The mechanosensitive TRPV2 calcium channel promotes human melanoma invasiveness and metastatic potential.

Melanoma is a highly aggressive cancer endowed with a unique capacity of rapidly metastasizing, which is fundamentally driven by aberrant cell motility behaviors. Discovering "migrastatics" targets, specifically controlling invasion and dissemination of melanoma cells during metastasis, is therefore of primary importance. Here, we uncover the prominent expression of the plasma membrane TRPV2 calcium channel as a distinctive feature of melanoma tumors, directly related to melanoma metastatic dissemination. In vitro as well as in vivo, TRPV2 activity is sufficient to confer both migratory and invasive potentials, while conversely TRPV2 silencing in highly metastatic melanoma cells prevents aggressive behavior. In invasive melanoma cells, TRPV2 channel localizes at the leading edge, in dynamic nascent adhesions, and regulates calcium-mediated activation of calpain and the ensuing cleavage of the adhesive protein talin, along with F-actin organization. In human melanoma tissues, TRPV2 overexpression correlates with advanced malignancy and poor prognosis, evoking a biomarker potential. Hence, by regulating adhesion and motility, the mechanosensitive TRPV2 channel controls melanoma cell invasiveness, highlighting a new therapeutic option for migrastatics in the treatment of metastatic melanoma.

Morphology of Abdominal Aortic Aneurysms and Correlation with Biomechanical Tests of Aneurysmal Wall Fragments.

BACKGROUND: Evaluate how specific morphologic aspects of abdominal aortic aneurysms (AAAs), including asymmetries, curvatures, tortuosities, and angulations, among others can influence the intrinsic biomechanical properties of the AAA's wall. This study analyzed the correlation of geometric measurements (1-dimensional, 2-dimensional, and 3-dimensional) of preoperative tomographic images of AAA with uniaxial biomechanical tests of the arterial wall fragments of these AAA obtained in open surgical repair of aneurysms. METHODS: It was a multicenter, experimental, and observational study, and initially 54 individuals were selected who underwent open surgical of AAA, with valid biomechanical tests of the anterior wall of the AAA. Seven individuals were excluded because they had poor preoperative quality computed tomography scans and/or artifacts that impeded image segmentation and extraction of AAA geometric indices. The aortic fragments were subjected to uniaxial biomechanical destructive tests to obtain the following data: maximum load, failure stress, failure tension, failure strain energy, strain, and fragment thickness. In the same patients, preoperative computed tomography scans were performed with the extraction of 26 geometric indices, subdivided into 9 1-dimensional indices, 6 2-dimensional indices, and 11 3-dimensional indices. Data were subjected to statistical analysis using SPSS version 28. RESULTS: Comparing ruptured and unruptured AAA, no statistical difference was observed between the biomechanical and geometric parameters. The fragment thickness of the ruptured AAA was lower than that of the unruptured AAA (P < 0.05). By comparing tomographic geometric indices and biomechanical parameters of the aortic fragments using Pearson's coefficient, positive and linear correlations (P < 0.05) were observed between the geometric variable maximum diameter (Dmax) of the AAA with maximum load (r = 0.408), failure tension (r = 0.372), and failure stress (r = 0.360). Positive and linear correlations were also observed between the variable diameter/height ratio (DHr) and the maximum load (r = 0.360), failure tension (r = 0.354), and failure stress (r = 0.289). The geometric variable DHr was dependent and correlated with Dmax. Simple regression analysis showed that R2 varied between 8.3% and 16.7%, and all models were significant (P < 0.05). CONCLUSIONS: Dmax and DHr were linearly and positively correlated with the resistance parameters (maximum load, failure tension, and failure stress) of the AAA fragments. The DHr variable is dependent and correlated with Dmax. There was no correlation between the other geometric indices and the biomechanical parameters of the AAA wall. The asymmetries did not globally influence the biomechanics of AAA wall; however, they may influence regionally. Larger AAAs were stronger than smaller ones. Therefore, such findings may point toward Dmax is still the main geometric parameter, which influences the anterior wall, and possibly globally in the AAA.

Natural History of Splanchnic Artery Aneurysms.

INTRODUCTION: Splanchnic artery aneurysms (SAAs) represent a rare and potential life-threatening disease with a documented incidence of 0.1-2.0%. The risk of rupture and the diameter to recommend surgery are still controversial. The purpose of this study was to review surveillance computed tomography scans (CTs) at a high-volume institution in order to better define the natural history of the SAA. METHODS: Between January 2000 and February 2019, all SAAs patients in follow-up at a single center institution were selected for analysis. CTs from patients managed nonoperatively and CTs before surgery from patients submitted to surgery were studied. The first CTs were used to determine aneurysm size, morphology, and anatomic characteristics, and the last CTs performed during nonoperative follow-up were used to compare the diameter with the previous CTs. Primary endpoint included growth rate for all SAAs location, and secondary endpoint included the clinical or anatomical characteristic associated with a faster growth rate. RESULTS: In total, 116 consecutive patients were identified with SAAs and 74 patients with 87 SAAs who had at least 2 CTs during follow-up were analyzed. From those 74 patients, 12 were submitted to surgery and only their preoperative CTs were analyzed. The SAAs' locations were: splenic (55.4%), hepatic (12.2%), superior mesenteric artery (17.6%), celiac trunk (27.0%), gastric and gastroepiploic arteries (1.4%), pancreaticoduodenal and gastroduodenal arteries (4.1%). The median follow-up for all patients was 46.7 months (±35.3), and the median of growth for all aneurysms was 0.63 mm/year (±2.19). Only the splenic aneurysms presented growth with statistic significance of 1.08 mm per/year (±1.99) (P < 0.001). Only portal hypertension showed statistically significance to splenic aneurysm growth (P = 0.002). Multivariate analysis for variables associated with splenic aneurysm growth ≥1 mm/year showed that portal hypertension was the only variable with statistical significance (P < 0.01, IC 95% 2.0-186.9, ß = 19.5). CONCLUSIONS: Although longer-term follow-up and larger sample size are needed to better understand the natural history of SAAs, the majority of SAAs tends to remain stable in size through follow-up. Portal hypertension was the only risk factor found for true splenic aneurysm growth, and so those patients must have a closer follow-up.

Store-Operated Calcium Entries Control Neural Stem Cell Self-Renewal in the Adult Brain Subventricular Zone.

The subventricular zone (SVZ) is the major stem cell niche in the brain of adult mammals. Within this region, neural stem cells (NSC) proliferate, self-renew and give birth to neurons and glial cells. Previous studies underlined enrichment in calcium signaling-related transcripts in adult NSC. Because of their ability to mobilize sustained calcium influxes in response to a wide range of extracellular factors, store-operated channels (SOC) appear to be, among calcium channels, relevant candidates to induce calcium signaling in NSC whose cellular activities are continuously adapted to physiological signals from the microenvironment. By Reverse Transcription Polymerase Chain Reaction (RT-PCR), Western blotting and immunocytochemistry experiments, we demonstrate that SVZ cells express molecular actors known to build up SOC, namely transient receptor potential canonical 1 (TRPC1) and Orai1, as well as their activator stromal interaction molecule 1 (STIM1). Calcium imaging reveals that SVZ cells display store-operated calcium entries. Pharmacological blockade of SOC with SKF-96365 or YM-58483 (also called BTP2) decreases proliferation, impairs self-renewal by shifting the type of SVZ stem cell division from symmetric proliferative to asymmetric, thereby reducing the stem cell population. Brain section immunostainings show that TRPC1, Orai1, and STIM1 are expressed in vivo, in SOX2-positive SVZ NSC. Injection of SKF-96365 in brain lateral ventricle diminishes SVZ cell proliferation and reduces the ability of SVZ cells to form neurospheres in vitro. The present study combining in vitro and in vivo approaches uncovers a major role for SOC in the control of SVZ NSC population and opens new fields of investigation for stem cell biology in health and disease. Stem Cells 2018;36:761-774.

Constitutive calcium entry and cancer: updated views and insights.

Tight control of basal cytosolic Ca2+ concentration is essential for cell survival and to fine-tune Ca2+-dependent cell functions. A way to control this basal cytosolic Ca2+ concentration is to regulate membrane Ca2+ channels including store-operated Ca2+ channels and secondary messenger-operated channels linked to G-protein-coupled or tyrosine kinase receptor activation. Orai, with or without its reticular STIM partner and Transient Receptor Potential (TRP) proteins, were considered to be the main Ca2+ channels involved. It is well accepted that, in response to cell stimulation, opening of these Ca2+ channels contributes to Ca2+ entry and the transient increase in cytosolic Ca2+ concentration involved in intracellular signaling. However, in various experimental conditions, Ca2+ entry and/or Ca2+ currents can be recorded at rest, without application of any experimental stimulation. This led to the proposition that some plasma membrane Ca2+ channels are already open/activated in basal condition, contributing therefore to constitutive Ca2+ entry. This article focuses on direct and indirect observations supporting constitutive activity of channels belonging to the Orai and TRP families and on the mechanisms underlying their basal/constitutive activities.

Plasma membrane calcium channels in cancer: Alterations and consequences for cell proliferation and migration.

The study of calcium channels in molecular mechanisms of cancer transformation is still a novel area of research. Several studies, mostly conducted on cancer cell lines, however support the idea that a diversity of plasma membrane channels participates in the remodeling of Ca2+ homeostasis, which regulates various cancer hallmarks such as uncontrolled multiplication and increase in migration and invasion abilities. However few is still understood concerning the intracellular signaling cascades mobilized by calcium influx participating to cancer cell behavior. This review intends to gather some of these pathways dependent on plasma membrane calcium channels and described in prostate, breast and lung cancer cell lines. In these cancer cell types, the calcium channels involved in calcium signaling pathways promoting cancer behaviors are mostly non-voltage activated calcium channels and belong to the TRP superfamily (TRPC, TPRPV and TRPM families) and the Orai family. TRP and Orai channels are part of many signaling cascades involving the activation of transmembrane receptors by extracellular ligand from the tumor environment. TRPV can sense changes in the physical and chemical environment of cancer cells and TRPM7 are stretch activated and sensitive to cholesterol. Changes in activation and or expression of plasma-membrane calcium channels affect calcium-dependent signaling processes relevant to tumorigenesis. The studies cited in this review suggest that an increase in plasma membrane calcium channel expression and/or activity sustain an elevated calcium entry (constitutive or under the control of extracellular signals) promoting higher cell proliferation and migration in most cases. A variety of non-voltage-operated calcium channels display change expression and/or activity in a same cancer type and cooperate to the same process relevant to cancer cell behavior, or can be involved in a different sequence of events during the tumorigenesis. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

Role of Scaffolding Proteins in the Regulation of TRPC-Dependent Calcium Entry.

Plasma membrane ion channels, and in particular TRPC channels need a specific membrane environment and association with scaffolding, signaling, and cytoskeleton proteins in order to play their important functional role. The molecular composition of TRPC channels is an important factor in determining channel activation mechanisms. TRPC proteins are incorporated in macromolecular complexes including several key Ca(2 +) signaling proteins as well as proteins involved in vesicle trafficking, cytoskeletal interactions, and scaffolding. Evidence has been provided for association of TRPC with calmodulin (CaM), IP3R, PMCA, Gq/11, RhoA, and a variety of scaffolding proteins. The interaction between TRPC channels with adaptor proteins, determines their mode of regulation as well as their cellular localization and function. Adaptor proteins do not display any enzymatic activity but act as scaffold for the building of signaling complexes. The scaffolding proteins are involved in the assembling of these Ca(2+) signaling complexes, the correct sub-cellular localization of protein partners, and the regulation of the TRPC channelosome. In particular, these proteins, via their multiple protein-protein interaction motifs, can interact with various ion channels involved in the transmembrane potential, and membrane excitability. Scaffolding proteins are key components for the functional organization of TRPC channelosomes that serves as a platform regulating slow Ca(2+) entry, spatially and temporally controlled [Ca(2+)]i signals and Ca(2+) -dependent cellular functions.

Dystrophin complex functions as a scaffold for signalling proteins.

Dystrophin is a 427kDa sub-membrane cytoskeletal protein, associated with the inner surface membrane and incorporated in a large macromolecular complex of proteins, the dystrophin-associated protein complex (DAPC). In addition to dystrophin the DAPC is composed of dystroglycans, sarcoglycans, sarcospan, dystrobrevins and syntrophin. This complex is thought to play a structural role in ensuring membrane stability and force transduction during muscle contraction. The multiple binding sites and domains present in the DAPC confer the scaffold of various signalling and channel proteins, which may implicate the DAPC in regulation of signalling processes. The DAPC is thought for instance to anchor a variety of signalling molecules near their sites of action. The dystroglycan complex may participate in the transduction of extracellular-mediated signals to the muscle cytoskeleton, and ß-dystroglycan was shown to be involved in MAPK and Rac1 small GTPase signalling. More generally, dystroglycan is view as a cell surface receptor for extracellular matrix proteins. The adaptor proteins syntrophin contribute to recruit and regulate various signalling proteins such as ion channels, into a macromolecular complex. Although dystrophin and dystroglycan can be directly involved in signalling pathways, syntrophins play a central role in organizing signalplex anchored to the dystrophin scaffold. The dystrophin associated complex, can bind up to four syntrophin through binding domains of dystrophin and dystrobrevin, allowing the scaffold of multiple signalling proteins in close proximity. Multiple interactions mediated by PH and PDZ domains of syntrophin also contribute to build a complete signalplex which may include ion channels, such as voltage-gated sodium channels or TRPC cation channels, together with, trimeric G protein, G protein-coupled receptor, plasma membrane calcium pump, and NOS, to enable efficient and regulated signal transduction and ion transport. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

p210bcr-abl induces amoeboid motility by recruiting ADF/destrin through RhoA/ROCK1.

We previously demonstrated that the Bcr-Abl oncogene, p210(bcr-abl), through its unique GEF domain, specifically activates RhoA and induces spontaneous amoeboid motility. We intend to study the pathways downstream RhoA controlling amoeboid motility. Mouse prolymphoblastic cells (Ba/F3 cell line) expressing different forms of Bcr-Abl were embedded in 3-dimensional (3D) Matrigel to study motility and explore the effects of inhibiting Rho pathway (inhibitors and siRNAs). The phosphorylation levels of cofilin-1 and destrin were analyzed by 2-dimensional electrophoresis. Composition of Bcr-Abl signalplex in different conditions was determined by coimmunoprecipitation. Ba/F3p190 and Ba/F3 expressing a mutant form of p210(bcr-abl) (unable to activate RhoA) cells presented a spontaneous motility, but not an amoeboid type. p210(bcr-abl)-induced amoeboid motility in a 3D matrix requires isoform-specific RhoA/ROCK-1/destrin signaling. Next to the conventional Rho/ROCK/MLC/myosin pathway, this pathway is a crucial determinant for amoeboid motility, specific for the destrin isoform (and not its coexpressed homologue cofilin-1). Also, the presence of destrin (and not cofilin-1) in the p210(bcr-abl) complex is dependent on ROCK1, and this signalplex is required for amoeboid motility. This underscores isoform-specific function within the ADF/cofilin family and provides new insight into Bcr-Abl signaling to amoeboid motility and possible impact on understanding chronic myeloid leukemia progression.

Involvement of TRPV2 and SOCE in calcium influx disorder in DMD primary human myotubes with a specific contribution of α1-syntrophin and PLC/PKC in SOCE regulation.

Calcium homeostasis is critical for several vital functions in excitable and nonexcitable cells and has been shown to be impaired in many pathologies including Duchenne muscular dystrophy (DMD). Various studies using murine models showed the implication of calcium entry in the dystrophic phenotype. However, alteration of store-operated calcium entry (SOCE) and transient receptor potential vanilloid 2 (TRPV2)-dependant cation entry has not been investigated yet in human skeletal muscle cells. We pharmacologically characterized basal and store-operated cation entries in primary cultures of myotubes prepared from muscle of normal and DMD patients and found, for the first time, an increased SOCE in DMD myotubes. Moreover, this increase cannot be explained by an over expression of the well-known SOCE actors: TRPC1/4, Orai1, and stromal interaction molecule 1 (STIM1) mRNA and proteins. Thus we investigated the modes of regulation of this cation entry. We firstly demonstrated the important role of the scaffolding protein α1-syntrophin, which regulates SOCE in primary human myotubes through its PDZ domain. We also studied the implication of phospholipase C (PLC) and protein kinase C (PKC) in SOCE and showed that their inhibition restores normal levels of SOCE in DMD human myotubes. In addition, the involvement of TRPV2 in calcium deregulation in DMD human myotubes was explored. We showed an abnormal elevation of TRPV2-dependant cation entry in dystrophic primary human myotubes compared with normal ones. These findings show that calcium homeostasis mishandling in DMD myotubes depends on SOCE under the influence of Ca(2+)/PLC/PKC pathway and α1-syntrophin regulation as well as on TRPV2-dependant cation influx.

Neural stem cell self-renewal stimulation by store-operated calcium entries in adult mouse area postrema: influence of leptin.

Neural stem cells (NSCs) persist in specific brain germinative niches and sustain neurogenesis throughout life in adult mammals. In addition to the two major stem cell niches in the subventricular zone and the hippocampal dentate gyrus, the area postrema located in the brainstem has been identified as a neurogenic zone as well. NSCs are regulated by signals from the microenvironment that adjust stem cell response to the needs of the organism. Evidence accumulated over the past decade indicates that Ca2+ channels play pivotal functions in NSC maintenance. In this study, we explored in area postrema NSCs the presence and roles of a subset of Ca2+ channels, the store-operated Ca2+ channels (SOCs) that have the capacity to transduce extracellular signals into Ca2+ signals. Our data show that NSCs derived from the area postrema express TRPC1 and Orai1, known to form SOCs, as well as their activator STIM1. Ca2+ imaging indicated that NSCs exhibit store-operated Ca2+ entries (SOCEs). Pharmacological blockade of SOCEs with SKF-96365, YM-58483 (also known as BTP2) or GSK-7975A resulted in decreased NSC proliferation and self-renewal, indicating a major role for SOCs in maintaining NSC activity within the area postrema. Furthermore, our results show that leptin, an adipose tissue-derived hormone whose ability to control energy homeostasis is dependent on the area postrema, decreased SOCEs and reduced self-renewal of NSCs in the area postrema. As aberrant SOC function has been linked to an increasing number of diseases, including brain disorders, our study opens new perspectives for NSCs in brain pathophysiology.

Challenges in glioblastoma research: focus on the tumor microenvironment.

Glioblastoma (GBM) is the most deadly type of malignant brain tumor, despite extensive molecular analyses of GBM cells. In recent years, the tumor microenvironment (TME) has been recognized as an important player and therapeutic target in GBM. However, there is a need for a full and integrated understanding of the different cellular and molecular components involved in the GBM TME and their interactions for the development of more efficient therapies. In this review, we provide a comprehensive report of the GBM TME, which assembles the contributions of physicians and translational researchers working on brain tumor pathology and therapy in France. We propose a holistic view of the subject by delineating the specific features of the GBM TME at the cellular, molecular, and therapeutic levels.

Tissue expression and actin binding of a novel N-terminal utrophin isoform.

Utrophin and dystrophin present two large proteins that link the intracellular actin cytoskeleton to the extracellular matrix via the C-terminal-associated protein complex. Here we describe a novel short N-terminal isoform of utrophin and its protein product in various rat tissues (N-utro, 62 kDa, amino acids 1-539, comprising the actin-binding domain plus the first two spectrin repeats). Using different N-terminal recombinant utrophin fragments, we show that actin binding exhibits pronounced negative cooperativity (affinity constants K(1) = -5 × 10(6) and K(2) =-1 × 10(5 )M(-1)) and is Ca(2+)-insensitive. Expression of the different fragments in COS7 cells and in myotubes indicates that the actin-binding domain alone binds exclusively to actin filaments. The recombinant N-utro analogue binds in vitro to actin and in the cells associates to the membranes. The results indicate that N-utro may be responsible for the anchoring of the cortical actin cytoskeleton to the membranes in muscle and other tissues.

Store-Operated Calcium Channels Control Proliferation and Self-Renewal of Cancer Stem Cells from Glioblastoma.

Glioblastoma is the most frequent and deadly form of primary brain tumors. Despite multimodal treatment, more than 90% of patients experience tumor recurrence. Glioblastoma contains a small population of cells, called glioblastoma stem cells (GSC) that are highly resistant to treatment and endowed with the ability to regenerate the tumor, which accounts for tumor recurrence. Transcriptomic studies disclosed an enrichment of calcium (Ca2+) signaling transcripts in GSC. In non-excitable cells, store-operated channels (SOC) represent a major route of Ca2+ influx. As SOC regulate the self-renewal of adult neural stem cells that are possible cells of origin of GSC, we analyzed the roles of SOC in cultures of GSC previously derived from five different glioblastoma surgical specimens. Immunoblotting and immunocytochemistry experiments showed that GSC express Orai1 and TRPC1, two core SOC proteins, along with their activator STIM1. Ca2+ imaging demonstrated that SOC support Ca2+ entries in GSC. Pharmacological inhibition of SOC-dependent Ca2+ entries decreased proliferation, impaired self-renewal, and reduced expression of the stem cell marker SOX2 in GSC. Our data showing the ability of SOC inhibitors to impede GSC self-renewal paves the way for a strategy to target the cells considered responsible for conveying resistance to treatment and tumor relapse.

Regulation of TRPC1 and TRPC4 cation channels requires an alpha1-syntrophin-dependent complex in skeletal mouse myotubes.

The dystrophin-associated protein complex (DAPC) is essential for skeletal muscle, and the lack of dystrophin in Duchenne muscular dystrophy results in a reduction of DAPC components such as syntrophins and in fiber necrosis. By anchoring various molecules, the syntrophins may confer a role in cell signaling to the DAPC. Calcium disorders and abnormally elevated cation influx in dystrophic muscle cells have suggested that the DAPC regulates some sarcolemmal cationic channels. We demonstrated previously that mini-dystrophin and alpha1-syntrophin restore normal cation entry in dystrophin-deficient myotubes and that sarcolemmal TRPC1 channels associate with dystrophin and the bound PDZ domain of alpha1-syntrophin. This study shows that small interfering RNA (siRNA) silencing of alpha1-syntrophin dysregulated cation influx in myotubes. Moreover, deletion of the PDZ-containing domain prevented restoration of normal cation entry by alpha1-syntrophin transfection in dystrophin-deficient myotubes. TRPC1 and TRPC4 channels are expressed at the sarcolemma of muscle cells; forced expression or siRNA silencing showed that cation influx regulated by alpha1-syntrophin is supported by TRPC1 and TRPC4. A molecular association was found between TRPC1 and TRPC4 channels and the alpha1-syntrophin-dystrophin complex. TRPC1 and TRPC4 channels may form sarcolemmal channels anchored to the DAPC, and alpha1-syntrophin is necessary to maintain the normal regulation of TRPC-supported cation entry in skeletal muscle. Cation channels with DAPC form a signaling complex that modulates cation entry and may be crucial for normal calcium homeostasis in skeletal muscles.

alpha-Skeletal muscle actin nemaline myopathy mutants cause cell death in cultured muscle cells.

Nemaline myopathy is a neuromuscular disorder, characterized by muscle weakness and hypotonia and is, in 20% of the cases, caused by mutations in the gene encoding alpha-skeletal muscle actin, ACTA1. It is a heterogeneous disease with various clinical phenotypes and severities. In patients the ultrastructure of muscle cells is often disturbed by nemaline rods and it is thought this is the cause for muscle weakness. To search for possible defects during muscle cell differentiation we expressed alpha-actin mutants in myoblasts and allowed these cells to differentiate into myotubes. Surprisingly, we observed two striking new phenotypes in differentiating myoblasts: rounding up of cells and bleb formation, two features reminiscent of apoptosis. Indeed expression of these mutants induced cell death with apoptotic features in muscle cell culture, using AIF and endonuclease G, in a caspase-independent but calpain-dependent pathway. This is the first report on a common cellular defect induced by NM causing actin mutants, independent of their biochemical phenotypes or rod and aggregate formation capacity. These data suggest that lack of type II fibers or atrophy observed in nemaline myopathy patients may be also due to an increased number of dying muscle cells.

alpha-Skeletal muscle actin mutants causing different congenital myopathies induce similar cytoskeletal defects in cell line cultures.

Central core disease (CCD), congenital fibre type disproportion (CFTD), and nemaline myopathy (NM) are earlyonset clinically heterogeneous congenital myopathies, characterized by generalized muscle weakness and hypotonia. All three diseases are associated with alpha-skeletal muscle actin mutations. We biochemically characterized the CCD and CFTD causing actin mutants and show that all mutants fold correctly and are stable. Expression studies in fibroblasts, myoblasts, and myotubes show that these mutants incorporate in filamentous structures. However they do not intercalate between the nascent z-lines in differentiating muscle cell cultures. We also show that the distribution of mitochondria and of the ryanodine receptors, and calcium release properties from ryanodine receptors, are unchanged in myotubes expressing the CCD causing mutants. CFTD causing mutants induce partly similar phenotypes as NM associated ones, such as rods and thickened actin fibers in cell culture. Our results suggest that molecular mechanisms behind CFTD and NM may be partly related.

Calcium Channels in Adult Brain Neural Stem Cells and in Glioblastoma Stem Cells.

The brain of adult mammals, including humans, contains neural stem cells (NSCs) located within specific niches of which the ventricular-subventricular zone (V-SVZ) is the largest one. Under physiological conditions, NSCs proliferate, self-renew and produce new neurons and glial cells. Several recent studies established that oncogenic mutations in adult NSCs of the V-SVZ are responsible for the emergence of malignant primary brain tumors called glioblastoma. These aggressive tumors contain a small subpopulation of cells, the glioblastoma stem cells (GSCs), that are endowed with proliferative and self-renewal abilities like NSCs from which they may arise. GSCs are thus considered as the cells that initiate and sustain tumor growth and, because of their resistance to current treatments, provoke tumor relapse. A growing body of studies supports that Ca2+ signaling controls a variety of processes in NSCs and GSCs. Ca2+ is a ubiquitous second messenger whose fluctuations of its intracellular concentrations are handled by channels, pumps, exchangers, and Ca2+ binding proteins. The concerted action of the Ca2+ toolkit components encodes specific Ca2+ signals with defined spatio-temporal characteristics that determine the cellular responses. In this review, after a general overview of the adult brain NSCs and GSCs, we focus on the multiple roles of the Ca2+ toolkit in NSCs and discuss how GSCs hijack these mechanisms to promote tumor growth. Extensive knowledge of the role of the Ca2+ toolkit in the management of essential functions in healthy and pathological stem cells of the adult brain should help to identify promising targets for clinical applications.

Negative modulation of inositol 1,4,5-trisphosphate type 1 receptor expression prevents dystrophin-deficient muscle cells death.

Evidence for a modulatory effect of cyclosporin A (CsA) on calcium signaling and cell survival in dystrophin-deficient cells is presented. Our previous works strongly supported the hypothesis of an overactivation of Ca(2+) release via inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) in dystrophin-deficient cells, both during membrane depolarization and at rest, through spontaneous Ca(2+) release events. Forced expression of mini-dystrophin in these cells contributed, during stimulation and in resting condition, to the recovery of a controlled calcium homeostasis. In the present work, we demonstrate that CsA exposure displayed a dual-modulator effect on calcium signaling in dystrophin-deficient cells. Short-time incubation induced a decrease of IP3-dependent calcium release, leading to patterns of release similar to those observed in myotubes expressing mini-dystrophin, whereas long-time incubation reduced the expression of the type I of IP3 receptors (IP3R-1) RNA levels. Moreover, both IP3R-1 knockdown and blockade through 2-aminoethoxydiphenyle borate or CsA induced improved survival of dystrophin-deficient myotubes, demonstrating the cell death dependence on the IP3-dependent calcium signaling as well as the protective effect of CsA. Inhibition of the IP3 pathway could be a very interesting approach for reducing the natural cell death of dystrophin-deficient cells in development.

Regulation by scaffolding proteins of canonical transient receptor potential channels in striated muscle.

Recent studies proposed a pivotal role of TRPC channels, in particular TRPC1, in the striated muscle tissue and in the development of calcium mishandling observed in dystrophin-deficient skeletal and cardiac muscle cells (Vandebrouck et al. in J Cell Biol 158:1089-1096, 2002; Williams and Allen in Am J Physiol Heart Circ Physiol 292:H846-H855, 2007; Stiber et al. in Mol Cell Biol 28:2637-2647, 2008). In skeletal muscle, TRPCs are proposed to function in a costameric macromolecular complex (Vandebrouck et al. in FASEB J 21:608-617, 2007; Gervasio et al. in J Cell Sci 121:2246-2255, 2008) in which scaffolding proteins and dystrophin are central components maintaining normal calcium entry (Stiber et al. in Mol Cell Biol 28:2637-2647, 2008; Sabourin et al. in J Biol Chem 284:36248-61, 2009). In this review, we shall summarize the roles played by scaffolding proteins in regulating the calcium entry through TRPC channels of skeletal muscle cells and the implications in muscle physiopathology. Interactions of TRPC1 with caveolin-3, Homer-1 and alpha-syntrophin will be addressed and these complexes will be compared with signalplex in other systems. The mechanosensitive function of scaffolding proteins will be discussed as well as interactions with TRPV2 channels regarding to calcium mishandling in Duchenne dystrophy.