Nesprin-1 LINC complexes recruit microtubule cytoskeleton proteins and drive pathology in Lmna-mutant striated muscle.

Mutations in LMNA, the gene encoding A-type lamins, cause laminopathies-diseases of striated muscle and other tissues. The aetiology of laminopathies has been attributed to perturbation of chromatin organization or structural weakening of the nuclear envelope (NE) such that the nucleus becomes more prone to mechanical damage. The latter model requires a conduit for force transmission to the nucleus. NE-associated Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes are one such pathway. Using clustered regularly interspaced short palindromic repeats to disrupt the Nesprin-1 KASH (Klarsicht, ANC-1, Syne Homology) domain, we identified this LINC complex protein as the predominant NE anchor for microtubule cytoskeleton components, including nucleation activities and motor complexes, in mouse cardiomyocytes. Loss of Nesprin-1 LINC complexes resulted in loss of microtubule cytoskeleton proteins at the nucleus and changes in nuclear morphology and positioning in striated muscle cells, but with no overt physiological defects. Disrupting the KASH domain of Nesprin-1 suppresses Lmna-linked cardiac pathology, likely by reducing microtubule cytoskeleton activities at the nucleus. Nesprin-1 LINC complexes thus represent a potential therapeutic target for striated muscle laminopathies.

GDF5 as a rejuvenating treatment for age-related neuromuscular failure.

Sarcopenia involves a progressive loss of skeletal muscle force, quality and mass during ageing, which results in increased inability and death; however, no cure has been established thus far. Growth differentiation factor 5 (GDF5) has been described to modulate muscle mass maintenance in various contexts. For our proof of concept, we overexpressed GDF5 by AAV vector injection in tibialis anterior muscle of adult aged (20 months) mice and performed molecular and functional analysis of skeletal muscle. We analysed human vastus lateralis muscle biopsies from adult young (21-42 years) and aged (77-80 years) donors, quantifying the molecular markers modified by GDF5 overexpression in mouse muscle. We validated the major effects of GDF5 overexpression using human immortalized myotubes and Schwann cells. We established a preclinical study by treating chronically (for 4 months) aged mice using recombinant GDF5 protein (rGDF5) in systemic administration and evaluated the long-term effect of this treatment on muscle mass and function. Here, we demonstrated that GDF5 overexpression in the old tibialis anterior muscle promoted an increase of 16.5% of muscle weight (P = 0.0471) associated with a higher percentage of 5000-6000 µm2 large fibres (P = 0.0211), without the induction of muscle regeneration. Muscle mass gain was associated with an amelioration of 26.8% of rate of force generation (P = 0.0330) and better neuromuscular connectivity (P = 0.0098). Moreover, GDF5 overexpression preserved neuromuscular junction morphology (38.5% of nerve terminal area increase, P < 0.0001) and stimulated the expression of reinnervation-related genes, in particular markers of Schwann cells (fold-change 3.19 for S100b gene expression, P = 0.0101). To characterize the molecular events induced by GDF5 overexpression during ageing, we performed a genome-wide transcriptomic analysis of treated muscles and showed that this factor leads to a 'rejuvenating' transcriptomic signature in aged mice, as 42% of the transcripts dysregulated by ageing reverted to youthful expression levels upon GDF5 overexpression (P < 0.05). Towards a preclinical approach, we performed a long-term systemic treatment using rGDF5 and showed its effectiveness in counteracting age-related muscle wasting, improving muscle function (17.8% of absolute maximal force increase, P = 0.0079), ensuring neuromuscular connectivity and preventing neuromuscular junction degeneration (7.96% of AchR area increase, P = 0.0125). In addition, in human muscle biopsies, we found the same age-related alterations than those observed in mice and improved by GDF5 and reproduced its major effects on human cells, suggesting this treatment as efficient in humans. Overall, these data provide a foundation to examine the curative potential of GDF5 drug in clinical trials for sarcopenia and, eventually, other neuromuscular diseases.

Science during lockdown - from virtual seminars to sustainable online communities.

The COVID-19 pandemic has disrupted traditional modes of scientific communication. In-person conferences and seminars have been cancelled and most scientists around the world have been confined to their homes. Although challenging, this situation has presented an opportunity to adopt new ways to communicate science and build scientific relationships within a digital environment, thereby reducing the environmental impact and increasing the inclusivity of scientific events. As a group of researchers who have recently created online seminar series for our respective research communities, we have come together to share our experiences and insights. Only a few weeks into this process, and often learning 'on the job', we have collectively encountered different problems and solutions. Here, we share our advice on formats and tools, security concerns, spreading the word to your community and creating a diverse, inclusive and collegial space online. We hope our experience will help others launch their own online initiatives, helping to shape the future of scientific communication as we move past the current crisis.

Nesprin-2 accumulates at the front of the nucleus during confined cell migration.

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.

A system for studying mechanisms of neuromuscular junction development and maintenance.

The neuromuscular junction (NMJ), a cellular synapse between a motor neuron and a skeletal muscle fiber, enables the translation of chemical cues into physical activity. The development of this special structure has been subject to numerous investigations, but its complexity renders in vivo studies particularly difficult to perform. In vitro modeling of the neuromuscular junction represents a powerful tool to delineate fully the fine tuning of events that lead to subcellular specialization at the pre-synaptic and post-synaptic sites. Here, we describe a novel heterologous co-culture in vitro method using rat spinal cord explants with dorsal root ganglia and murine primary myoblasts to study neuromuscular junctions. This system allows the formation and long-term survival of highly differentiated myofibers, motor neurons, supporting glial cells and functional neuromuscular junctions with post-synaptic specialization. Therefore, fundamental aspects of NMJ formation and maintenance can be studied using the described system, which can be adapted to model multiple NMJ-associated disorders.

MAP and kinesin-dependent nuclear positioning is required for skeletal muscle function.

The basic unit of skeletal muscle in all metazoans is the multinucleate myofibre, within which individual nuclei are regularly positioned. The molecular machinery responsible for myonuclear positioning is not known. Improperly positioned nuclei are a hallmark of numerous diseases of muscle, including centronuclear myopathies, but it is unclear whether correct nuclear positioning is necessary for muscle function. Here we identify the microtubule-associated protein ensconsin (Ens)/microtubule-associated protein 7 (MAP7) and kinesin heavy chain (Khc)/Kif5b as essential, evolutionarily conserved regulators of myonuclear positioning in Drosophila and cultured mammalian myotubes. We find that these proteins interact physically and that expression of the Kif5b motor domain fused to the MAP7 microtubule-binding domain rescues nuclear positioning defects in MAP7-depleted cells. This suggests that MAP7 links Kif5b to the microtubule cytoskeleton to promote nuclear positioning. Finally, we show that myonuclear positioning is physiologically important. Drosophila ens mutant larvae have decreased locomotion and incorrect myonuclear positioning, and these phenotypes are rescued by muscle-specific expression of Ens. We conclude that improper nuclear positioning contributes to muscle dysfunction in a cell-autonomous fashion.

Nuclear movement during myotube formation is microtubule and dynein dependent and is regulated by Cdc42, Par6 and Par3.

Cells actively position their nucleus within the cytoplasm. One striking example is observed during skeletal myogenesis. Differentiated myoblasts fuse to form a multinucleated myotube with nuclei positioned in the centre of the syncytium by an unknown mechanism. Here, we describe that the nucleus of a myoblast moves rapidly after fusion towards the central myotube nuclei. This movement is driven by microtubules and dynein/dynactin complex, and requires Cdc42, Par6 and Par3. We found that Par6ß and dynactin accumulate at the nuclear envelope of differentiated myoblasts and myotubes, and this accumulation is dependent on Par6 and Par3 proteins but not on microtubules. These results suggest a mechanism where nuclear movement after fusion is driven by microtubules that emanate from one nucleus that are pulled by dynein/dynactin complex anchored to the nuclear envelope of another nucleus.

Bioengineering a miniaturized in vitro 3D myotube contraction monitoring chip to model muscular dystrophies.

Quantification of skeletal muscle functional contraction is essential to assess the outcomes of therapeutic procedures for neuromuscular disorders. Muscle three-dimensional "Organ-on-chip" models usually require a substantial amount of biological material, which rarely can be obtained from patient biopsies. Here, we developed a miniaturized 3D myotube culture chip with contraction monitoring capacity at the single cell level. Optimized micropatterned substrate design enabled to obtain high culture yields in tightly controlled microenvironments, with myotubes derived from primary human myoblasts displaying spontaneous contractions. Analysis of nuclear morphology confirmed similar myonuclei structure between obtained myotubes and in vivo myofibers, as compared to 2D monolayers. LMNA-related Congenital Muscular Dystrophy (L-CMD) was modeled with successful development of diseased 3D myotubes displaying reduced contraction. The miniaturized myotube technology can thus be used to study contraction characteristics and evaluate how diseases affect muscle organization and force generation. Importantly, it requires significantly fewer starting materials than current systems, which should substantially improve drug screening capability.

Caveolae and Bin1 form ring-shaped platforms for T-tubule initiation.

Excitation-contraction coupling requires a highly specialized membrane structure, the triad, composed of a plasma membrane invagination, the T-tubule, surrounded by two sarcoplasmic reticulum terminal cisternae. Although the precise mechanisms governing T-tubule biogenesis and triad formation remain largely unknown, studies have shown that caveolae participate in T-tubule formation and mutations of several of their constituents induce muscle weakness and myopathies. Here, we demonstrate that, at the plasma membrane, Bin1 and caveolae composed of caveolin-3 assemble into ring-like structures from which emerge tubes enriched in the dihydropyridine receptor. Bin1 expression lead to the formation of both rings and tubes and we show that Bin1 forms scaffolds on which caveolae accumulate to form the initial T-tubule. Cav3 deficiency caused by either gene silencing or pathogenic mutations results in defective ring formation and perturbed Bin1-mediated tubulation that may explain defective T-tubule organization in mature muscles. Our results uncover new pathophysiological mechanisms that may prove relevant to myopathies caused by Cav3 or Bin1 dysfunction.

Development of versatile allele-specific siRNAs able to silence all the dominant dynamin 2 mutations.

Dominant centronuclear myopathy (CNM) is a rare form of congenital myopathy associated with a wide clinical spectrum, from severe neonatal to milder adult forms. There is no available treatment for this disease due to heterozygous mutations in the DNM2 gene encoding Dynamin 2 (DNM2). Dominant DNM2 mutations also cause rare forms of Charcot-Marie-Tooth disease and hereditary spastic paraplegia, and deleterious DNM2 overexpression was noticed in several diseases. The proof of concept for therapy by allele-specific RNA interference devoted to silence the mutated mRNA without affecting the normal allele was previously achieved in a mouse model and patient-derived cells, both expressing the most frequent DNM2 mutation in CNM. In order to have versatile small interfering RNAs (siRNAs) usable regardless of the mutation, we have developed allele-specific siRNAs against two non-pathogenic single-nucleotide polymorphisms (SNPs) frequently heterozygous in the population. In addition, allele-specific siRNAs against the p.S619L DNM2 mutation, a mutation frequently associated with severe neonatal cases, were developed. The beneficial effects of these new siRNAs are reported for a panel of defects occurring in patient-derived cell lines. The development of these new molecules allows targeting the large majority of the patients harboring DNM2 mutations or overexpression by only a few siRNAs.

Reconstituting the Interaction Between Purified Nuclei and Microtubule Network.

The nucleus is the stiffest organelle in the cell. Several morphogenetic processes depend on its deformation such as cell migration, cell differentiation, or senescence. Recent studies have revealed various mechanisms involved in the regulation of nucleus stiffness and deformation. The implication of chromatin swelling, lamin density, actin filament, and microtubule network revealed that nucleus shape is the outcome of a fine balance between various sources of external forces and numerous means of internal resistance. In adherent cells, the actin network is the dominant player in external force production, whereas in nonadherent cells microtubules seem to take over. It is therefore important to set up reconstitution assays in order to decipher the exact contribution of each player in this mechanical balance. In this method, we describe a nucleus purification protocol that is suitable for nonadherent cells. We also show that purified nuclei can interact with microtubules and that nuclei purified from distinct cell types get differentially wrapped into the array of microtubules. A combination with a microtubule gliding assay offers the possibility to counterbalance the binding to the nucleus membrane by active motor-based forces pulling on microtubules. So this protocol allows an in-depth study of microtubule-nucleus interactions in vitro.

Actin-microtubule cytoskeletal interplay mediated by MRTF-A/SRF signaling promotes dilated cardiomyopathy caused by LMNA mutations.

Mutations in the lamin A/C gene (LMNA) cause dilated cardiomyopathy associated with increased activity of ERK1/2 in the heart. We recently showed that ERK1/2 phosphorylates cofilin-1 on threonine 25 (phospho(T25)-cofilin-1) that in turn disassembles the actin cytoskeleton. Here, we show that in muscle cells carrying a cardiomyopathy-causing LMNA mutation, phospho(T25)-cofilin-1 binds to myocardin-related transcription factor A (MRTF-A) in the cytoplasm, thus preventing the stimulation of serum response factor (SRF) in the nucleus. Inhibiting the MRTF-A/SRF axis leads to decreased α-tubulin acetylation by reducing the expression of ATAT1 gene encoding α-tubulin acetyltransferase 1. Hence, tubulin acetylation is decreased in cardiomyocytes derived from male patients with LMNA mutations and in heart and isolated cardiomyocytes from Lmnap.H222P/H222P male mice. In Atat1 knockout mice, deficient for acetylated α-tubulin, we observe left ventricular dilation and mislocalization of Connexin 43 (Cx43) in heart. Increasing α-tubulin acetylation levels in Lmnap.H222P/H222P mice with tubastatin A treatment restores the proper localization of Cx43 and improves cardiac function. In summary, we show for the first time an actin-microtubule cytoskeletal interplay mediated by cofilin-1 and MRTF-A/SRF, promoting the dilated cardiomyopathy caused by LMNA mutations. Our findings suggest that modulating α-tubulin acetylation levels is a feasible strategy for improving cardiac function.

Severe ACTA1-related nemaline myopathy: intranuclear rods, cytoplasmic bodies, and enlarged perinuclear space as characteristic pathological features on muscle biopsies.

Nemaline myopathy (NM) is a muscle disorder with broad clinical and genetic heterogeneity. The clinical presentation of affected individuals ranges from severe perinatal muscle weakness to milder childhood-onset forms, and the disease course and prognosis depends on the gene and mutation type. To date, 14 causative genes have been identified, and ACTA1 accounts for more than half of the severe NM cases. ACTA1 encodes α-actin, one of the principal components of the contractile units in skeletal muscle. We established a homogenous cohort of ten unreported families with severe NM, and we provide clinical, genetic, histological, and ultrastructural data. The patients manifested antenatal or neonatal muscle weakness requiring permanent respiratory assistance, and most deceased within the first months of life. DNA sequencing identified known or novel ACTA1 mutations in all. Morphological analyses of the muscle biopsy specimens showed characteristic features of NM histopathology including cytoplasmic and intranuclear rods, cytoplasmic bodies, and major myofibrillar disorganization. We also detected structural anomalies of the perinuclear space, emphasizing a physiological contribution of skeletal muscle α-actin to nuclear shape. In-depth investigations of the nuclei confirmed an abnormal localization of lamin A/C, Nesprin-1, and Nesprin-2, forming the main constituents of the nuclear lamina and the LINC complex and ensuring nuclear envelope integrity. To validate the relevance of our findings, we examined muscle samples from three previously reported ACTA1 cases, and we identified the same set of structural aberrations. Moreover, we measured an increased expression of cardiac α-actin in the muscle samples from the patients with longer lifespan, indicating a potential compensatory effect. Overall, this study expands the genetic and morphological spectrum of severe ACTA1-related nemaline myopathy, improves molecular diagnosis, highlights the enlargement of the perinuclear space as an ultrastructural hallmark, and indicates a potential genotype/phenotype correlation.

Actin on and around the Nucleus.

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.

Ctdnep1 and Eps8L2 regulate dorsal actin cables for nuclear positioning during cell migration.

Cells actively position their nuclei within the cytoplasm for multiple cellular and physiological functions.1-3 Consequently, nuclear mispositioning is usually associated with cell dysfunction and disease, from muscular disorders to cancer metastasis.4-7 Different cell types position their nuclei away from the leading edge during cell migration.8-11 In migrating fibroblasts, nuclear positioning is driven by an actin retrograde flow originated at the leading edge that drives dorsal actin cables away from the leading edge. The dorsal actin cables connect to the nuclear envelope by the linker of nucleoskeleton and cytoskeleton (LINC) complex on transmembrane actin-associated nuclear (TAN) lines.12-14 Dorsal actin cables are required for the formation of TAN lines. How dorsal actin cables are organized to promote TAN lines formation is unknown. Here, we report a role for Ctdnep1/Dullard, a nuclear envelope phosphatase,15-22 and the actin regulator Eps8L223-25 on nuclear positioning and cell migration. We demonstrate that Ctdnep1 and Eps8L2 directly interact, and this interaction is important for nuclear positioning and cell migration. We also show that Ctdnep1 and Eps8L2 are involved in the formation and thickness of dorsal actin cables required for TAN lines engagement during nuclear movement. We propose that Ctdnep1-Eps8L2 interaction regulates dorsal actin cables for nuclear movement during cell migration.

TGFß signaling curbs cell fusion and muscle regeneration.

Muscle cell fusion is a multistep process involving cell migration, adhesion, membrane remodeling and actin-nucleation pathways to generate multinucleated myotubes. However, molecular brakes restraining cell-cell fusion events have remained elusive. Here we show that transforming growth factor beta (TGFß) pathway is active in adult muscle cells throughout fusion. We find TGFß signaling reduces cell fusion, regardless of the cells' ability to move and establish cell-cell contacts. In contrast, inhibition of TGFß signaling enhances cell fusion and promotes branching between myotubes in mouse and human. Exogenous addition of TGFß protein in vivo during muscle regeneration results in a loss of muscle function while inhibition of TGFßR2 induces the formation of giant myofibers. Transcriptome analyses and functional assays reveal that TGFß controls the expression of actin-related genes to reduce cell spreading. TGFß signaling is therefore requisite to limit mammalian myoblast fusion, determining myonuclei numbers and myofiber size.

Nesprins are mechanotransducers that discriminate epithelial-mesenchymal transition programs.

LINC complexes are transmembrane protein assemblies that physically connect the nucleoskeleton and cytoskeleton through the nuclear envelope. Dysfunctions of LINC complexes are associated with pathologies such as cancer and muscular disorders. The mechanical roles of LINC complexes are poorly understood. To address this, we used genetically encoded FRET biosensors of molecular tension in a nesprin protein of the LINC complex of fibroblastic and epithelial cells in culture. We exposed cells to mechanical, genetic, and pharmacological perturbations, mimicking a range of physiological and pathological situations. We show that nesprin experiences tension generated by the cytoskeleton and acts as a mechanical sensor of cell packing. Moreover, nesprin discriminates between inductions of partial and complete epithelial-mesenchymal transitions. We identify the implicated mechanisms, which involve α-catenin capture at the nuclear envelope by nesprin upon its relaxation, thereby regulating ß-catenin transcription. Our data thus implicate LINC complex proteins as mechanotransducers that fine-tune ß-catenin signaling in a manner dependent on the epithelial-mesenchymal transition program.

Dullard-mediated Smad1/5/8 inhibition controls mouse cardiac neural crest cells condensation and outflow tract septation.

The establishment of separated pulmonary and systemic circulation in vertebrates, via cardiac outflow tract (OFT) septation, is a sensitive developmental process accounting for 10% of all congenital anomalies. Neural Crest Cells (NCC) colonising the heart condensate along the primitive endocardial tube and force its scission into two tubes. Here, we show that NCC aggregation progressively decreases along the OFT distal-proximal axis following a BMP signalling gradient. Dullard, a nuclear phosphatase, tunes the BMP gradient amplitude and prevents NCC premature condensation. Dullard maintains transcriptional programs providing NCC with mesenchymal traits. It attenuates the expression of the aggregation factor Sema3c and conversely promotes that of the epithelial-mesenchymal transition driver Twist1. Altogether, Dullard-mediated fine-tuning of BMP signalling ensures the timed and progressive zipper-like closure of the OFT by the NCC and prevents the formation of a heart carrying the congenital abnormalities defining the tetralogy of Fallot.

An embryonic CaVß1 isoform promotes muscle mass maintenance via GDF5 signaling in adult mouse.

Deciphering the mechanisms that govern skeletal muscle plasticity is essential to understand its pathophysiological processes, including age-related sarcopenia. The voltage-gated calcium channel CaV1.1 has a central role in excitation-contraction coupling (ECC), raising the possibility that it may also initiate the adaptive response to changes during muscle activity. Here, we revealed the existence of a gene transcription switch of the CaV1.1 ß subunit (CaVß1) that is dependent on the innervation state of the muscle in mice. In a mouse model of sciatic denervation, we showed increased expression of an embryonic isoform of the subunit that we called CaVß1E. CaVß1E boosts downstream growth differentiation factor 5 (GDF5) signaling to counteract muscle loss after denervation in mice. We further reported that aged mouse muscle expressed lower quantity of CaVß1E compared with young muscle, displaying an altered GDF5-dependent response to denervation. Conversely, CaVß1E overexpression improved mass wasting in aging muscle in mice by increasing GDF5 expression. We also identified the human CaVß1E analogous and show a correlation between CaVß1E expression in human muscles and age-related muscle mass decline. These results suggest that strategies targeting CaVß1E or GDF5 might be effective in reducing muscle mass loss in aging.