located in exons 1 and 6, and metabolic disease-associated LMNA variants are frequently found in the tail of lamin A/C. Additionally, exon 6 of the emerin gene, EMD, may be a mutation "hot-spot", and diseases related to SYNE1, encoding nesprin-1, are most often caused by nonsense type mutations. These results provide insight into the diverse roles of LINC-complex proteins in human disease and provide direction for future gene-targeted therapy development. [ABSTRACT FROM AUTHOR]

Mechanical force plays a pivotal role in the pathogenesis of hypertrophic scar (HTS). Dermal fibroblasts and myofibroblasts are the key cells involved in HTS. Myofibroblasts in HTS possess different biochemical and biophysical characteristics by which myofibroblasts are often distinguished from fibroblasts. The role of mechanotransducers outside the nucleus in the pathogenesis of HTS has been reported in many studies. However, the role of Nesprin‐2 in HTS is not clear. Hence, we aim to construct a cell model of HTS and explore the role of Nesprin‐2 in this process. Myofibroblasts and fibroblasts were isolated from HTS and healthy skin tissues of the same patient. Fibroblasts were exposed to cyclic stretch with 10% magnitude and a frequency of 0.1 Hz for 3 days, 5 days, and 7 days, respectively. After the cell model was confirmed, fibroblasts transfected with siRNA targeting human Nesprin‐2 were exposed to cyclic stretch. The mechanical behaviour and biochemical reaction of the dermal fibroblasts were analysed. The stretched fibroblasts at day 5 showed the same mechanotransductive and biochemical features as unstretched myofibroblasts. Mechanical strain could induce the myofibroblasts differentiation and a cell model of HTS was established successfully at day 5. The expressions of lamin A/C, alpha‐smooth muscle actin, transforming growth factor beta 1, and collagen type I in fibroblasts were reduced by the silencing of Nesprin‐2. Mechanical strain could induce the myofibroblasts differentiation and silencing of Nesprin‐2 could block the mechanical stimulation of terminal myofibroblasts differentiation. Nesprin‐2 might be a potential target to treat the HTS. [ABSTRACT FROM AUTHOR]

Nuclear positioning is important for the functionality of many cell types and is mediated by interactions of cytoskeletal elements and nucleoskeleton proteins. Nesprin proteins, part of the linker of nucleoskeleton and cytoskeleton (LINC) complex, have been shown to participate in nuclear positioning in multiple cell types. Outer hair cells (OHCs) in the inner ear are specialized sensory epithelial cells that utilize somatic electromotility to amplify auditory signals in the cochlea. Recently, Nesprin-4 (encoded by Syne4) was shown to play a crucial role in nuclear positioning in OHCs. Syne4 deficiency in humans and mice leads to mislocalization of the OHC nuclei and cell death resulting in deafness. However, it is unknown how Nesprin-4 mediates the position of the nucleus, and which other molecular components are involved in this process. Here, we show that the interaction of Nesprin-4 and the microtubule motor kinesin-1 is mediated by a conserved 4 amino-acid motif. Using in vivo AAV gene delivery, we show that this interaction is critical for nuclear positioning and hearing in mice. Nuclear mislocalization and cell death of OHCs coincide with the onset of hearing and electromotility and are solely restricted to outer, but not inner, hair cells. Likewise, the C. elegans functional homolog of Nesprin-4, UNC-83, uses a similar motif to mediate interactions between migrating nuclei and kinesin-1. Overall, our results suggest that OHCs require unique cellular machinery for proper nuclear positioning at the onset of electromotility. This machinery relies on the interaction between Nesprin-4 and kinesin-1 motors supporting a microtubule cargo model for nuclear positioning.

Pathogenic variants in the SYNE1 gene are associated with a phenotypic spectrum spanning from late-onset, slowly progressive, relatively pure ataxia to early-onset, fast progressive multisystemic disease. Since its first description in 2007 as an adult-onset ataxia in French Canadian families, subsequent identification of patients worldwide has widened the clinical spectrum and increased the number of identified pathogenic variants. We report a 20-year-old Faroese female with early-onset progressive gait problems, weakness, dysphagia, slurred speech, orthostatic dizziness, and urge incontinence. Neurological examination revealed mild cognitive deficits, dysarthria, broken slow pursuit, hypometric saccades, weakness with spasticity, hyperreflexia, absent ankle reflexes, ataxia, and wide-based, spastic gait. Magnetic resonance imaging displayed atrophy of the cerebellum, brainstem, and spinal cord. Severely prolonged central motor conduction time and lower motor neuron involvement was demonstrated electrophysiologically. Fluorodeoxyglucose-positron emission tomography (FDG-PET) scan showed hypometabolism of the cerebellum and right frontal lobe. Muscle biopsy revealed chronic neurogenic changes and near-absent immunostaining for Nesprin-1. Next-generation sequencing revealed a previously undescribed homozygous truncating, likely pathogenic variant in the SYNE1 gene. The patient's mother and paternal grandfather were heterozygous carriers of the variant. Her father's genotype was unobtainable. We expand the list of likely pathogenic variants in SYNE1 ataxia with a novel homozygous truncating variant with proximity to the C-terminus and relate it to a phenotype comprising early-onset cerebellar deficits, upper and lower motor neuron involvement and cognitive deficits. Also, we report novel findings of focally reduced frontal lobe FDG-PET uptake and motor evoked potential abnormalities suggestive of central demyelination. [ABSTRACT FROM AUTHOR]

The nuclear envelope consists of 2 membranes separated by 30-50 nm, but how the 2 membranes are evenly spaced has been an open question in the field. Nuclear envelope bridges composed of inner nuclear membrane SUN proteins and outer nuclear membrane KASH proteins have been proposed to set and regulate nuclear envelope spacing. We tested this hypothesis directly by examining nuclear envelope spacing in Caenorhabditis elegans animals lacking UNC-84, the sole somatic SUN protein. SUN/KASH bridges are not required to maintain even nuclear envelope spacing in most tissues. However, UNC-84 is required for even spacing in body wall muscle nuclei. Shortening UNC-84 by 300 amino acids did not narrow the nuclear envelope space. While SUN proteins may play a role in maintaining nuclear envelope spacing in cells experiencing forces, our data suggest they are dispensable in most cells.

Abstract Mesenchymal stem cells (MSCs) show tremendous promise as a cell source for tissue engineering and regenerative medicine, and are understood to be mechanosensitive to external mechanical environments. In recent years, increasing evidence points to nuclear envelope proteins as a key player in sensing and relaying mechanical signals in MSCs to modulate cellular form, function, and differentiation. Of particular interest is the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex that includes nesprin and SUN. In this review, the way in which cells can sense external mechanical environments through an intact nuclear envelope and LINC complex proteins will be briefly described. Then, we will highlight the current body of literature on the role of the LINC complex in regulating MSC function and fate decision, without and with external mechanical loading conditions. Our review and suggested future perspective may provide a new insight into the understanding of MSC mechanobiology and related functional tissue engineering applications.

Syne2b/nesprin-2 is a giant protein implicated in tethering the nucleus to the cytoskeleton and plays an important role in maintaining cellular architecture. Epiboly is a conserved morphogenetic movement that involves extensive spreading and thinning of the epithelial blastoderm to shape the embryo and organize the three germ layers. Dynamic cytoskeletal organization is critical for this process, but how it is regulated remains elusive. Here we generated a zebrafish syne2b mutant line and analyzed the effects of impaired Syne2b function during early development. By CRISPR/Cas9-mediated genome editing, we obtained a large deletion in the syne2b locus, predicted to cause truncation of the nuclear localization KASH domain in the translated protein. Maternal and zygotic syne2b embryos showed delayed epiboly initiation and progression without defects in embryonic patterning. Remarkably, disruption of Syne2b function severely impaired cytoskeletal organization across the embryo, leading to aberrant clustering of F-actin at multiple cell contact regions and abnormal cell shape changes. These caused disintegration of the epithelial blastoderm before the end of gastrulation in most severely affected embryos. Moreover, the migration of yolk nuclear syncytium also became defective, likely due to disorganized cytoskeletal networks at the blastoderm margin and in the yolk cell. These findings demonstrate an essential function of Syne2b in maintaining cytoskeletal architecture and epithelial integrity during epiboly movement.

Experiments in C. elegans reveal new insights into how the ANC-1 protein helps to anchor the nucleus and other organelles in place.

KASH proteins in the outer nuclear membrane comprise the cytoplasmic half of linker of nucleoskeleton and cytoskeleton (LINC) complexes that connect nuclei to the cytoskeleton. Caenorhabditis elegans ANC-1, an ortholog of Nesprin-1/2, contains actin-binding and KASH domains at opposite ends of a long spectrin-like region. Deletion of either the KASH or calponin homology (CH) domains does not completely disrupt nuclear positioning, suggesting neither KASH nor CH domains are essential. Deletions in the spectrin-like region of ANC-1 led to significant defects, but only recapitulated the null phenotype in combination with mutations in the transmembrane (TM) span. In anc-1 mutants, the endoplasmic reticulum ER, mitochondria, and lipid droplets were unanchored, moving throughout the cytoplasm. The data presented here support a cytoplasmic integrity model where ANC-1 localizes to the ER membrane and extends into the cytoplasm to position nuclei, ER, mitochondria, and other organelles in place.

Actin plays roles in many important cellular processes, including cell motility, organelle movement, and cell signaling. The discovery of transmembrane actin-binding proteins at the outer nuclear membrane (ONM) raises the exciting possibility that actin can play a role in direct force transmission to the nucleus and the genome at its interior. Actin-dependent nucleus displacement was first described a decade ago. We are now gaining a more detailed understanding of its mechanisms, as well as new roles for actin during mitosis and meiosis, for gene expression, and in the cell's response to mechanical stimuli. Here we review these recent developments, the actin-binding proteins involved, the tissue specificity of these mechanisms, and methods developed to reconstitute and study this interaction in vitro. Direct connections between the actin cytoskeleton and the nucleus govern nuclear positioning, nuclear movement during cell polarization and migration, nuclear movement before and after mitosis and meiosis, nuclear envelope breakdown, mechanotransduction, and gene expression. The nucleus directly connects to actin through transmembrane proteins (LINC complex proteins) complemented by a large family of actin-associated proteins that interact with proteins of the nuclear envelope. Actin can act indirectly on the nucleus to displace it, to protect it from external forces, and to dampen mechanical stimuli. However, the compression exerted by actin can cause nuclear rupture. Interactions between the nucleus and actin are studied in vitro using isolated nuclei and reconstituted actin networks. [ABSTRACT FROM AUTHOR]

The mechanisms by which cells exert forces on their nuclei to migrate through openings smaller than the nuclear diameter remain unclear. We use CRISPR/Cas9 to fluorescently label nesprin‐2 giant, which links the cytoskeleton to the nuclear interior. We demonstrate that nesprin‐2 accumulates at the front of the nucleus during nuclear deformation through narrow constrictions, independently of the nuclear lamina. We find that nesprins are mobile at time scales similar to the accumulation. Using artificial constructs, we show that the actin‐binding domain of nesprin‐2 is necessary and sufficient for this accumulation. Actin filaments are organized in a barrel structure around the nucleus in the direction of movement. Using two‐photon ablation and cytoskeleton‐inhibiting drugs, we demonstrate an actomyosin‐dependent pulling force on the nucleus from the front of the cell. The elastic recoil upon ablation is dampened when nesprins are reduced at the nuclear envelope. We thus show that actin redistributes nesprin‐2 giant toward the front of the nucleus and contributes to pulling the nucleus through narrow constrictions, in concert with myosin. Synopsis: The outer nuclear envelope protein nesprin‐2 accumulates at the front of the nucleus during migration through narrow constrictions via its actin domain. Actomyosin contractility pulls the nucleus through constrictions, implicating that giant nesprin‐2 is a force transducer between actin and the nucleus during 3‐D migration. Nesprin redistribution at the front of the nucleus depends on its actin‐binding domain, but not on A‐type lamins at the nucleoskeleton.Actomyosin contractility is necessary to translocate nuclei through narrow constrictions.Nuclei migrating through constrictions are subjected to a pulling force from the front of the cell. [ABSTRACT FROM AUTHOR]

• EDMD is caused by mutations in the genes encoding emerin, lamin A/C, nesprins, or FHL1 • Emerin, lamin A/C, nesprins, and FHL1B are integral to the LINC complex • Emerin is involved in regulation of myogenic differentiation signalling pathways • Nesprin1/2, SUN1/2, and lamin A/C are necessary for correct nuclear positioning • Future work should identify therapeutic targets downstream from defective genes Emery-Dreifuss muscular dystrophy (EDMD) is a rare genetic disorder characterised by the early development of muscle contractures, progressive muscle weakness, and heart abnormalities. The latter may result in serious complications, or in severe cases, sudden death. Currently, there are very few effective treatment options available for EDMD and so there is a high clinical need for new therapies. Various genetic mutations have been identified in the development and causation of EDMD, each encoding proteins that are components of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, which spans the nuclear envelope and serves to connect the nuclear lamina to the cytoskeleton. Within this review, we examine how mutations in the genes encoding these proteins, including lamins A/C, emerin, nesprins 1/2, FHL1, and SUN1/2 lead to muscle cell differentiation and development pathway defects. Further work to identify conserved molecular pathways downstream of these defective proteins may reveal potential targets for therapy design. [ABSTRACT FROM AUTHOR]

Nuclear positioning plays an essential role in defining cell architecture and behaviour in both development and disease, and nuclear location frequently adjusts according to internal and external cues. For instance, during periods of migration in many cell types, the nucleus may be actively repositioned behind the microtubule-organising centre. Nuclear movement, for the most part, is dependent upon coupling of the cytoskeleton to the nuclear periphery. This is accomplished largely through SUN and KASH domain proteins, which together assemble to form LINC (linker of the nucleoskeleton and cytoskeleton) complexes spanning the nuclear envelope. SUN proteins of the inner nuclear membrane provide a connection to nuclear structures while acting as a tether for outer nuclear membrane KASH proteins. The latter contain binding sites for diverse cytoskeletal components. Recent publications highlight new aspects of LINC complex regulation revealing that the interplay between SUN and KASH partners can strongly influence how the nucleus functionally engages with different branches of the cytoskeleton.

The autophagy-lysosomal pathway enables the controlled degradation of cellular contents. Nucleophagy is the selective autophagic recycling of nuclear components upon delivery to the lysosome. Although methods to monitor and quantify autophagy as well as selective types of autophagy have been developed and implemented in cells and in vivo, methods monitoring nucleophagy remain scarce. Here, we describe a procedure to monitor the autophagic engagement of an endogenous nuclear envelope component, i.e., ANC-1, the nematode homologue of the mammalian Nesprins in vivo, utilizing super-resolution microscopy., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mechanotransduction is defined as the ability of cells to sense mechanical stimuli from their surroundings and translate them into biochemical signals. Epidermal keratinocytes respond to mechanical cues by altering their proliferation, migration, and differentiation. In vitro cell culture, however, utilises tissue culture plastic, which is significantly stiffer than the in vivo environment. Current epidermal models fail to consider the effects of culturing keratinocytes on plastic prior to setting up three-dimensional cultures, so the impact of this non-physiological exposure on epidermal assembly is largely overlooked. In this study, primary keratinocytes cultured on plastic were compared with those grown on 4, 8, and 50 kPa stiff biomimetic hydrogels that have similar mechanical properties to skin. Our data show that keratinocytes cultured on biomimetic hydrogels exhibited major changes in cellular architecture, cell density, nuclear biomechanics, and mechanoprotein expression, such as specific Linker of Nucleoskeleton and Cytoskeleton (LINC) complex constituents. Mechanical conditioning of keratinocytes on 50 kPa biomimetic hydrogels improved the thickness and organisation of 3D epidermal models. In summary, the current study demonstrates that the effects of extracellular mechanics on keratinocyte cell biology are significant and therefore should be harnessed in skin research to ensure the successful production of physiologically relevant skin models.

Highlights • Migrating neurons generate traction forces in the proximal leading process. • Actomyosin generates the traction forces during neuronal migration. • The LINC complex transmits contractile forces to the nucleus. Abstract Somal translocation in long bipolar neurons is regulated by actomyosin contractile forces, yet the precise spatiotemporal sites of force generation are unknown. Here we investigate the force dynamics generated during somal translocation using traction force microscopy. Neurons with a short leading process generated a traction force in the growth cone and counteracting forces in the leading and trailing processes. In contrast, neurons with a long leading process generated a force dipole with opposing traction forces in the proximal leading process during nuclear translocation. Transient accumulation of actin filaments was observed at the dipole center of the two opposing forces, which was abolished by inhibition of myosin II activity. A swelling in the leading process emerged and generated a traction force that pulled the nucleus when nuclear translocation was physically hampered. The traction force in the leading process swelling was uncoupled from somal translocation in neurons expressing a dominant negative mutant of the KASH protein, which disrupts the interaction between cytoskeletal components and the nuclear envelope. Our results suggest that the leading process is the site of generation of actomyosin-dependent traction force in long bipolar neurons, and that the traction force is transmitted to the nucleus via KASH proteins. [ABSTRACT FROM AUTHOR]

Abstract Aims To define the neurological and neuropathological alterations caused by SYNE1 mutations. Methods We describe 5 patients (3 males, 2 females; age 3–24 years) from 3 families. The diagnostic work-up included three muscle biopsies and two nerve biopsies in three of the cases. Results Three different phenotypes were discerned. Two patients showed progressive ataxia, mental retardation, neuropathy and radially deviated thumbs (spinocerebellar ataxia, SCAR, type 8 phenotype). Two patients had mild congenital myopathy with restrictive lung disease, clubfeet and thumb anomalies (myopathic arthrogryposis). One patient had congenital myopathy with dilated cardiomyopathy and adducted thumbs (Emery-Dreifuss Muscular Dystrophy, EDMD, type 4). Light microscopy of the three muscle biopsies revealed chronic non-necrotizing myopathy without rimmed vacuoles in all cases combined with neurogenic atrophy in one case. The two nerve biopsies showed predominantly axonal neuropathy with demyelinating features. Nuclear alterations, most notably lobulation and focal widening of the space between inner and outer leaflet of the nuclear envelope, were a prominent consistent feature of myonuclei and Schwann cell nuclei in each of the three muscle specimens and one nerve specimen that could be examined by electron microscopy. Conclusion Thumb abnormalities and nuclear envelope alterations are characteristic for SYNE 1 mutations. Schwann cell nuclei are affected, indicating that such nuclear envelope changes in glial cells contribute to the neurodegenerative phenotype in human nesprinopathies. Highlights • Thumb abnormalities in patients with SYNE1 mutations. • Lobulated myonuclei were a consistent ultrastructural feature. • Nuclear envelope pathology not only in myonuclei but also in Schwann cells nuclei. • Schwann cell nuclei are affected, indicating the neurodegenerative phenotype. [ABSTRACT FROM AUTHOR]

Skeletal muscles are large cells with multiple nuclei that are precisely positioned. The importance of the correct nuclear position is highlighted by the correlation between mispositioned nuclei and muscle disease (Spiro et al., 1966; Gueneau et al., 2009). Myonuclei are generally considered to be equivalent and therefore how far nuclei are from their nearest neighbor is the primary measurement of nuclear positioning. However, skeletal muscles have two specialized cell-cell contacts, the neuromuscular (NMJ) and the myotendinous junction (MTJ). Using these cell-cell contacts as reference points, we have determined that there are at least two distinct populations of myonuclei whose position is uniquely regulated. The post-synaptic myonuclei (PSMs) near the NMJ, and the myonuclei near the myotendinous junction myonuclei (MJMs) have different spacing requirements compared to other myonuclei. The correct positioning of pairs of PSMs depends on the specific action of dynein and kinesin. Positions of the PSMs and MJMs relative to the junctions that define them depend on the KASH-domain protein, Klar. We also found that MJMs are positioned close to the MTJ as a consequence of muscle stretching. Our study defines for the first time that nuclei in skeletal muscles are not all equally positioned, and that subsets of distinct myonuclei have specialized rules that dictate their spacing.

Abstract The positioning and movement of the nucleus has recently emerged as an important aspect of cell migration. Understanding of nuclear positioning and movement has reached an apogee in studies of fibroblast migration. Specific nuclear positioning and movements have been described in the polarization of fibroblast for cell migration and in active migration in 2D and 3D environments. Here, we review recent studies that have uncovered novel molecular mechanisms that contribute to these events in fibroblasts. Many of these involve a connection between the nucleus and the cytoskeleton through the LINC complex composed of outer nuclear membrane nesprins and inner nuclear membrane SUN proteins. We consider evidence that appropriate nuclear positioning contributes to efficient fibroblast polarization and migration and the possible mechanism through which the nucleus affects cell migration. [ABSTRACT FROM AUTHOR]

βcatenin acts as a primary intracellular signal transducer for mechanical and Wnt signaling pathways to control cell function and fate. Regulation of βcatenin in the cytoplasm has been well studied but βcatenin nuclear trafficking and function remains unclear. In a previous study we showed that, in mesenchymal stem cells (MSC), mechanical blockade of adipogenesis relied on inhibition of βcatenin destruction complex element GSK3β (glycogen synthase kinase 3β) to increase nuclear βcatenin as well as the function of Linker of Cytoskeleton and Nucleoskeleton (LINC) complexes, suggesting that these two mechanisms may be linked. Here we show that shortly after inactivation of GSK3β due to either low intensity vibration (LIV), substrate strain or pharmacologic inhibition, βcatenin associates with the nucleoskeleton, defined as the insoluble nuclear fraction that provides structure to the integrated nuclear envelope, nuclear lamina and chromatin. Co-depleting LINC elements Sun-1 and Sun-2 interfered with both nucleoskeletal association and nuclear entry of βcatenin, resulting in decreased nuclear βcatenin levels. Our findings reveal that the insoluble structural nucleoskeleton actively participates in βcatenin dynamics. As the cytoskeleton transmits applied mechanical force to the nuclear surface to influence the nucleoskeleton and its LINC mediated interaction, our results suggest a pathway by which LINC mediated connectivity may play a role in signaling pathways that depend on nuclear access of βcatenin. [ABSTRACT FROM AUTHOR]

The cytoskeleton provides structure and plays an important role in cellular function such as migration, resisting compression forces, and transport. The cytoskeleton also reacts to physical cues such as fluid shear stress or extracellular matrix remodeling by reorganizing filament associations, most commonly focal adhesions and cell-cell cadherin junctions. These mechanical stimuli can result in genome-level changes, and the physical connection of the cytoskeleton to the nucleus provides an optimal conduit for signal transduction by interfacing with nuclear envelope proteins, called nesprins, within the LINC (linker of the nucleus to the cytoskeleton) complex. Using single-molecule on single nuclei assays, we report that the interactions between the nucleus and the cytoskeleton, thought to be nesprin-cytoskeleton interactions, are highly sensitive to force magnitude and direction depending on whether cells are historically interfaced with the matrix or with cell aggregates. Application of ∼10–30 pN forces to these nesprin linkages yielded structural transitions, with a base transition size of 5–6 nm, which are speculated to be associated with partial unfoldings of the spectrin domains of the nesprins and/or structural changes of histones within the nucleus. [ABSTRACT FROM PUBLISHER]

Nesprins (nuclear envelope spectrin repeat proteins) are multi-isomeric scaffolding proteins. Giant nesprin-1 and -2 localise to the outer nuclear membrane, interact with SUN (Sad1p/UNC-84) domain-containing proteins at the inner nuclear membrane to form the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex, which, in association with lamin A/C and emerin, mechanically couples the nucleus to the cytoskeleton. Despite ubiquitous expression of nesprin giant isoforms, pathogenic mutations in nesprin-1 and -2 are associated with tissue-specific disorders, particularly related to striated muscle such as dilated cardiomyopathy and Emery-Dreifuss muscular dystrophy. Recent evidence suggests this muscle-specificity might be attributable in part, to the small muscle specific isoform, nesprin-1α2, which has a novel role in striated muscle function. Our current understanding of muscle-specific functions of nesprin-1 and its isoforms will be summarised in this review to provide insight into potential pathological mechanisms of nesprin-related muscle disease and may inform potential targets of therapeutic modulation., (© 2023 The Author(s).)

Myotubes are syncytial cells generated by fusion of myoblasts. Among the numerous nuclei in myotubes of skeletal muscle fibres, the majority are equidistantly positioned at the periphery, except for clusters of multiple nuclei underneath the motor endplate. The correct positioning of nuclei is thought to be important for muscle function and requires nesprin-1 (also known as SYNE1), a protein of the nuclear envelope. Consistent with this, mice lacking functional nesprin-1 show defective nuclear positioning and present aspects of Emery-Dreifuss muscular dystrophy. In this study, we perform small interfering RNA (siRNA) experiments in C2C12 myoblasts undergoing differentiation, demonstrating that the positioning of nuclei requires PCM-1, a protein of the centrosome that relocalizes to the nuclear envelope at the onset of differentiation in a manner that is dependent on the presence of nesprin-1. PCM-1 itself is required for recruiting proteins of the dynein-dynactin complex and of kinesin motor complexes. This suggests that microtubule motors that are attached to the nuclear envelope support the movement of nuclei along microtubules, to ensure their correct positioning in the myotube. [ABSTRACT FROM AUTHOR]

A bstract Numerous factors including chemical, hormonal, spatial, and physical cues determine stem cell fate. While the regulation of stem cell differentiation by soluble factors is well-characterized, the role of mechanical force in the determination of lineage fate is just beginning to be understood. Investigation of the role of force on cell function has largely focused on 'outside-in' signaling, initiated at the plasma membrane. When interfaced with the extracellular matrix, the cell uses integral membrane proteins, such as those found in focal adhesion complexes to translate force into biochemical signals. Akin to these outside-in connections, the internal cytoskeleton is physically linked to the nucleus, via proteins that span the nuclear membrane. Although structurally and biochemically distinct, these two forms of mechanical coupling influence stem cell lineage fate and, when disrupted, often lead to disease. Here we provide an overview of how mechanical coupling occurs at the plasma and nuclear membranes. We also discuss the role of force on stem cell differentiation, with focus on the biochemical signals generated at the cell membrane and the nucleus, and how those signals influence various diseases. While the interaction of stem cells with their physical environment and how they respond to force is complex, an understanding of the mechanical regulation of these cells is critical in the design of novel therapeutics to combat diseases associated with aging, cancer, and osteoporosis. S tem C ells 2016;34:1455-1463 [ABSTRACT FROM AUTHOR]

Nonsense mutations across the whole coding sequence of Syne1 / Nesprin1 have been linked to autosomal recessive cerebellar ataxia Type I (ARCA1). However, nothing is known about the molecular etiology of this late-onset debilitating pathology. In this work, we report that Nesprin1 giant is specifically expressed in CNS tissues. We also identified a CNS-specific splicing event that leads to the abundant expression of a KASH-LESS variant of Nesprin1 giant (KLNes1g) in the cerebellum. KLNes1g displayed a noncanonical localization at glomeruli of cerebellar mossy fibers whereas Nesprin2 exclusively decorated the nuclear envelope of all cerebellar neurons. In immunogold electron microscopy, KLNes1g colocalized both with synaptic vesicles within mossy fibers and with dendritic membranes of cerebellar granule neurons. We further identified vesicle- and membrane-associated proteins in KLNes1g immunoprecipitates. Together, our results suggest that the loss of function of KLNes1g resulting from Nesprin1 nonsense mutations underlies the molecular etiology of ARCA1. [ABSTRACT FROM AUTHOR]

A cell's ability to recognize and adapt to the physical environment is central to its survival and function, but how mechanical cues are perceived and transduced into intracellular signals remains unclear. In mesenchymal stem cells (MSCs), high-magnitude substrate strain (HMS, ≥2%) effectively suppresses adipogenesis via induction of focal adhesion (FA) kinase (FAK)/mTORC2/Akt signaling generated at FAs. Physiologic systems also rely on a persistent barrage of low-level signals to regulate behavior. Exposing MSC to extremely low-magnitude mechanical signals (LMS) suppresses adipocyte formation despite the virtual absence of substrate strain (<0.001%), suggesting that LMS-induced dynamic accelerations can generate force within the cell. Here, we show that MSC response to LMS is enabled through mechanical coupling between the cytoskeleton and the nucleus, in turn activating FAK and Akt signaling followed by FAK-dependent induction of RhoA. While LMS and HMS synergistically regulated FAK activity at the FAs, LMS-induced actin remodeling was concentrated at the perinuclear domain. Preventing nuclear-actin cytoskeleton mechanocoupling by disrupting linker of nucleoskeleton and cytoskeleton (LINC) complexes inhibited these LMS-induced signals as well as prevented LMS repression of adipogenic differentiation, highlighting that LINC connections are critical for sensing LMS. In contrast, FAK activation by HMS was unaffected by LINC decoupling, consistent with signal initiation at the FA mechanosome. These results indicate that the MSC responds to its dynamic physical environment not only with 'outside-in' signaling initiated by substrate strain, but vibratory signals enacted through the LINC complex enable matrix independent 'inside-inside' signaling. S tem C ells 2013;33:2063-2076 [ABSTRACT FROM AUTHOR]