Dynamics of membrane tubulation coupled with fission by a two-component module.

Membrane tubulation coupled with fission (MTCF) is a widespread phenomenon but mechanisms for their coordination remain unclear, partly because of the lack of assays to monitor dynamics of membrane tubulation and subsequent fission. Using polymer cushioned bilayer islands, we analyze the membrane tubulator Bridging Integrator 1 (BIN1) mixed with the fission catalyst dynamin2 (Dyn2). Our results reveal this mixture to constitute a minimal two-component module that demonstrates MTCF. MTCF is an emergent property and arises because BIN1 facilitates recruitment but inhibits membrane binding of Dyn2 in a dose-dependent manner. MTCF is therefore apparent only at high Dyn2 to BIN1 ratios. Because of their mutual involvement in T-tubules biogenesis, mutations in BIN1 and Dyn2 are associated with centronuclear myopathies and our analysis links the pathology with aberrant MTCF. Together, our results establish cushioned bilayer islands as a facile template for the analysis of membrane tubulation and inform of mechanisms that coordinate MTCF.

Myofibrillar myopathy hallmarks associated with ZAK deficiency.

The ZAK gene encodes two functionally distinct kinases, ZAKα and ZAKß. Homozygous loss of function mutations affecting both isoforms causes a congenital muscle disease. ZAKß is the only isoform expressed in skeletal muscle and is activated by muscle contraction and cellular compression. The ZAKß substrates in skeletal muscle or the mechanism whereby ZAKß senses mechanical stress remains to be determined. To gain insights into the pathogenic mechanism, we exploited ZAK-deficient cell lines, zebrafish, mice and a human biopsy. ZAK-deficient mice and zebrafish show a mild phenotype. In mice, comparative histopathology data from regeneration, overloading, ageing and sex conditions indicate that while age and activity are drivers of the pathology, ZAKß appears to have a marginal role in myoblast fusion in vitro or muscle regeneration in vivo. The presence of SYNPO2, BAG3 and Filamin C (FLNC) in a phosphoproteomics assay and extended analyses suggested a role for ZAKß in the turnover of FLNC. Immunofluorescence analysis of muscle sections from mice and a human biopsy showed evidence of FLNC and BAG3 accumulations as well as other myofibrillar myopathy markers. Moreover, endogenous overloading of skeletal muscle exacerbated the presence of fibres with FLNC accumulations in mice, indicating that ZAKß signalling is necessary for an adaptive turnover of FLNC that allows for the normal physiological response to sustained mechanical stress. We suggest that accumulation of mislocalized FLNC and BAG3 in highly immunoreactive fibres contributes to the pathogenic mechanism of ZAK deficiency.

Common and Key Differential Pathogenic Pathways in Desminopathy and Titinopathy.

Myofibrillar myopathy (MFM) is a group of hereditary myopathies that mainly involves striated muscles. This study aimed to use tandem mass tag (TMT)-based proteomics to investigate the underlying pathomechanisms of two of the most common MFM subtypes, desminopathy and titinopathy. Muscles from 7 patients with desminopathy, 5 with titinopathy and 5 control individuals were included. Samples were labelled with TMT and then underwent high-resolution liquid chromatography-mass spectrometry analysis. Compared with control samples, there were 436 differentially abundant proteins (DAPs) in the desminopathy group and 269 in the titinopathy group. When comparing the desminopathy with the titinopathy group, there were 113 DAPs. In desminopathy, mitochondrial ATP production, muscle contraction, and cytoskeleton organization were significantly suppressed. Activated cellular components and pathways were mostly related to extracellular matrix (ECM). In titinopathy, mitochondrial-related pathways and the cellular component ECM were downregulated, while gluconeogenesis was activated. Direct comparison between desminopathy and titinopathy revealed hub genes that were all involved in glycolytic process. The disparity in glycolysis in the two MFM subtypes is likely due to fiber type switching. This study has revealed disorganization of cytoskeleton and mitochondrial dysfunction as the common pathophysiological processes in MFM, and glycolysis and ECM as the differential pathomechanism between desminopathy and titinopathy. This offers a future direction for targeted therapy for MFM.

SPEG binds with desmin and its deficiency causes defects in triad and focal adhesion proteins.

Striated preferentially expressed gene (SPEG), a member of the myosin light chain kinase family, is localized at the level of triad surrounding myofibrils in skeletal muscles. In humans, SPEG mutations are associated with centronuclear myopathy and cardiomyopathy. Using a striated muscle-specific Speg-knockout (KO) mouse model, we have previously shown that SPEG is critical for triad maintenance and calcium handling. Here, we further examined the molecular function of SPEG and characterized the effects of SPEG deficiency on triad and focal adhesion proteins. We used yeast two-hybrid assay, and identified desmin, an intermediate filament protein, to interact with SPEG and confirmed this interaction by co-immunoprecipitation. Using domain-mapping assay, we defined that Ig-like and fibronectin III domains of SPEG interact with rod domain of desmin. In skeletal muscles, SPEG depletion leads to desmin aggregates in vivo and a shift in desmin equilibrium from soluble to insoluble fraction. We also profiled the expression and localization of triadic proteins in Speg-KO mice using western blot and immunofluorescence. The amount of RyR1 and triadin were markedly reduced, whereas DHPRα1, SERCA1 and triadin were abnormally accumulated in discrete areas of Speg-KO myofibers. In addition, Speg-KO muscles exhibited internalized vinculin and ß1 integrin, both of which are critical components of the focal adhesion complex. Further, ß1 integrin was abnormally accumulated in early endosomes of Speg-KO myofibers. These results demonstrate that SPEG-deficient skeletal muscles exhibit several pathological features similar to those seen in MTM1 deficiency. Defects of shared cellular pathways may underlie these structural and functional abnormalities in both types of diseases.

Regulation of myonuclear positioning and muscle function by the skeletal muscle-specific CIP protein.

The appropriate arrangement of myonuclei within skeletal muscle myofibers is of critical importance for normal muscle function, and improper myonuclear localization has been linked to a variety of skeletal muscle diseases, such as centronuclear myopathy and muscular dystrophies. However, the molecules that govern myonuclear positioning remain elusive. Here, we report that skeletal muscle-specific CIP (sk-CIP) is a regulator of nuclear positioning. Genetic deletion of sk-CIP in mice results in misalignment of myonuclei along the myofibers and at specialized structures such as neuromuscular junctions (NMJs) and myotendinous junctions (MTJs) in vivo, impairing myonuclear positioning after muscle regeneration, leading to severe muscle dystrophy in mdx mice, a mouse model of Duchenne muscular dystrophy. sk-CIP is localized to the centrosome in myoblasts and relocates to the outer nuclear envelope in myotubes upon differentiation. Mechanistically, we found that sk-CIP interacts with the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex and the centriole Microtubule Organizing Center (MTOC) proteins to coordinately modulate myonuclear positioning and alignment. These findings indicate that sk-CIP may function as a muscle-specific anchoring protein to regulate nuclear position in multinucleated muscle cells.

Genetic compensation prevents myopathy and heart failure in an in vivo model of Bag3 deficiency.

Mutations in the molecular co-chaperone Bcl2-associated athanogene 3 (BAG3) are found to cause dilated cardiomyopathy (DCM), resulting in systolic dysfunction and heart failure, as well as myofibrillar myopathy (MFM), which is characterized by protein aggregation and myofibrillar disintegration in skeletal muscle cells. Here, we generated a CRISPR/Cas9-induced Bag3 knockout zebrafish line and found the complete preservation of heart and skeletal muscle structure and function during embryonic development, in contrast to morpholino-mediated knockdown of Bag3. Intriguingly, genetic compensation, a process of transcriptional adaptation which acts independent of protein feedback loops, was found to prevent heart and skeletal muscle damage in our Bag3 knockout model. Proteomic profiling and quantitative real-time PCR analyses identified Bag2, another member of the Bag protein family, significantly upregulated on a transcript and protein level in bag3-/- mutants. This implied that the decay of bag3 mutant mRNA in homozygous bag3-/- embryos caused the transcriptional upregulation of bag2 expression. We further demonstrated that morpholino-mediated knockdown of Bag2 in bag3-/- embryos evoked severe functional and structural heart and skeletal muscle defects, which are similar to Bag3 morphants. However, Bag2 knockdown in bag3+/+ or bag3+/- embryos did not result in (cardio-)myopathy. Finally, we found that inhibition of the nonsense-mediated mRNA decay (NMD) machinery by knockdown of upf1, an essential NMD factor, caused severe heart and skeletal muscle defects in bag3-/- mutants due to the blockade of transcriptional adaptation of bag2 expression. Our findings provide evidence that genetic compensation might vitally influence the penetrance of disease-causing bag3 mutations in vivo.

Deep Characterization of a Greek Patient with Desmin-Related Myofibrillar Myopathy and Cardiomyopathy.

Desmin is a class III intermediate filament protein highly expressed in cardiac, smooth and striated muscle. Autosomal dominant or recessive mutations in the desmin gene (DES) result in a variety of diseases, including cardiomyopathies and myofibrillar myopathy, collectively called desminopathies. Here we describe the clinical, histological and radiological features of a Greek patient with a myofibrillar myopathy and cardiomyopathy linked to the c.734A>G,p.(Glu245Gly) heterozygous variant in the DES gene. Moreover, through ribonucleic acid sequencing analysis in skeletal muscle we show that this variant provokes a defect in exon 3 splicing and thus should be considered clearly pathogenic.

Molecular Mechanisms of Deregulation of Muscle Contractility Caused by the R168H Mutation in TPM3 and Its Attenuation by Therapeutic Agents.

The substitution for Arg168His (R168H) in γ-tropomyosin (TPM3 gene, Tpm3.12 isoform) is associated with congenital muscle fiber type disproportion (CFTD) and muscle weakness. It is still unclear what molecular mechanisms underlie the muscle dysfunction seen in CFTD. The aim of this work was to study the effect of the R168H mutation in Tpm3.12 on the critical conformational changes that myosin, actin, troponin, and tropomyosin undergo during the ATPase cycle. We used polarized fluorescence microscopy and ghost muscle fibers containing regulated thin filaments and myosin heads (myosin subfragment-1) modified with the 1,5-IAEDANS fluorescent probe. Analysis of the data obtained revealed that a sequential interdependent conformational-functional rearrangement of tropomyosin, actin and myosin heads takes place when modeling the ATPase cycle in the presence of wild-type tropomyosin. A multistep shift of the tropomyosin strands from the outer to the inner domain of actin occurs during the transition from weak to strong binding of myosin to actin. Each tropomyosin position determines the corresponding balance between switched-on and switched-off actin monomers and between the strongly and weakly bound myosin heads. At low Ca2+, the R168H mutation was shown to switch some extra actin monomers on and increase the persistence length of tropomyosin, demonstrating the freezing of the R168HTpm strands close to the open position and disruption of the regulatory function of troponin. Instead of reducing the formation of strong bonds between myosin heads and F-actin, troponin activated it. However, at high Ca2+, troponin decreased the amount of strongly bound myosin heads instead of promoting their formation. Abnormally high sensitivity of thin filaments to Ca2+, inhibition of muscle fiber relaxation due to the appearance of the myosin heads strongly associated with F-actin, and distinct activation of the contractile system at submaximal concentrations of Ca2+ can lead to muscle inefficiency and weakness. Modulators of troponin (tirasemtiv and epigallocatechin-3-gallate) and myosin (omecamtiv mecarbil and 2,3-butanedione monoxime) have been shown to more or less attenuate the negative effects of the tropomyosin R168H mutant. Tirasemtiv and epigallocatechin-3-gallate may be used to prevent muscle dysfunction.

Human Mutated MYOT and CRYAB Genes Cause a Myopathic Phenotype in Zebrafish.

Myofibrillar myopathies (MFMs) are a group of hereditary neuromuscular disorders sharing common histological features, such as myofibrillar derangement, Z-disk disintegration, and the accumulation of degradation products into protein aggregates. They are caused by mutations in several genes that encode either structural proteins or molecular chaperones. Nevertheless, the mechanisms by which mutated genes result in protein aggregation are still unknown. To unveil the role of myotilin and αB-crystallin in the pathogenesis of MFM, we injected zebrafish fertilized eggs at the one-cell stage with expression plasmids harboring cDNA sequences of human wildtype or mutated MYOT (p.Ser95Ile) and human wildtype or mutated CRYAB (p.Gly154Ser). We evaluated the effects on fish survival, motor behavior, muscle structure and development. We found that transgenic zebrafish showed morphological defects that were more severe in those overexpressing mutant genes. which developed a myopathic phenotype consistent with that of human myofibrillar myopathy, including the formation of protein aggregates. Results indicate that pathogenic mutations in myotilin and αB-crystallin genes associated with MFM cause a structural and functional impairment of the skeletal muscle in zebrafish, thereby making this non-mammalian organism a powerful model to dissect disease pathogenesis and find possible druggable targets.

Mutant BIN1-Dynamin 2 complexes dysregulate membrane remodeling in the pathogenesis of centronuclear myopathy.

Membrane remodeling is required for dynamic cellular processes such as cell division, polarization, and motility. BAR domain proteins and dynamins are key molecules in membrane remodeling that work together for membrane deformation and fission. In striated muscles, sarcolemmal invaginations termed T-tubules are required for excitation-contraction coupling. BIN1 and DNM2, which encode a BAR domain protein BIN1 and dynamin 2, respectively, have been reported to be causative genes of centronuclear myopathy (CNM), a hereditary degenerative disease of skeletal muscle, and deformation of T-tubules is often observed in the CNM patients. However, it remains unclear how BIN1 and dynamin 2 are implicated in T-tubule biogenesis and how mutations in these molecules cause CNM to develop. Here, using an in cellulo reconstitution assay, we demonstrate that dynamin 2 is required for stabilization of membranous structures equivalent to T-tubules. GTPase activity of wild-type dynamin 2 is suppressed through interaction with BIN1, whereas that of the disease-associated mutant dynamin 2 remains active due to lack of the BIN1-mediated regulation, thus causing aberrant membrane remodeling. Finally, we show that in cellulo aberrant membrane remodeling by mutant dynamin 2 variants is correlated with their enhanced membrane fission activities, and the results can explain severity of the symptoms in patients. Thus, this study provides molecular insights into dysregulated membrane remodeling triggering the pathogenesis of DNM2-related CNM.

X-linked myotubular myopathy is associated with epigenetic alterations and is ameliorated by HDAC inhibition.

X-linked myotubular myopathy (XLMTM) is a fatal neuromuscular disorder caused by loss of function mutations in MTM1. At present, there are no directed therapies for XLMTM, and incomplete understanding of disease pathomechanisms. To address these knowledge gaps, we performed a drug screen in mtm1 mutant zebrafish and identified four positive hits, including valproic acid, which functions as a potent suppressor of the mtm1 zebrafish phenotype via HDAC inhibition. We translated these findings to a mouse XLMTM model, and showed that valproic acid ameliorates the murine phenotype. These observations led us to interrogate the epigenome in Mtm1 knockout mice; we found increased DNA methylation, which is normalized with valproic acid, and likely mediated through aberrant 1-carbon metabolism. Finally, we made the unexpected observation that XLMTM patients share a distinct DNA methylation signature, suggesting that epigenetic alteration is a conserved disease feature amenable to therapeutic intervention.

Multi-omics comparisons of different forms of centronuclear myopathies and the effects of several therapeutic strategies.

Omics analyses are powerful methods to obtain an integrated view of complex biological processes, disease progression, or therapy efficiency. However, few studies have compared different disease forms and different therapy strategies to define the common molecular signatures representing the most significant implicated pathways. In this study, we used RNA sequencing and mass spectrometry to profile the transcriptomes and proteomes of mouse models for three forms of centronuclear myopathies (CNMs), untreated or treated with either a drug (tamoxifen), antisense oligonucleotides reducing the level of dynamin 2 (DNM2), or following modulation of DNM2 or amphiphysin 2 (BIN1) through genetic crosses. Unsupervised analysis and differential gene and protein expression were performed to retrieve CNM molecular signatures. Longitudinal studies before, at, and after disease onset highlighted potential disease causes and consequences. Main pathways in the common CNM disease signature include muscle contraction, regeneration and inflammation. The common therapy signature revealed novel potential therapeutic targets, including the calcium regulator sarcolipin. We identified several novel biomarkers validated in muscle and/or plasma through RNA quantification, western blotting, and enzyme-linked immunosorbent assay (ELISA) assays, including ANXA2 and IGFBP2. This study validates the concept of using multi-omics approaches to identify molecular signatures common to different disease forms and therapeutic strategies.

Gain-of-Function Dynamin-2 Mutations Linked to Centronuclear Myopathy Impair Ca2+-Induced Exocytosis in Human Myoblasts.

Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to a gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as a reporter of the GLUT4 vesicle trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 µM ionomycin, indicating that in addition to impairing exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.

Centronuclear Myopathy Caused by Defective Membrane Remodelling of Dynamin 2 and BIN1 Variants.

Centronuclear myopathy (CNM) is a congenital myopathy characterised by centralised nuclei in skeletal myofibers. T-tubules, sarcolemmal invaginations required for excitation-contraction coupling, are disorganised in the skeletal muscles of CNM patients. Previous studies showed that various endocytic proteins are involved in T-tubule biogenesis and their dysfunction is tightly associated with CNM pathogenesis. DNM2 and BIN1 are two causative genes for CNM that encode essential membrane remodelling proteins in endocytosis, dynamin 2 and BIN1, respectively. In this review, we overview the functions of dynamin 2 and BIN1 in T-tubule biogenesis and discuss how their dysfunction in membrane remodelling leads to CNM pathogenesis.

Silencing of the Ca2+ Channel ORAI1 Improves the Multi-Systemic Phenotype of Tubular Aggregate Myopathy (TAM) and Stormorken Syndrome (STRMK) in Mice.

Tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) form a clinical continuum associating progressive muscle weakness with additional multi-systemic anomalies of the bones, skin, spleen, and platelets. TAM/STRMK arises from excessive extracellular Ca2+ entry due to gain-of-function mutations in the Ca2+ sensor STIM1 or the Ca2+ channel ORAI1. Currently, no treatment is available. Here we assessed the therapeutic potential of ORAI1 downregulation to anticipate and reverse disease development in a faithful mouse model carrying the most common TAM/STRMK mutation and recapitulating the main signs of the human disorder. To this aim, we crossed Stim1R304W/+ mice with Orai1+/- mice expressing 50% of ORAI1. Systematic phenotyping of the offspring revealed that the Stim1R304W/+Orai1+/- mice were born with a normalized ratio and showed improved postnatal growth, bone architecture, and partly ameliorated muscle function and structure compared with their Stim1R304W/+ littermates. We also produced AAV particles containing Orai1-specific shRNAs, and intramuscular injections of Stim1R304W/+ mice improved the skeletal muscle contraction and relaxation properties, while muscle histology remained unchanged. Altogether, we provide the proof-of-concept that Orai1 silencing partially prevents the development of the multi-systemic TAM/STRMK phenotype in mice, and we also established an approach to target Orai1 expression in postnatal tissues.

Different in vivo impacts of dynamin 2 mutations implicated in Charcot-Marie-Tooth neuropathy or centronuclear myopathy.

Dynamin 2 (DNM2) is a ubiquitously expressed GTPase implicated in many cellular functions such as membrane trafficking and cytoskeleton regulation. Dominant mutations in DNM2 result in tissue-specific diseases affecting peripheral nerves (Charcot-Marie-Tooth neuropathy, CMT) or skeletal muscles (centronuclear myopathy, CNM). However, the reason for this tissue specificity is unknown, and it remains unclear if these diseases share a common pathomechanism. To compare the disease pathophysiological mechanisms in skeletal muscle, we exogenously expressed wild-type DNM2 (WT-DNM2), the DNM2-CMT mutation K562E or DNM2-CNM mutations R465W and S619L causing adult and neonatal forms, respectively, by intramuscular adeno-associated virus (AAV) injections. All muscles expressing exogenous WT-DNM2 and CNM or CMT mutations exhibited reduced muscle force. However, only expression of CNM mutations and WT-DNM2 correlated with CNM-like histopathological hallmarks of nuclei centralization and reduced fiber size. The extent of alterations correlated with clinical severity in patients. Ultrastructural and immunofluorescence analyses highlighted defects of the triads, mitochondria and costameres as major causes of the CNM phenotype. Despite the reduction in force upon expression of the DNM2-CMT mutation, muscle histology and ultrastructure were almost normal. However, the neuromuscular junction was affected in all DNM2-injected muscles, with the DNM2-CMT mutation inducing the most severe alterations, potentially explaining the reduction in force observed with this mutant. In conclusion, expression of WT and CNM mutants recreate a CNM-like phenotype, suggesting CNM mutations are gain-of-function. Histological, ultrastructural and molecular analyses pointed to key pathways uncovering the different pathomechanisms involved in centronuclear myopathy or Charcot-Marie-Tooth neuropathy linked to DNM2 mutations.

Common Pathogenic Mechanisms in Centronuclear and Myotubular Myopathies and Latest Treatment Advances.

Centronuclear myopathies (CNM) are rare congenital disorders characterized by muscle weakness and structural defects including fiber hypotrophy and organelle mispositioning. The main CNM forms are caused by mutations in: the MTM1 gene encoding the phosphoinositide phosphatase myotubularin (myotubular myopathy), the DNM2 gene encoding the mechanoenzyme dynamin 2, the BIN1 gene encoding the membrane curvature sensing amphiphysin 2, and the RYR1 gene encoding the skeletal muscle calcium release channel/ryanodine receptor. MTM1, BIN1, and DNM2 proteins are involved in membrane remodeling and trafficking, while RyR1 directly regulates excitation-contraction coupling (ECC). Several CNM animal models have been generated or identified, which confirm shared pathological anomalies in T-tubule remodeling, ECC, organelle mispositioning, protein homeostasis, neuromuscular junction, and muscle regeneration. Dynamin 2 plays a crucial role in CNM physiopathology and has been validated as a common therapeutic target for three CNM forms. Indeed, the promising results in preclinical models set up the basis for ongoing clinical trials. Another two clinical trials to treat myotubular myopathy by MTM1 gene therapy or tamoxifen repurposing are also ongoing. Here, we review the contribution of the different CNM models to understanding physiopathology and therapy development with a focus on the commonly dysregulated pathways and current therapeutic targets.

SPEG-deficient skeletal muscles exhibit abnormal triad and defective calcium handling.

Centronuclear myopathies (CNM) are a subtype of congenital myopathies (CM) characterized by skeletal muscle weakness and an increase in the number of central myonuclei. We have previously identified three CNM probands, two with associated dilated cardiomyopathy, carrying striated preferentially expressed gene (SPEG) mutations. Currently, the role of SPEG in skeletal muscle function is unclear as constitutive SPEG-deficient mice developed severe dilated cardiomyopathy and died in utero. We have generated a conditional Speg-KO mouse model and excised Speg by crosses with striated muscle-specific cre-expressing mice (MCK-Cre). The resulting litters had a delay in Speg excision consistent with cre expression starting in early postnatal life and, therefore, an extended lifespan up to a few months. KO mice were significantly smaller and weaker than their littermate-matched controls. Histopathological skeletal muscle analysis revealed smaller myofibers, marked fiber-size variability, and poor integrity and low number of triads. Further, SPEG-deficient muscle fibers were weaker by physiological and in vitro studies and exhibited abnormal Ca2+ handling and excitation-contraction (E-C) coupling. Overall, SPEG deficiency in skeletal muscle is associated with fewer and abnormal triads, and defective calcium handling and excitation-contraction coupling, suggesting that therapies targeting calcium signaling may be beneficial in such patients.

Functional analyses of STIM1 mutations reveal a common pathomechanism for tubular aggregate myopathy and Stormorken syndrome.

Tubular aggregate myopathy (TAM) is a progressive disorder characterized by muscle weakness, cramps, and myalgia. TAM clinically overlaps with Stormorken syndrome (STRMK), combining TAM with miosis, thrombocytopenia, hyposplenism, ichthyosis, short stature, and dyslexia. TAM and STRMK arise from gain-of-function mutations in STIM1 (stromal interaction molecule 1) or ORAI1, both encoding key regulators of Ca2+ homeostasis, and mutations in either gene result in excessive extracellular Ca2+ entry. The pathomechanistic similarities and differences between TAM and STRMK are only partially understood. Here we provide functional in vitro experiments demonstrating that STIM1 harboring the TAM D84G or the STRMK R304W mutation similarly cluster and exert a dominant effect on the wild-type protein. Both mutants recruit ORAI1 to the clusters, increase cytosolic Ca2+ levels, promote major nuclear import of the Ca2+ -dependent transcription factor NFAT (nuclear factor of activated T cells), and trigger the formation of circular membrane stacks. In conclusion, the analyzed TAM and STRMK mutations have a comparable impact on STIM1 protein function and downstream effects of excessive Ca2+ entry, highlighting that TAM and STRMK involve a common pathomechanism.

Luminal STIM1 Mutants that Cause Tubular Aggregate Myopathy Promote Autophagic Processes.

Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca2+ sensor protein that induces permeation of Orai Ca2+ channels upon endoplasmic reticulum Ca2+-store depletion. A drop in luminal Ca2+ causes partial unfolding of the N-terminal STIM1 domains and thus initial STIM1 activation. We compared the STIM1 structure upon Ca2+ depletion from our molecular dynamics (MD) simulations with a recent 2D NMR structure. Simulation- and structure-based results showed unfolding of two α-helices in the canonical and in the non-canonical EF-hand. Further, we structurally and functionally evaluated mutations in the non-canonical EF-hand that have been shown to cause tubular aggregate myopathy. We found these mutations to cause full constitutive activation of Ca2+-release-activated Ca2+ currents (ICRAC) and to promote autophagic processes. Specifically, heterologously expressed STIM1 mutations in the non-canonical EF-hand promoted translocation of the autophagy transcription factors microphthalmia-associated transcription factor (MITF) and transcription factor EB (TFEB) into the nucleus. These STIM1 mutations additionally stimulated an enhanced production of autophagosomes. In summary, mutations in STIM1 that cause structural unfolding promoted Ca2+ down-stream activation of autophagic processes.