Nuclear Envelope Proteins Research Articles

A network of nuclear envelope proteins and cytoskeletal force generators mediates movements of and within nuclei throughout Caenorhabditis elegans development.

Nuclear migration and anchorage, together referred to as nuclear positioning, are central to many cellular and developmental events. Nuclear positioning is mediated by a conserved network of nuclear envelope proteins that interacts with force generators in the cytoskeleton. At the heart of this network are linker of nucleoskeleton and cytoskeleton (LINC) complexes made of Sad1 and UNC-84 (SUN) proteins at the inner nuclear membrane and Klarsicht, ANC-1, and Syne homology (KASH) proteins in the outer nuclear membrane. LINC complexes span the nuclear envelope, maintain nuclear envelope architecture, designate the surface of nuclei distinctly from the contiguous endoplasmic reticulum, and were instrumental in the early evolution of eukaryotes. LINC complexes interact with lamins in the nucleus and with various cytoplasmic KASH effectors from the surface of nuclei. These effectors regulate the cytoskeleton, leading to a variety of cellular outputs including pronuclear migration, nuclear migration through constricted spaces, nuclear anchorage, centrosome attachment to nuclei, meiotic chromosome movements, and DNA damage repair. How LINC complexes are regulated and how they function are reviewed here. The focus is on recent studies elucidating the best-understood network of LINC complexes, those used throughout Caenorhabditis elegans development.Impact statementDefects in nuclear positioning disrupt development in many mammalian tissues. In human development, LINC complexes play important cellular functions including nuclear positioning, homolog pairing in meiosis, DNA damage repair, wound healing, and gonadogenesis. The topics reviewed here are relevant to public health because defects in nuclear positioning and mutations in LINC components are associated with a wide variety of human diseases including muscular dystrophies, neurological disorders, progeria, aneurysms, hearing loss, blindness, sterility, and multiple cancers. Although this review focuses on findings in the model nematode Caenorhabditis elegans, the studies are relevant because almost all the findings originally made in C. elegans are conserved to humans. Furthermore, C. elegans remains the best described network for how LINC complexes are regulated and function.

The NAE Pathway: Autobahn to the Nucleus for Cell Surface Receptors.

Various growth factors and full-length cell surface receptors such as EGFR are translocated from the cell surface to the nucleoplasm, baffling cell biologists to the mechanisms and functions of this process. Elevated levels of nuclear EGFR correlate with poor prognosis in various cancers. In recent years, nuclear EGFR has been implicated in regulating gene transcription, cell proliferation and DNA damage repair. Different models have been proposed to explain how the receptors are transported into the nucleus. However, a clear consensus has yet to be reached. Recently, we described the nuclear envelope associated endosomes (NAE) pathway, which delivers EGFR from the cell surface to the nucleus. This pathway involves transport, docking and fusion of NAEs with the outer membrane of the nuclear envelope. EGFR is then presumed to be transported through the nuclear pore complex, extracted from membranes and solubilised. The SUN1/2 nuclear envelope proteins, Importin-beta, nuclear pore complex proteins and the Sec61 translocon have been implicated in the process. While this framework can explain the cell surface to nucleus traffic of EGFR and other cell surface receptors, it raises several questions that we consider in this review, together with implications for health and disease.

Dilated cardiomyopathy: from epidemiologic to genetic phenotypes: A translational review of current literature.

Dilated cardiomyopathy (DCM) is characterized by left ventricular dilatation and, consecutively, contractile dysfunction. The causes of DCM are heterogeneous. DCM often results from myocarditis, exposure to alcohol, drugs or other toxins and metabolic or endocrine disturbances. In about 35% of patients, genetic mutations can be identified that usually involve genes responsible for cytoskeletal, sarcomere and nuclear envelope proteins. Due to its heterogeneity, a detailed diagnostic work-up is necessary to identify the specific underlying cause and exclude other conditions with phenotype overlap. Patients with DCM show typical systolic heart failure symptoms, but, with progress of the disease, diastolic dysfunction is present as well. Depending on the underlying pathology, DCM patients also become apparent through arrhythmias, thromboembolic events or cardiogenic shock. Disease progression and prognosis are mostly driven by disease severity and reverse remodelling within the heart. The worst prognosis is seen in patients with lowest ejection fractions or severe diastolic dysfunction, leading to terminal heart failure with subsequent need for left ventricular assist device implantation or heart transplantation. Guideline-based heart failure medication and device therapy reduces the frequency of heart failure hospitalizations and improves survival.

Stabilizing heterochromatin by DGCR8 alleviates senescence and osteoarthritis

DiGeorge syndrome critical region 8 (DGCR8) is a critical component of the canonical microprocessor complex for microRNA biogenesis. However, the non-canonical functions of DGCR8 have not been studied. Here, we demonstrate that DGCR8 plays an important role in maintaining heterochromatin organization and attenuating aging. An N-terminal-truncated version of DGCR8 (DR8dex2) accelerated senescence in human mesenchymal stem cells (hMSCs) independent of its microRNA-processing activity. Further studies revealed that DGCR8 maintained heterochromatin organization by interacting with the nuclear envelope protein Lamin B1, and heterochromatin-associated proteins, KAP1 and HP1γ. Overexpression of any of these proteins, including DGCR8, reversed premature senescent phenotypes in DR8dex2 hMSCs. Finally, DGCR8 was downregulated in pathologically and naturally aged hMSCs, whereas DGCR8 overexpression alleviated hMSC aging and mouse osteoarthritis. Taken together, these analyses uncovered a novel, microRNA processing-independent role in maintaining heterochromatin organization and attenuating senescence by DGCR8, thus representing a new therapeutic target for alleviating human aging-related disorders.

Pathogenic mutations in genes encoding nuclear envelope proteins and defective nucleocytoplasmic connections.

Mutations in genes encoding nuclear lamins and associated nuclear envelope proteins have been linked to a broad range of inherited diseases affecting different tissues and organs. These diseases are often referred to as laminopathies. Scientists have yet to elucidate exactly how pathogenic mutations leading to alteration of a nuclear envelope protein cause disease. Our relatively recent research has shown that pathogenic mutations in genes encoding nuclear envelope proteins lead to defective nucleocytoplasmic connections that disrupt proper functioning of the linker of nucleoskeleton and cytoskeleton complex in the establishment of cell polarity. These defects may explain, at least in part, pathogenic mechanisms underlying laminopathies.Impact statementMutations in genes encoding nuclear lamins and associated nuclear envelope proteins have been linked to several diseases affecting different tissues and organs. The pathogenic mechanisms underlying these diseases, often called laminopathies, remain poorly understood. Increased knowledge of the functions of different nuclear envelope proteins and the interactions between them is crucial to elucidate these disease mechanisms. Our research has shown that pathogenic mutations in genes encoding nuclear envelope proteins lead to defective nucleocytoplasmic connections that disrupt proper functioning of the linker of nucleoskeleton and cytoskeleton (LINC) complex in the establishment of cell polarity. These defects may contribute to the pathogenesis of laminopathies and provide novel targets for therapeutics.

Mechanosensing at the Nuclear Envelope by Nuclear Pore Complex Stretch Activation and Its Effect in Physiology and Pathology.

Cell fate is correlated to mechanotransduction, in which forces transmitted by the cytoskeleton filaments alter the nuclear shape, affecting transcription factor import/export, cells transcription activity and chromatin distribution. There is in fact evidence that stem cells cultured in 3D environments mimicking the native niche are able to maintain their stemness or modulate their cellular function. However, the molecular and biophysical mechanisms underlying cellular mechanosensing are still largely unclear. The propagation of mechanical stimuli via a direct pathway from cell membrane integrins to SUN proteins residing in the nuclear envelop has been demonstrated, but we suggest that the cells’ fate is mainly affected by the force distribution at the nuclear envelope level, where the SUN protein transmits the stimuli via its mechanical connection to several cell structures such as chromatin, lamina and the nuclear pore complex (NPC). In this review, we analyze the NPC structure and organization, which have not as yet been fully investigated, and its plausible involvement in cell fate. NPC is a multiprotein complex that spans the nuclear envelope, and is involved in several key cellular processes such as bidirectional nucleocytoplasmic exchange, cell cycle regulation, kinetochore organization, and regulation of gene expression. As several connections between the NPC and the nuclear envelope, chromatin and other transmembrane proteins have been identified, it is reasonable to suppose that nuclear deformations can alter the NPC structure. We provide evidence that the transmission of mechanical forces may significantly affects the basket conformation via the Nup153-SUN1 connection, both altering the passage of molecules through it and influencing the state of chromatin packing. Finally, we review the known correlations between a pathological NPC structure and diseases such as cancer, autoimmune disease, aging and laminopathies.

Nuclear Organization in Stress and Aging.

The eukaryotic nucleus controls most cellular processes. It is isolated from the cytoplasm by the nuclear envelope, which plays a prominent role in the structural organization of the cell, including nucleocytoplasmic communication, chromatin positioning, and gene expression. Alterations in nuclear composition and function are eminently pronounced upon stress and during premature and physiological aging. These alterations are often accompanied by epigenetic changes in histone modifications. We review, here, the role of nuclear envelope proteins and histone modifiers in the 3-dimensional organization of the genome and the implications for gene expression. In particular, we focus on the nuclear lamins and the chromatin-associated protein BAF, which are linked to Hutchinson–Gilford and Nestor–Guillermo progeria syndromes, respectively. We also discuss alterations in nuclear organization and the epigenetic landscapes during normal aging and various stress conditions, ranging from yeast to humans.

Impairments in contractility and cytoskeletal organisation cause nuclear defects in nemaline myopathy

Nemaline myopathy (NM) is a skeletal muscle disorder caused by mutations in genes that are generally involved in muscle contraction, in particular those related to the structure and/or regulation of the thin filament. Many pathogenic aspects of this disease remain largely unclear. Here, we report novel pathological defects in skeletal muscle fibres of mouse models and patients with NM: irregular spacing and morphology of nuclei; disrupted nuclear envelope; altered chromatin arrangement; and disorganisation of the cortical cytoskeleton. Impairments in contractility are the primary cause of these nuclear defects. We also establish the role of microtubule organisation in determining nuclear morphology, a phenomenon which is likely to contribute to nuclear alterations in this disease. Our results overlap with findings in diseases caused directly by mutations in nuclear envelope or cytoskeletal proteins. Given the important role of nuclear shape and envelope in regulating gene expression, and the cytoskeleton in maintaining muscle fibre integrity, our findings are likely to explain some of the hallmarks of NM, including contractile filament disarray, altered mechanical properties and broad transcriptional alterations.

An Emerin LEM-Domain Mutation Impairs Cell Response to Mechanical Stress.

Emerin is a nuclear envelope protein that contributes to genome organization and cell mechanics. Through its N-terminal LAP2-emerin-MAN1 (LEM)-domain, emerin interacts with the DNA-binding protein barrier-to-autointegration (BAF). Emerin also binds to members of the linker of the nucleoskeleton and cytoskeleton (LINC) complex. Mutations in the gene encoding emerin are responsible for the majority of cases of X-linked Emery-Dreifuss muscular dystrophy (X-EDMD). Most of these mutations lead to an absence of emerin. A few missense and short deletion mutations in the disordered region of emerin are also associated with X-EDMD. More recently, missense and short deletion mutations P22L, ∆K37 and T43I were discovered in emerin LEM-domain, associated with isolated atrial cardiac defects (ACD). Here we reveal which defects, at both the molecular and cellular levels, are elicited by these LEM-domain mutations. Whereas ΔK37 mutation impaired the correct folding of the LEM-domain, P22L and T43I had no impact on the 3D structure of emerin. Surprisingly, all three mutants bound to BAF, albeit with a weaker affinity in the case of ΔK37. In human myofibroblasts derived from a patient’s fibroblasts, emerin ∆K37 was correctly localized at the inner nuclear membrane, but was present at a significantly lower level, indicating that this mutant is abnormally degraded. Moreover, SUN2 was reduced, and these cells were defective in producing actin stress fibers when grown on a stiff substrate and after cyclic stretches. Altogether, our data suggest that the main effect of mutation ΔK37 is to perturb emerin function within the LINC complex in response to mechanical stress.

Accumulation of Progerin Affects the Symmetry of Cell Division and Is Associated with Impaired Wnt Signaling and the Mislocalization of Nuclear Envelope Proteins

Hutchinson-Gilford progeria syndrome (HGPS) is the result of a defective form of the lamin A protein called progerin. While progerin is known to disrupt the properties of the nuclear lamina, the underlying mechanisms responsible for the pathophysiology of HGPS remain less clear. Previous studies in our laboratory have shown that progerin expression in murine epidermal basal cells results in impaired stratification and halted development of the skin. Stratification and differentiation of the epidermis is regulated by asymmetric stem cell division. Here, we show that expression of progerin impairs the ability of stem cells to maintain tissue homeostasis as a result of altered cell division. Quantification of basal skin cells showed an increase in symmetric cell division that correlated with progerin accumulation in HGPS mice. Investigation of the mechanisms underlying this phenomenon revealed a putative role of Wnt/β-catenin signaling. Further analysis suggested an alteration in the nuclear translocation of β-catenin involving the inner and outer nuclear membrane proteins, emerin and nesprin-2. Taken together, our results suggest a direct involvement of progerin in the transmission of Wnt signaling and normal stem cell division. These insights into the molecular mechanisms of progerin may help develop new treatment strategies for HGPS.

Disruption of valosin-containing protein activity causes cardiomyopathy and reveals pleiotropic functions in cardiac homeostasis

Valosin-containing protein (VCP), also known as p97, is an ATPase with diverse cellular functions, although the most highly characterized is targeting of misfolded or aggregated proteins to degradation pathways, including the endoplasmic reticulum-associated degradation (ERAD) pathway. However, how VCP functions in the heart has not been carefully examined despite the fact that human mutations in VCP cause Paget disease of bone and frontotemporal dementia, an autosomal dominant multisystem proteinopathy that includes disease in the heart, skeletal muscle, brain, and bone. Here we generated heart-specific transgenic mice overexpressing WT VCP or a VCPK524A mutant with deficient ATPase activity. Transgenic mice overexpressing WT VCP exhibit normal cardiac structure and function, whereas mutant VCP-overexpressing mice develop cardiomyopathy. Mechanistically, mutant VCP-overexpressing hearts up-regulate ERAD complex components and have elevated levels of ubiquitinated proteins prior to manifestation of cardiomyopathy, suggesting dysregulation of ERAD and inefficient clearance of proteins targeted for proteasomal degradation. The hearts of mutant VCP transgenic mice also exhibit profound defects in cardiomyocyte nuclear morphology with increased nuclear envelope proteins and nuclear lamins. Proteomics revealed overwhelming interactions of endogenous VCP with ribosomal, ribosome-associated, and RNA-binding proteins in the heart, and impairment of cardiac VCP activity resulted in aggregation of large ribosomal subunit proteins. These data identify multifactorial functions and diverse mechanisms whereby VCP regulates cardiomyocyte protein and RNA quality control that are critical for cardiac homeostasis, suggesting how human VCP mutations negatively affect the heart.

SPAG4L/SPAG4L interacts with Nesprin2 to participate in the meiosis of spermatogenesis

SUN domain proteins are identified as a novel family of nuclear envelope proteins which are involved in spermatogenesis. SPAG4L is identified as the fifth member of this family. Previous studies have revealed that SPAG4L is involved in spermatogenesis and the mutations occurring in SPAG4L will lead to male infertility. However, the transcriptions of SPAG4L and its interacting proteins in the testis are still unclear. In this study, we identified a shorter transcript variant of SPAG4L, named SPAG4Lβ, in human testis by northern blot and reverse transcription-polymerase chain reaction. Bioinformatics analysis showed that it encodes a protein consisting of 311 amino acids, and subcellular localization analysis revealed that it is mainly expressed in the cytoplasm. In situ hybridization and immunofluorescence assay revealed that SPAG4L/SPAG4Lβ is involved in meiosis. Furthermore, co-IP results demonstrated that SPAG4L/SPAG4Lβ interacts with Nesprin2, a KASH domain protein to form the LINC (linker of nucleoskeleton and cytoskeleton) complexes. Immunofluorescence results revealed that the LINC complexes of Spag4l/Nesprin2 in mouse are involved in spermatocyte division. Our data indicated that SPAG4L/SPAG4Lβ may play an important role in the meiotic process.

Characterization of a Unique Form of Arrhythmic Cardiomyopathy Caused by Recessive Mutation in LEMD2

Nuclear envelope proteins have been shown to play an important role in the pathogenesis of inherited dilated cardiomyopathy. Here, we present a remarkable cardiac phenotype caused by a homozygous LEMD2 mutation in patients of the Hutterite population with juvenile cataract. Mutation carriers develop arrhythmic cardiomyopathy with mild impairment of left ventricular systolic function but severe ventricular arrhythmias leading to sudden cardiac death. Affected cardiac tissue from a deceased patient and fibroblasts exhibit elongated nuclei with abnormal condensed heterochromatin at the periphery. The patient fibroblasts demonstrate cellular senescence and reduced proliferation capacity, which may suggest an involvement of LEM domain containing protein 2 in chromatin remodeling processes and premature aging.

The Driving Force: Nuclear Mechanotransduction in Cellular Function, Fate, and Disease.

Cellular behavior is continuously affected by microenvironmental forces through the process of mechanotransduction, in which mechanical stimuli are rapidly converted to biochemical responses. Mounting evidence suggests that the nucleus itself is a mechanoresponsive element, reacting to cytoskeletal forces and mediating downstream biochemical responses. The nucleus responds through a host of mechanisms, including partial unfolding, conformational changes, and phosphorylation of nuclear envelope proteins; modulation of nuclear import/export; and altered chromatin organization, resulting in transcriptional changes. It is unclear which of these events present direct mechanotransduction processes and which are downstream of other mechanotransduction pathways. We critically review and discuss the current evidence for nuclear mechanotransduction, particularly in the context of stem cell fate, a largely unexplored topic, and in disease, where an improved understanding of nuclear mechanotransduction is beginning to open new treatment avenues. Finally, we discuss innovative technological developments that will allow outstanding questions in the rapidly growing field of nuclear mechanotransduction to be answered.

The Discovery of a LEMD2-Associated Nuclear Envelopathy with Early Progeroid Appearance Suggests Advanced Applications for AI-Driven Facial Phenotyping

Over a relatively short period of time, the clinical geneticist's "toolbox" has been expanded by machine-learning algorithms for image analysis, which can be applied to the task of syndrome identification on the basis of facial photographs, but these technologies harbor potential beyond the recognition of established phenotypes. Here, we comprehensively characterized two individuals with a hitherto unknown genetic disorder caused by the same de novo mutation in LEMD2 (c.1436C>T;p.Ser479Phe), the gene which encodes the nuclear envelope protein LEM domain-containing protein 2 (LEMD2). Despite different ages and ethnic backgrounds, both individuals share a progeria-like facial phenotype and a distinct combination of physical and neurologic anomalies, such as growth retardation; hypoplastic jaws crowded with multiple supernumerary, yet unerupted, teeth; and cerebellar intention tremor. Immunofluorescence analyses of patient fibroblasts revealed mutation-induced disturbance of nuclear architecture, recapitulating previously published data in LEMD2-deficient cell lines, and additional experiments suggested mislocalization of mutant LEMD2 protein within the nuclear lamina. Computational analysis of facial features with two different deep neural networks showed phenotypic proximity to other nuclear envelopathies. One of the algorithms, when trained to recognize syndromic similarity (rather than specific syndromes) in an unsupervised approach, clustered both individuals closely together, providing hypothesis-free hints for a common genetic etiology. We show that a recurrent de novo mutation in LEMD2 causes a nuclear envelopathy whose prognosis in adolescence is relatively good in comparison to that of classical Hutchinson-Gilford progeria syndrome, and we suggest that the application of artificial intelligence to the analysis of patient images can facilitate the discovery of new genetic disorders.

Research progress on lamin and Hutchinson-Gilford progeria syndrome

Many studies have shown that abnormal expression and modification of lamin are closely related to aging.Hutchinson-Gilford progeria syndrome(HGPS)is a rare and severe premature aging disease caused by mutations in the gene encoding nuclear envelope proteins of A-type lamins(LMNA). The pathogenesis of HGPS is similar to the aging process of normal individuals, thus research on HGPS will be helpful for understanding the mechanisms of senescence and developing anti-aging drugs.This paper reviews recent advances in lamin and the pathogenesis and treatment of HGPS, in order to provide a reference for further basic and clinical research on HGPS. Key words: Aging; Lamin; Hutchinson-Gilford progeria syndrome

Combined loss of LAP1B and LAP1C results in an early onset multisystemic nuclear envelopathy

Nuclear envelopathies comprise a heterogeneous group of diseases caused by mutations in genes encoding nuclear envelope proteins. Mutations affecting lamina-associated polypeptide 1 (LAP1) result in two discrete phenotypes of muscular dystrophy and progressive dystonia with cerebellar atrophy. We report 7 patients presenting at birth with severe progressive neurological impairment, bilateral cataract, growth retardation and early lethality. All the patients are homozygous for a nonsense mutation in the TOR1AIP1 gene resulting in the loss of both protein isoforms LAP1B and LAP1C. Patient-derived fibroblasts exhibit changes in nuclear envelope morphology and large nuclear-spanning channels containing trapped cytoplasmic organelles. Decreased and inefficient cellular motility is also observed in these fibroblasts. Our study describes the complete absence of both major human LAP1 isoforms, underscoring their crucial role in early development and organogenesis. LAP1-associated defects may thus comprise a broad clinical spectrum depending on the availability of both isoforms in the nuclear envelope throughout life.

Challenges and Opportunities for Characterizing the Assembly of Nuclear Envelope Proteins by Fluorescence Fluctuation Spectroscopy

The nuclear envelope (NE), consisting of two membranes separated by a thin perinuclear space, has proven to be a challenging environment for measuring protein dynamics in living cells. First, exogenously expressed, fluorescently tagged proteins must be properly localized to the NE. Furthermore, undulations of the nuclear membranes complicate the application of fluorescence fluctuation spectroscopy (FFS) to proteins located within the perinuclear space by adding a slowly fluctuating signal. While this signal is absent for proteins associated with the nuclear membranes, distinguishing soluble from membrane-associated protein complexes by FFS presents a significant challenge since the diffusion time depends strongly on molecular size within the perinuclear space. Some proteins, such as the AAA+ ATPase TorsinA, may exist in both a membrane-associated and soluble form depending on the conditions in the cell. Determining the extent to which a protein associates with the membrane can provide valuable insight into its function within the cell. While diffusion time alone is insufficient to differentiate these populations, we explore whether the dependence of diffusion time on temperature can provide insight into a protein's localization within the NE. In addition, we explore how spatial correlations can be used to study the dynamics of proteins within the NE and the membrane undulations we previously reported. Finally, z-scan FFS provides a means to quantify the localization of proteins to the NE. These examples highlight the ways in which FFS techniques can be used to overcome the challenges of quantifying protein behavior in the NE of living cells. This work has been supported by a grant from the National Institutes of Health (R01 GM64589).

Illuminating SUN2/KASH Hetero-Complex Formation Within the Nuclear Envelope of Living Cells with Dual-Color Time-Shifted MSQ

The nuclear envelope (NE) is a cellular compartment consisting of an inner and outer nuclear membrane separated by the perinuclear space. Research on cellular mechanotransduction identified the nucleus as a mechanosensor, which responds to the mechanical properties of the extracellular environment. Force transmission across the NE into the nucleoplasm is facilitated by linker of nucleoskeleton-and-cytoskeleton (LINC) complexes, which are conserved molecular bridges formed by the transluminal physical interaction of the outer and inner nuclear membrane KASH and the SUN proteins, respectively. Mammals encode six KASH proteins and five SUN proteins. In vitro, KASH proteins bind to the domain interfaces of trimeric SUN2 proteins resulting in the formation of a SUN2/KASH hetero-hexamer. Recently, we extended the application of single-color fluorescence fluctuation spectroscopy (FFS) to quantify protein oligomerization within the NE of living cells by developing mean segmented Q-factor (MSQ) and time-shifted MSQ (tsMSQ) to address experimental challenges unique to the environment of the NE. Using FFS, we demonstrated that SUN2 trimerizes in vivo, whereas the luminal SUN-binding KASH peptide of the KASH protein nesprin-2 (KASH2) remains monomeric. To be able to identify the oligomerization of SUN2/KASH within the NE of living cells, we describe here the development of dual-color (DC) tsMSQ. We first validated DC tsMSQ using nuclear envelope proteins tagged with EGFP and mCherry as model systems. Finally, we applied DC tsMSQ to detect the formation of hetero-complexes of EGFP-tagged SUN2 and mCherry-tagged KASH2. The development of DC tsMSQ will enable future efforts aimed at the mechanistic dissection of LINC complex assembly and its regulation within the NE as well as investigations of binary protein systems within this important sub-cellular compartment. This work has been supported by a grant from the NIH (R01 GM64589).

Nesprin-2G knockout fibroblasts exhibit reduced migration, changes in focal adhesion composition, and reduced ability to generate traction forces.

The nuclear envelope protein nesprin-2G is a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex and is responsible for mechanical and signaling crosstalk between the nucleus and cytoskeleton. A prior study has demonstrated that nesprin-2G knockout mice show delayed wound healing. The goal was to elucidate the mechanism underlying the delayed wound closure in this mouse model. Primary fibroblasts from wild-type and knockout neonatal mice were isolated. Knockout cells exhibited decreased focal adhesion (FA) size, number, and intensity. Consistent with this result, FA protein expression levels were decreased in knockout cells. Additionally, knockout fibroblasts displayed an abnormal actin cytoskeleton, as evidenced by loss of TAN line formation and both cytoplasmic and peri-nuclear actin staining. Using collective and single cell motility assays, it was found that knockout cells exhibited a reduction in both speed and directed migration. Traction force microscopy revealed that knockout fibroblasts generated fewer traction forces compared with WT fibroblasts. In summary, the data indicated that changes in actin organization and defects in FAs result in a reduced ability of knockout fibroblasts to generate traction forces needed for efficient motility.