Berthier Christine - Academia.edu (original) (raw)

Papers by Berthier Christine

Journal of Biological Chemistry

Junctophilins (JPs) anchor the endo/sarcoplasmic reticulum (ER/SR) to the plasma membrane, thus c... more Junctophilins (JPs) anchor the endo/sarcoplasmic reticulum (ER/SR) to the plasma membrane, thus contributing to the assembly of junctional membrane complexes in striated muscles and neurons. Recent reports have shown that JPs may be also involved in regulating Ca2+ homeostasis. Here, we report that in skeletal muscle JP1 and JP2 are part of a complex that, in addition to Ryanodine Receptor 1 (RyR1), includes Caveolin 3 and the dihydropyridine receptor (DHPR). The interaction between JPs and DHPR was mediated by a region encompassing amino acids 230-369 and amino acids 216-399 in JP1 and JP2, respectively. Immunofluorescence studies revealed that the pattern of DHPR and RyR signals in C2C12 cells knockdown for JP1 and JP2 was rather diffused and characterized by smaller puncta in contrast to that observed in control cells. Functional experiments revealed that down-regulation of JPs in differentiated C2C12 cells resulted in a reduction of intramembrane charge movement and the L-type C...

The Journal of general physiology, 2015

Phosphoinositides act as signaling molecules in numerous cellular transduction processes, and pho... more Phosphoinositides act as signaling molecules in numerous cellular transduction processes, and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) regulates the function of several types of plasma membrane ion channels. We investigated the potential role of PtdIns(4,5)P2 in Ca(2+) homeostasis and excitation-contraction (E-C) coupling of mouse muscle fibers using in vivo expression of the voltage-sensing phosphatases (VSPs) Ciona intestinalis VSP (Ci-VSP) or Danio rerio VSP (Dr-VSP). Confocal images of enhanced green fluorescent protein-tagged Dr-VSP revealed a banded pattern consistent with VSP localization within the transverse tubule membrane. Rhod-2 Ca(2+) transients generated by 0.5-s-long voltage-clamp depolarizing pulses sufficient to elicit Ca(2+) release from the sarcoplasmic reticulum (SR) but below the range at which VSPs are activated were unaffected by the presence of the VSPs. However, in Ci-VSP-expressing fibers challenged by 5-s-long depolarizing pulses, the Ca(2+) l...

Journal of cell science, 1999

Vertebrate tropomyosins (TMs) are expressed from four genes, and at least 18 distinct isoforms ar... more Vertebrate tropomyosins (TMs) are expressed from four genes, and at least 18 distinct isoforms are generated via a complex pattern of alternative RNA splicing and alternative promoters. The functional significance of this isoform diversity is largely unknown and it remains to be determined whether specific isoforms are required for assembly and integration into distinct actin-containing structures. The ability of nonmuscle (TM-1, -2, -3, -4, -5(NM1), -5a or -5b) and striated muscle (skeletal muscle (&agr;)-TM) isoforms to incorporate into actin filaments of neonatal rat cardiomyocytes (NRCs) was studied using expression plasmids containing TM-fusions with GFP (green fluorescent protein) as well as with VSV- or HA-epitope tags. All isoforms, except of fibroblast TM-4, were able to incorporate into the I-band of NRCs. When TM-4 was co-transfected with other low molecular weight (LMW) isoforms of TM (TM-5, TM-5a and TM-5b), it was able to incorporate into sarcomeres of NRCs. This resul...

The Journal of Physiology, 2007

The physiological properties and role of the type 3 ryanodine receptor (RyR3), a calcium release ... more The physiological properties and role of the type 3 ryanodine receptor (RyR3), a calcium release channel expressed in a wide variety of cell types, remain mysterious. We forced, in vivo, the expression of RyR3 in adult mouse skeletal muscle fibres using a GFP-RyR3 DNA construct. GFP fluorescence was found within spatially restricted regions of muscle fibres where it exhibited a sarcomere-related banded pattern consistent with a localization within or near the junctional sarcoplasmic reticulum membrane. Immunostaining confirmed the presence of RyR3 together with RyR1 within the GFP-positive areas. In ∼90% of RyR3-positive fibres microinjected with the calcium indicator fluo-3, we detected repetitive spontaneous transient elevations of intracellular Ca 2+ that persisted when fibres were voltage-clamped at −80 mV. These Ca 2+ transients remained essentially confined to the RyR3 expression region. They ranged from wide local events to propagating Ca 2+ waves and were in some cases associated with local contractile activity. When voltage-clamp depolarizations were applied while fluo-3 or rhod-2 fluorescence was measured within the RyR3-expressing region, no voltage-evoked 'spark-like' elementary Ca 2+ release event could be detected. Still global voltage-activated Ca 2+ release exhibited a prominent early peak within the RyR3-expressing regions. Measurements were also taken from muscles fibres expressing a GFP-RyR1 construct; positive fibres also yielded a local banded pattern of GFP fluorescence but exhibited no spontaneous Ca 2+ release. Results demonstrate that RyR3 is a very potent source of voltage-independent Ca 2+ release activity. Conversely we find no evidence that it could contribute to the production of discrete voltage-activated Ca 2+ release events in differentiated mammalian skeletal muscle.

The Journal of Physiology, 2007

Caveolins are membrane scaffolding proteins that associate with and regulate a variety of signall... more Caveolins are membrane scaffolding proteins that associate with and regulate a variety of signalling proteins, including ion channels. A deficiency in caveolin-3 (Cav-3), the major striated muscle isoform, is responsible for skeletal muscle disorders, such as limb-girdle muscular dystrophy 1C (LGMD 1C). The molecular mechanisms leading to the muscle wasting that characterizes this pathology are poorly understood. Here we show that a loss of Cav-3 induced by the expression of the LGMD 1C-associated mutant P104L (Cav-3 P104L ) provokes a reduction by half of the maximal conductance of the voltage-dependent L-type Ca 2+ channel in mouse primary cultured myotubes and fetal skeletal muscle fibres. Confocal immunomiscrocopy indicated a colocalization of Cav-3 and Ca v 1.1, the pore-forming subunit of the L-type Ca 2+ channel, at the surface membrane and in the developing T-tubule network in control myotubes and fetal fibres. In myotubes expressing Cav-3 P104L , the loss of Cav-3 was accompanied by a 66% reduction in Ca v 1.1 mean labelling intensity. Our results suggest that Cav-3 is involved in L-type Ca 2+ channel membrane function and localization in skeletal muscle cells and that an alteration of L-type Ca 2+ channels could be involved in the physiopathological mechanisms of caveolinopathies.

The Journal of Physiology, 2004

Caveolae and transverse (T-) tubules are membrane structures enriched in cholesterol and glycosph... more Caveolae and transverse (T-) tubules are membrane structures enriched in cholesterol and glycosphingolipids. They play an important role in receptor signalling and myogenesis. The Tsystemisalsohighlyenrichedindihydropyridinereceptors(DHPRs),whichcontrolexcitationcontraction (E-C) coupling. Recent results have shown that a depletion of membrane cholesterol alters caveolae and T-tubules, yet detailed functional studies of DHPR expression are lacking. Here we studied electrophysiological and morphological effects of methyl-βcyclodextrin (MβCD), a cholesterol-sequestering drug, on freshly isolated fetal skeletal muscle cells. Exposure of fetal myofibres to 1-3 mm MβCD for 1 h at 37 • C led to a significant reduction in caveolae and T-tubule areas and to a decrease in cell membrane electrical capacitance. In whole-cell voltage-clamp experiments, the L-type Ca 2+ current amplitude was significantly reduced, and its voltage dependence was shifted ∼15 mV towards more positive potentials. Activation and inactivation kinetics were slower in treated cells than in control cells and stimulation by a saturating concentration of Bay K 8644 was enhanced. In addition, intramembrane charge movement and Ca 2+ transients evoked by a depolarization were reduced without a shift of the midpoint, indicating a weakening of E-C coupling. In contrast, T-type Ca 2+ current was not affected by MβCD treatment. Most of the L-type Ca 2+ conductance reduction and E-C coupling weakening could be explained by a decrease of the number of DHPRs due to the disruption of caveolae and T-tubules. However, the effects on L-type channel gating kinetics suggest that membrane cholesterol content modulates DHPR function. Moreover, the significant shift of the voltage dependence of L-type current without any change in the voltage dependence of charge movement and Ca 2+ transients suggests that cholesterol differentially regulates the two functions of the DHPR.

Pfl�gers Archiv European Journal of Physiology, 2001

The membrane cytoskeleton is increasingly considered as both an anchor and a functional modulator... more The membrane cytoskeleton is increasingly considered as both an anchor and a functional modulator for ion channels. The cytoskeletal disruptions that occur in the absence of dystrophin led us to investigate the voltage-gated sodium channel (SkM1) content in the extensor digitorum longus (EDL) muscle of the dystrophindeficient mdx mouse. Levels of SkM1 mRNA were determined by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). A C-terminal portion of the mouse-specific SkM1 α-subunit cDNA (mScn4a) was identified first. SkM1 mRNA levels were as abundant in mdx as in normal muscle, thus suggesting that the transcriptional rate of SkM1 remains unchanged in mdx muscle. However, SkM1 density in the extrajunctional sarcolemma was shown to be significantly reduced in mdx muscle, using confocal immunofluorescence image analysis. This decrease was found to be associated with a reduction in the number of SkM1-rich fast-twitch IIb fibres in mdx muscle. In addition, lowered SkM1 sarcolemmal labelling was found in all mdx fibres regardless of their metabolic type. These results suggest the existence of a perturbation of SkM1 anchorage to the plasma membrane. Such an alteration is likely to be related to the 50% decrease in mdx muscle of the dystrophinassociated syntrophins, which are presumed to be involved in SkM1 anchorage. However, the moderate reduction in SkM1 density (-12.7%) observed in mdx muscle argues in favour of a non-exclusive role of syntrophins in SkM1 anchorage and suggests that other membrane-associated proteins are probably also involved.

Molecular Biology of the Cell, 1999

Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca 2ϩ -calmodulin-regulated ... more Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca 2ϩ -calmodulin-regulated manner. Although a nonmuscle isoform of caldesmon is widely expressed, its functional role has not yet been elucidated. We studied the effects of nonmuscle caldesmon on cellular contractility, actin cytoskeletal organization, and the formation of focal adhesions in fibroblasts. Transient transfection of nonmuscle caldesmon prevents myosin II-dependent cell contractility and induces a decrease in the number and size of tyrosine-phosphorylated focal adhesions. Expression of caldesmon interferes with Rho A-V14-mediated formation of focal adhesions and stress fibers as well as with formation of focal adhesions induced by microtubule disruption. This inhibitory effect depends on the actin-and myosin-binding regions of caldesmon, because a truncated variant lacking both of these regions is inactive. The effects of caldesmon are blocked by the ionophore A23187, thapsigargin, and membrane depolarization, presumably because of the ability of Ca 2ϩ -calmodulin or Ca 2ϩ -S100 proteins to antagonize the inhibitory function of caldesmon on actomyosin contraction. These results indicate a role for nonmuscle caldesmon in the physiological regulation of actomyosin contractility and adhesion-dependent signaling and further demonstrate the involvement of contractility in focal adhesion formation.

Journal of Muscle Research and Cell Motility, 1995

The ultrastructural organization of the highly interconnected filamentous network underneath the ... more The ultrastructural organization of the highly interconnected filamentous network underneath the sarcolemma as well as the role played by the muscle protein dystrophin within this cytoskeleton remain yet unclear. More accurate information has been obtained by using a method which provides three-dimensional en face views of large membrane areas applied to mouse cultured myotubes and isolated adult skeletal muscle fibres. Two levels have been distinguished in the cytoskeleton underlying the sarcolemma: the submembranous level, partly integrated into the membrane, and the cortical level, invading the proximal cytoplasmic space. Few differences have been found between the membrane cytoskeletons of myotubes issued from 14-day-old cultures and those of adult fibres. The comparison was done with cells where dystrophin is missing (mdx mouse muscle): surprisingly, the lack of dystrophin does not induce obvious or dramatic ultrastructural disorganization, either in the cortical cytoskeletal network or in the submembranous one. Immunogold labelling of either the central-rod or the C-terminal domain of dystrophin is not located among the cortical network. This study provides additional data on the spatial ordering of subsarcolemmal cytoskeletal elements: dystrophin does not appear as a filamentous structure entirely located among subsarcolemmal cytoskeleton but seems partly embedded in membranous material.

The Journal of General Physiology, 2012

Journal of Biological Chemistry, 2011

Background: Junctophilins form junctional membrane complexes between the sarcoplasmic reticulum a... more Background: Junctophilins form junctional membrane complexes between the sarcoplasmic reticulum and the plasma membrane in skeletal muscle. Results: Junctophilins interact with both the dihydropyridine receptors and the ryanodine receptor Ca 2ϩ channels. Conclusion: Assembly of junctional proteins in junctional membrane complexes is facilitated by Junctophilins. Significance: This suggests that junctophilins have multiple roles in the assembly of Ca 2ϩ release units in muscle cells.

Biophysical Journal, 2011

receptor is primarily activated by the tachykinin peptide hormone Neurokinin B (NKB), the most po... more receptor is primarily activated by the tachykinin peptide hormone Neurokinin B (NKB), the most potent natural agonist for the NK-3 receptor. NKB has been reported to play a vital role in the normal human reproduction pathway, potentially life threatening disease such as pre eclampsia and as a neuroprotective agent in case of neurodegenerative diseases. Agonist binding to the receptor is a crucial event in initiating signaling and therefore characterization of the structural features of the agonists can reveal the molecular basis of receptor activation and help in rational design of novel therapeutics. In this study a molecular model for the interaction of the primary ligand NKB with its G-protein coupled receptor NK-3 has been developed. A three-dimensional model for the NK-3 receptor has been generated by homology modeling using rhodopsin as a template. A knowledge based docking of the NMR derived bioactive conformation of NKB to the receptor has been performed utilizing limited ligand binding data obtained from the photo-affinity labeling and site-directed mutagenesis studies. A molecular model for the NKB-NK-3 receptor complex thus obtained sheds light on the topographical features of the binding pocket of the receptor and provides insight into the biochemical data currently available for the receptor. The results of the receptor modeling studies have been used to discuss the molecular determinants for NK-3 receptor selectivity. Junctophilins (JPs) play a central role in anchoring the sarcoplasmic reticulum (SR) to the sarcolemma, but also interact with several proteins of the SR and the plasma membrane. We have observed an interaction between JP1 and JP2 and the dihydropyridine receptor (DHPR) in both co-immunoprecipitation and pulldown experiments. This was further supported by immunofluorescence experiments, where Knockdown of JP1 and JP2 appeared to affect the assembly of junctional proteins. Functional experiments performed under voltage-clamp conditions revealed that Knockdown of JPs in cultured myotubes heavily depressed the Ca2þ channel function of the DHPR: JPs Knockdown-positive cells yielded a reduction of the L-type Ca2þ current density that corresponded to an average 50 % reduction of the peak conductance and was accompanied by an~10 mV right-shift of the voltage-dependence of channel activation. Interestingly, measurements of intracellular Ca2þ transients with the dye rhod-2 showed that there was no substantial alteration of the peak SR Ca2þ release following Knock-down of JPs. Knockdown of the JP proteins in adult fibers also appeared to alter the Ca2þ channel function of the DHPR, though to a lesser extent than in myotubes. The decrease in adult fibers corresponded to an average~25 % reduction of the peak conductance. As in the myotubes, voltage-activated Ca2þ transients appeared unaffected. Altogether these results suggest that JPs are involved in the organization of a structural and functional platform necessary for the assembly of the e-c coupling components, where their interaction with RyR1 and DHPR may be relevant to the functional cross-talk between plasma membrane channels and intracellular channels in ensuring proper function of the e-c coupling machinery.

Biophysical Journal, 2013

to determine how these processes are involved during long-lasting depolarizations of isolated mou... more to determine how these processes are involved during long-lasting depolarizations of isolated mouse muscle fibers under voltage control by measuring cytosolic Ca 2þ changes using fura-2 or luminal SR Ca 2þ changes using fluo5N in the presence of 50 mM internal EGTA. Decays of cytosolic Ca 2þ signals elicited by 50-s duration depolarizations became more marked and faster with depolarization amplitude. Pre-depolarizations of 2-min duration and of increasing amplitude induced a reduction of voltage-activated cytosolic Ca 2þ signals with a mean voltage of À50 mV inducing half-maximum reduction. A comparable protocol applied to fibers loaded with fluo5N showed that low voltage depolarizing prepulses induced a marked SR Ca 2þ depletion that contributed to reduce a subsequent voltage-activated SR Ca 2þ change with a mean voltage of À50 mV inducing half-maximum reduction. Measuring SR Ca 2þ changes in response to long-lasting depolarizations indicated that SR Ca 2þ release channels inactivated in response to much higher depolarizations with a mean halfmaximum inactivation voltage of À20 mV. Finally, trains of action potential of 50 s duration produced cytosolic Ca 2þ signals that decayed with time, whereas SR Ca 2þ changes did not display any sign of inactivation. These results indicate that the decline in SR Ca 2þ release during long-lasting depolarizations mainly results from SR Ca 2þ depletion. The work was supported by AFM, CNRS and University Lyon 1. of. Stromal interaction molecule 1 (STIM1) mediates store-operated Ca 2þ entry (SOCE) in skeletal muscle. However, the direct role(s) of STIM1 in the innate skeletal muscle event such as the Ca 2þ release from the sarcoplasmic reticulum (SR) for muscle contraction have not been identified. In the present study, wildtype STIM1 and two STIM1 mutants (the Triple mutant, missing Ca 2þ -sensing residues, and E136X, missing the C-terminus) were over-expressed in mouse primary skeletal myotubes. The wild-type STIM1 increased SOCE, while neither mutant had an effect on SOCE. Interestingly, the development of puncta by endogenous STIM1 and Orai1 was detected without any stimulus during the differentiation of myoblasts to myotubes, and increased puncta formation was observed in the triple mutant as well as the wild-type STIM1, suggesting that, in skeletal muscle, the formation of puncta is part of the differentiation process and not the necessary and sufficient condition for SOCE. On the other hand, the Triple mutant, but not E136X, decreased the Ca 2þ release from the SR in response to KCl in a dominant-negative manner without affecting the SR Ca 2þ amount or resting Ca 2þ level. STIM1 was co-immunoprecipitated with the dihydropyridine receptor (DHRP). These results suggest that STIM1 could negatively regulate the Ca 2þ release from the SR, possibly via its C-terminal interaction with DHPR. Mitsugumin 53 (MG53) is a member of membrane repair system in skeletal muscle. However, role(s) of MG53 in unique functions of skeletal muscle has not been addressed although MG53 is expressed only in skeletal and cardiac muscle. In the present study, MG53-binding proteins were searched among proteins mediating skeletal muscle contraction and relaxation using the binding assays of various MG53 domains and quadrupole time-of-flight mass spectrometry. MG53 binds to sarcoplasmic reticulum Ca 2þ -ATPase 1a (SERCA1a) via its tripartite motif (TRIM) and PRY domains. The binding was confirmed in rabbit skeletal muscle and mouse primary skeletal myotubes by coimmunoprecipitation and immunocytochemistry. MG53 knock-down in mouse primary skeletal myotubes increased Ca 2þ -uptake through SERCA1a (more than 35%) at micromolar Ca 2þ but not at nanomolar Ca 2þ , suggesting that MG53 attenuates SERCA1a activity possibly during skeletal muscle contraction or relaxation but not during the resting state of skeletal muscle. In-silico studies suggest that the binding of MG53 to SERCA1a is mediated by unique ways compared with bindings by other proteins containing TRIM or PRY domains.

Biology of the Cell, 1997

This review is focused on the composition and organization of the junctional subsarcolemmal cytos... more This review is focused on the composition and organization of the junctional subsarcolemmal cytoskeleton of adult muscle fibers. The cytoskeleton of muscle fibers is organized in functionally distinct compartments and the subsarcolemmal cytoskeleton itself can be broadly divided into junctional (myotendinous junction, neuromuscular junction and costameres) and non-junctional domains. In junctional zones three different multimolecular cytoskeletal complexes coexist: the focal adhesion-type, the spectrin-based and the dystrophin vs utrophin-based membrane skeleton systems. These complexes extend over several levels, from intracytoplasmic to subsarcolemmal and transmembranous; their common feature is the anchorage of actin filaments emanating from the intracytoplasmic level. The different cytoskeletal proteins, their putative roles and their interactions with various signaling pathways are presented here in detail. The subsarcolemmal cytoskeleton complexes are thought to play distinct physiological roles (membrane stabilization, force transmission to extracellular matrix, ionic channel anchorage, etc) but their colocalization on the three sarcolemmal junctional domains strongly suggests interrelated or common functions.

Biology of the Cell, 1992

Biology of the Cell, 1993

The Journal of Physiology, 2004

In skeletal muscle, nitric oxide (NO) is commonly referred to as a modulator of the activity of t... more In skeletal muscle, nitric oxide (NO) is commonly referred to as a modulator of the activity of the ryanodine receptor (RyR) calcium release channel. However the reported effects of NO on isolated sarcoplasmic reticulum (SR) preparations and single ryanodine receptor (RyR) activity are diverse, and how NO affects SR calcium release and intracellular calcium homeostasis under physiological conditions remains poorly documented and hardly predictable. Here, we studied the effects of NO donors on membrane current and intracellular [Ca 2+ ] in single skeletal muscle fibres from mouse, under voltage-clamp conditions. When fibres were chronically exposed to millimolar levels of sodium nitroprusside (SNP) and challenged by short membrane depolarizations, there was a progressive increase in the resting [Ca 2+ ] level. This effect was use-dependent with the slope of rise in resting [Ca 2+ ] being increased two-fold when the depolarizing pulse level was raised from −20 to +10 mV. Analysis of the decay of the [Ca 2+ ] transients suggested that cytoplasmic Ca 2+ removal processes were largely unaffected by the presence of SNP. Also the functional properties of the dihydropyridine receptor were very similar under control conditions and in the presence of SNP. The resting [Ca 2+ ] elevation due to SNP was accompanied by a depression of the peak calcium release elicited by pulses to +10 mV. The effects of SNP could be reproduced by the chemically distinct NO donor NOC-12. They could be reversed upon exposure of the fibres to the thiol reducing agent dithiothreitol. Results suggest that large levels of NO produce a redox-sensitive continuous leak of Ca 2+ from the SR, through a limited number of release channels that do not close once they are activated by membrane depolarization. This SR Ca 2+ leak and the resulting increase in resting [Ca 2+ ] may be important in mediating the effects of excess NO on voltage-activated calcium release.

Journal of Biological Chemistry

Junctophilins (JPs) anchor the endo/sarcoplasmic reticulum (ER/SR) to the plasma membrane, thus c... more Junctophilins (JPs) anchor the endo/sarcoplasmic reticulum (ER/SR) to the plasma membrane, thus contributing to the assembly of junctional membrane complexes in striated muscles and neurons. Recent reports have shown that JPs may be also involved in regulating Ca2+ homeostasis. Here, we report that in skeletal muscle JP1 and JP2 are part of a complex that, in addition to Ryanodine Receptor 1 (RyR1), includes Caveolin 3 and the dihydropyridine receptor (DHPR). The interaction between JPs and DHPR was mediated by a region encompassing amino acids 230-369 and amino acids 216-399 in JP1 and JP2, respectively. Immunofluorescence studies revealed that the pattern of DHPR and RyR signals in C2C12 cells knockdown for JP1 and JP2 was rather diffused and characterized by smaller puncta in contrast to that observed in control cells. Functional experiments revealed that down-regulation of JPs in differentiated C2C12 cells resulted in a reduction of intramembrane charge movement and the L-type C...

The Journal of general physiology, 2015

Phosphoinositides act as signaling molecules in numerous cellular transduction processes, and pho... more Phosphoinositides act as signaling molecules in numerous cellular transduction processes, and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) regulates the function of several types of plasma membrane ion channels. We investigated the potential role of PtdIns(4,5)P2 in Ca(2+) homeostasis and excitation-contraction (E-C) coupling of mouse muscle fibers using in vivo expression of the voltage-sensing phosphatases (VSPs) Ciona intestinalis VSP (Ci-VSP) or Danio rerio VSP (Dr-VSP). Confocal images of enhanced green fluorescent protein-tagged Dr-VSP revealed a banded pattern consistent with VSP localization within the transverse tubule membrane. Rhod-2 Ca(2+) transients generated by 0.5-s-long voltage-clamp depolarizing pulses sufficient to elicit Ca(2+) release from the sarcoplasmic reticulum (SR) but below the range at which VSPs are activated were unaffected by the presence of the VSPs. However, in Ci-VSP-expressing fibers challenged by 5-s-long depolarizing pulses, the Ca(2+) l...

Journal of cell science, 1999

Vertebrate tropomyosins (TMs) are expressed from four genes, and at least 18 distinct isoforms ar... more Vertebrate tropomyosins (TMs) are expressed from four genes, and at least 18 distinct isoforms are generated via a complex pattern of alternative RNA splicing and alternative promoters. The functional significance of this isoform diversity is largely unknown and it remains to be determined whether specific isoforms are required for assembly and integration into distinct actin-containing structures. The ability of nonmuscle (TM-1, -2, -3, -4, -5(NM1), -5a or -5b) and striated muscle (skeletal muscle (&agr;)-TM) isoforms to incorporate into actin filaments of neonatal rat cardiomyocytes (NRCs) was studied using expression plasmids containing TM-fusions with GFP (green fluorescent protein) as well as with VSV- or HA-epitope tags. All isoforms, except of fibroblast TM-4, were able to incorporate into the I-band of NRCs. When TM-4 was co-transfected with other low molecular weight (LMW) isoforms of TM (TM-5, TM-5a and TM-5b), it was able to incorporate into sarcomeres of NRCs. This resul...

The Journal of Physiology, 2007

The physiological properties and role of the type 3 ryanodine receptor (RyR3), a calcium release ... more The physiological properties and role of the type 3 ryanodine receptor (RyR3), a calcium release channel expressed in a wide variety of cell types, remain mysterious. We forced, in vivo, the expression of RyR3 in adult mouse skeletal muscle fibres using a GFP-RyR3 DNA construct. GFP fluorescence was found within spatially restricted regions of muscle fibres where it exhibited a sarcomere-related banded pattern consistent with a localization within or near the junctional sarcoplasmic reticulum membrane. Immunostaining confirmed the presence of RyR3 together with RyR1 within the GFP-positive areas. In ∼90% of RyR3-positive fibres microinjected with the calcium indicator fluo-3, we detected repetitive spontaneous transient elevations of intracellular Ca 2+ that persisted when fibres were voltage-clamped at −80 mV. These Ca 2+ transients remained essentially confined to the RyR3 expression region. They ranged from wide local events to propagating Ca 2+ waves and were in some cases associated with local contractile activity. When voltage-clamp depolarizations were applied while fluo-3 or rhod-2 fluorescence was measured within the RyR3-expressing region, no voltage-evoked 'spark-like' elementary Ca 2+ release event could be detected. Still global voltage-activated Ca 2+ release exhibited a prominent early peak within the RyR3-expressing regions. Measurements were also taken from muscles fibres expressing a GFP-RyR1 construct; positive fibres also yielded a local banded pattern of GFP fluorescence but exhibited no spontaneous Ca 2+ release. Results demonstrate that RyR3 is a very potent source of voltage-independent Ca 2+ release activity. Conversely we find no evidence that it could contribute to the production of discrete voltage-activated Ca 2+ release events in differentiated mammalian skeletal muscle.

The Journal of Physiology, 2007

Caveolins are membrane scaffolding proteins that associate with and regulate a variety of signall... more Caveolins are membrane scaffolding proteins that associate with and regulate a variety of signalling proteins, including ion channels. A deficiency in caveolin-3 (Cav-3), the major striated muscle isoform, is responsible for skeletal muscle disorders, such as limb-girdle muscular dystrophy 1C (LGMD 1C). The molecular mechanisms leading to the muscle wasting that characterizes this pathology are poorly understood. Here we show that a loss of Cav-3 induced by the expression of the LGMD 1C-associated mutant P104L (Cav-3 P104L ) provokes a reduction by half of the maximal conductance of the voltage-dependent L-type Ca 2+ channel in mouse primary cultured myotubes and fetal skeletal muscle fibres. Confocal immunomiscrocopy indicated a colocalization of Cav-3 and Ca v 1.1, the pore-forming subunit of the L-type Ca 2+ channel, at the surface membrane and in the developing T-tubule network in control myotubes and fetal fibres. In myotubes expressing Cav-3 P104L , the loss of Cav-3 was accompanied by a 66% reduction in Ca v 1.1 mean labelling intensity. Our results suggest that Cav-3 is involved in L-type Ca 2+ channel membrane function and localization in skeletal muscle cells and that an alteration of L-type Ca 2+ channels could be involved in the physiopathological mechanisms of caveolinopathies.

The Journal of Physiology, 2004

Caveolae and transverse (T-) tubules are membrane structures enriched in cholesterol and glycosph... more Caveolae and transverse (T-) tubules are membrane structures enriched in cholesterol and glycosphingolipids. They play an important role in receptor signalling and myogenesis. The Tsystemisalsohighlyenrichedindihydropyridinereceptors(DHPRs),whichcontrolexcitationcontraction (E-C) coupling. Recent results have shown that a depletion of membrane cholesterol alters caveolae and T-tubules, yet detailed functional studies of DHPR expression are lacking. Here we studied electrophysiological and morphological effects of methyl-βcyclodextrin (MβCD), a cholesterol-sequestering drug, on freshly isolated fetal skeletal muscle cells. Exposure of fetal myofibres to 1-3 mm MβCD for 1 h at 37 • C led to a significant reduction in caveolae and T-tubule areas and to a decrease in cell membrane electrical capacitance. In whole-cell voltage-clamp experiments, the L-type Ca 2+ current amplitude was significantly reduced, and its voltage dependence was shifted ∼15 mV towards more positive potentials. Activation and inactivation kinetics were slower in treated cells than in control cells and stimulation by a saturating concentration of Bay K 8644 was enhanced. In addition, intramembrane charge movement and Ca 2+ transients evoked by a depolarization were reduced without a shift of the midpoint, indicating a weakening of E-C coupling. In contrast, T-type Ca 2+ current was not affected by MβCD treatment. Most of the L-type Ca 2+ conductance reduction and E-C coupling weakening could be explained by a decrease of the number of DHPRs due to the disruption of caveolae and T-tubules. However, the effects on L-type channel gating kinetics suggest that membrane cholesterol content modulates DHPR function. Moreover, the significant shift of the voltage dependence of L-type current without any change in the voltage dependence of charge movement and Ca 2+ transients suggests that cholesterol differentially regulates the two functions of the DHPR.

Pfl�gers Archiv European Journal of Physiology, 2001

The membrane cytoskeleton is increasingly considered as both an anchor and a functional modulator... more The membrane cytoskeleton is increasingly considered as both an anchor and a functional modulator for ion channels. The cytoskeletal disruptions that occur in the absence of dystrophin led us to investigate the voltage-gated sodium channel (SkM1) content in the extensor digitorum longus (EDL) muscle of the dystrophindeficient mdx mouse. Levels of SkM1 mRNA were determined by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). A C-terminal portion of the mouse-specific SkM1 α-subunit cDNA (mScn4a) was identified first. SkM1 mRNA levels were as abundant in mdx as in normal muscle, thus suggesting that the transcriptional rate of SkM1 remains unchanged in mdx muscle. However, SkM1 density in the extrajunctional sarcolemma was shown to be significantly reduced in mdx muscle, using confocal immunofluorescence image analysis. This decrease was found to be associated with a reduction in the number of SkM1-rich fast-twitch IIb fibres in mdx muscle. In addition, lowered SkM1 sarcolemmal labelling was found in all mdx fibres regardless of their metabolic type. These results suggest the existence of a perturbation of SkM1 anchorage to the plasma membrane. Such an alteration is likely to be related to the 50% decrease in mdx muscle of the dystrophinassociated syntrophins, which are presumed to be involved in SkM1 anchorage. However, the moderate reduction in SkM1 density (-12.7%) observed in mdx muscle argues in favour of a non-exclusive role of syntrophins in SkM1 anchorage and suggests that other membrane-associated proteins are probably also involved.

Molecular Biology of the Cell, 1999

Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca 2ϩ -calmodulin-regulated ... more Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca 2ϩ -calmodulin-regulated manner. Although a nonmuscle isoform of caldesmon is widely expressed, its functional role has not yet been elucidated. We studied the effects of nonmuscle caldesmon on cellular contractility, actin cytoskeletal organization, and the formation of focal adhesions in fibroblasts. Transient transfection of nonmuscle caldesmon prevents myosin II-dependent cell contractility and induces a decrease in the number and size of tyrosine-phosphorylated focal adhesions. Expression of caldesmon interferes with Rho A-V14-mediated formation of focal adhesions and stress fibers as well as with formation of focal adhesions induced by microtubule disruption. This inhibitory effect depends on the actin-and myosin-binding regions of caldesmon, because a truncated variant lacking both of these regions is inactive. The effects of caldesmon are blocked by the ionophore A23187, thapsigargin, and membrane depolarization, presumably because of the ability of Ca 2ϩ -calmodulin or Ca 2ϩ -S100 proteins to antagonize the inhibitory function of caldesmon on actomyosin contraction. These results indicate a role for nonmuscle caldesmon in the physiological regulation of actomyosin contractility and adhesion-dependent signaling and further demonstrate the involvement of contractility in focal adhesion formation.

Journal of Muscle Research and Cell Motility, 1995

The ultrastructural organization of the highly interconnected filamentous network underneath the ... more The ultrastructural organization of the highly interconnected filamentous network underneath the sarcolemma as well as the role played by the muscle protein dystrophin within this cytoskeleton remain yet unclear. More accurate information has been obtained by using a method which provides three-dimensional en face views of large membrane areas applied to mouse cultured myotubes and isolated adult skeletal muscle fibres. Two levels have been distinguished in the cytoskeleton underlying the sarcolemma: the submembranous level, partly integrated into the membrane, and the cortical level, invading the proximal cytoplasmic space. Few differences have been found between the membrane cytoskeletons of myotubes issued from 14-day-old cultures and those of adult fibres. The comparison was done with cells where dystrophin is missing (mdx mouse muscle): surprisingly, the lack of dystrophin does not induce obvious or dramatic ultrastructural disorganization, either in the cortical cytoskeletal network or in the submembranous one. Immunogold labelling of either the central-rod or the C-terminal domain of dystrophin is not located among the cortical network. This study provides additional data on the spatial ordering of subsarcolemmal cytoskeletal elements: dystrophin does not appear as a filamentous structure entirely located among subsarcolemmal cytoskeleton but seems partly embedded in membranous material.

The Journal of General Physiology, 2012

Journal of Biological Chemistry, 2011

Background: Junctophilins form junctional membrane complexes between the sarcoplasmic reticulum a... more Background: Junctophilins form junctional membrane complexes between the sarcoplasmic reticulum and the plasma membrane in skeletal muscle. Results: Junctophilins interact with both the dihydropyridine receptors and the ryanodine receptor Ca 2ϩ channels. Conclusion: Assembly of junctional proteins in junctional membrane complexes is facilitated by Junctophilins. Significance: This suggests that junctophilins have multiple roles in the assembly of Ca 2ϩ release units in muscle cells.

Biophysical Journal, 2011

receptor is primarily activated by the tachykinin peptide hormone Neurokinin B (NKB), the most po... more receptor is primarily activated by the tachykinin peptide hormone Neurokinin B (NKB), the most potent natural agonist for the NK-3 receptor. NKB has been reported to play a vital role in the normal human reproduction pathway, potentially life threatening disease such as pre eclampsia and as a neuroprotective agent in case of neurodegenerative diseases. Agonist binding to the receptor is a crucial event in initiating signaling and therefore characterization of the structural features of the agonists can reveal the molecular basis of receptor activation and help in rational design of novel therapeutics. In this study a molecular model for the interaction of the primary ligand NKB with its G-protein coupled receptor NK-3 has been developed. A three-dimensional model for the NK-3 receptor has been generated by homology modeling using rhodopsin as a template. A knowledge based docking of the NMR derived bioactive conformation of NKB to the receptor has been performed utilizing limited ligand binding data obtained from the photo-affinity labeling and site-directed mutagenesis studies. A molecular model for the NKB-NK-3 receptor complex thus obtained sheds light on the topographical features of the binding pocket of the receptor and provides insight into the biochemical data currently available for the receptor. The results of the receptor modeling studies have been used to discuss the molecular determinants for NK-3 receptor selectivity. Junctophilins (JPs) play a central role in anchoring the sarcoplasmic reticulum (SR) to the sarcolemma, but also interact with several proteins of the SR and the plasma membrane. We have observed an interaction between JP1 and JP2 and the dihydropyridine receptor (DHPR) in both co-immunoprecipitation and pulldown experiments. This was further supported by immunofluorescence experiments, where Knockdown of JP1 and JP2 appeared to affect the assembly of junctional proteins. Functional experiments performed under voltage-clamp conditions revealed that Knockdown of JPs in cultured myotubes heavily depressed the Ca2þ channel function of the DHPR: JPs Knockdown-positive cells yielded a reduction of the L-type Ca2þ current density that corresponded to an average 50 % reduction of the peak conductance and was accompanied by an~10 mV right-shift of the voltage-dependence of channel activation. Interestingly, measurements of intracellular Ca2þ transients with the dye rhod-2 showed that there was no substantial alteration of the peak SR Ca2þ release following Knock-down of JPs. Knockdown of the JP proteins in adult fibers also appeared to alter the Ca2þ channel function of the DHPR, though to a lesser extent than in myotubes. The decrease in adult fibers corresponded to an average~25 % reduction of the peak conductance. As in the myotubes, voltage-activated Ca2þ transients appeared unaffected. Altogether these results suggest that JPs are involved in the organization of a structural and functional platform necessary for the assembly of the e-c coupling components, where their interaction with RyR1 and DHPR may be relevant to the functional cross-talk between plasma membrane channels and intracellular channels in ensuring proper function of the e-c coupling machinery.

Biophysical Journal, 2013

to determine how these processes are involved during long-lasting depolarizations of isolated mou... more to determine how these processes are involved during long-lasting depolarizations of isolated mouse muscle fibers under voltage control by measuring cytosolic Ca 2þ changes using fura-2 or luminal SR Ca 2þ changes using fluo5N in the presence of 50 mM internal EGTA. Decays of cytosolic Ca 2þ signals elicited by 50-s duration depolarizations became more marked and faster with depolarization amplitude. Pre-depolarizations of 2-min duration and of increasing amplitude induced a reduction of voltage-activated cytosolic Ca 2þ signals with a mean voltage of À50 mV inducing half-maximum reduction. A comparable protocol applied to fibers loaded with fluo5N showed that low voltage depolarizing prepulses induced a marked SR Ca 2þ depletion that contributed to reduce a subsequent voltage-activated SR Ca 2þ change with a mean voltage of À50 mV inducing half-maximum reduction. Measuring SR Ca 2þ changes in response to long-lasting depolarizations indicated that SR Ca 2þ release channels inactivated in response to much higher depolarizations with a mean halfmaximum inactivation voltage of À20 mV. Finally, trains of action potential of 50 s duration produced cytosolic Ca 2þ signals that decayed with time, whereas SR Ca 2þ changes did not display any sign of inactivation. These results indicate that the decline in SR Ca 2þ release during long-lasting depolarizations mainly results from SR Ca 2þ depletion. The work was supported by AFM, CNRS and University Lyon 1. of. Stromal interaction molecule 1 (STIM1) mediates store-operated Ca 2þ entry (SOCE) in skeletal muscle. However, the direct role(s) of STIM1 in the innate skeletal muscle event such as the Ca 2þ release from the sarcoplasmic reticulum (SR) for muscle contraction have not been identified. In the present study, wildtype STIM1 and two STIM1 mutants (the Triple mutant, missing Ca 2þ -sensing residues, and E136X, missing the C-terminus) were over-expressed in mouse primary skeletal myotubes. The wild-type STIM1 increased SOCE, while neither mutant had an effect on SOCE. Interestingly, the development of puncta by endogenous STIM1 and Orai1 was detected without any stimulus during the differentiation of myoblasts to myotubes, and increased puncta formation was observed in the triple mutant as well as the wild-type STIM1, suggesting that, in skeletal muscle, the formation of puncta is part of the differentiation process and not the necessary and sufficient condition for SOCE. On the other hand, the Triple mutant, but not E136X, decreased the Ca 2þ release from the SR in response to KCl in a dominant-negative manner without affecting the SR Ca 2þ amount or resting Ca 2þ level. STIM1 was co-immunoprecipitated with the dihydropyridine receptor (DHRP). These results suggest that STIM1 could negatively regulate the Ca 2þ release from the SR, possibly via its C-terminal interaction with DHPR. Mitsugumin 53 (MG53) is a member of membrane repair system in skeletal muscle. However, role(s) of MG53 in unique functions of skeletal muscle has not been addressed although MG53 is expressed only in skeletal and cardiac muscle. In the present study, MG53-binding proteins were searched among proteins mediating skeletal muscle contraction and relaxation using the binding assays of various MG53 domains and quadrupole time-of-flight mass spectrometry. MG53 binds to sarcoplasmic reticulum Ca 2þ -ATPase 1a (SERCA1a) via its tripartite motif (TRIM) and PRY domains. The binding was confirmed in rabbit skeletal muscle and mouse primary skeletal myotubes by coimmunoprecipitation and immunocytochemistry. MG53 knock-down in mouse primary skeletal myotubes increased Ca 2þ -uptake through SERCA1a (more than 35%) at micromolar Ca 2þ but not at nanomolar Ca 2þ , suggesting that MG53 attenuates SERCA1a activity possibly during skeletal muscle contraction or relaxation but not during the resting state of skeletal muscle. In-silico studies suggest that the binding of MG53 to SERCA1a is mediated by unique ways compared with bindings by other proteins containing TRIM or PRY domains.

Biology of the Cell, 1997

This review is focused on the composition and organization of the junctional subsarcolemmal cytos... more This review is focused on the composition and organization of the junctional subsarcolemmal cytoskeleton of adult muscle fibers. The cytoskeleton of muscle fibers is organized in functionally distinct compartments and the subsarcolemmal cytoskeleton itself can be broadly divided into junctional (myotendinous junction, neuromuscular junction and costameres) and non-junctional domains. In junctional zones three different multimolecular cytoskeletal complexes coexist: the focal adhesion-type, the spectrin-based and the dystrophin vs utrophin-based membrane skeleton systems. These complexes extend over several levels, from intracytoplasmic to subsarcolemmal and transmembranous; their common feature is the anchorage of actin filaments emanating from the intracytoplasmic level. The different cytoskeletal proteins, their putative roles and their interactions with various signaling pathways are presented here in detail. The subsarcolemmal cytoskeleton complexes are thought to play distinct physiological roles (membrane stabilization, force transmission to extracellular matrix, ionic channel anchorage, etc) but their colocalization on the three sarcolemmal junctional domains strongly suggests interrelated or common functions.

Biology of the Cell, 1992

Biology of the Cell, 1993

The Journal of Physiology, 2004

In skeletal muscle, nitric oxide (NO) is commonly referred to as a modulator of the activity of t... more In skeletal muscle, nitric oxide (NO) is commonly referred to as a modulator of the activity of the ryanodine receptor (RyR) calcium release channel. However the reported effects of NO on isolated sarcoplasmic reticulum (SR) preparations and single ryanodine receptor (RyR) activity are diverse, and how NO affects SR calcium release and intracellular calcium homeostasis under physiological conditions remains poorly documented and hardly predictable. Here, we studied the effects of NO donors on membrane current and intracellular [Ca 2+ ] in single skeletal muscle fibres from mouse, under voltage-clamp conditions. When fibres were chronically exposed to millimolar levels of sodium nitroprusside (SNP) and challenged by short membrane depolarizations, there was a progressive increase in the resting [Ca 2+ ] level. This effect was use-dependent with the slope of rise in resting [Ca 2+ ] being increased two-fold when the depolarizing pulse level was raised from −20 to +10 mV. Analysis of the decay of the [Ca 2+ ] transients suggested that cytoplasmic Ca 2+ removal processes were largely unaffected by the presence of SNP. Also the functional properties of the dihydropyridine receptor were very similar under control conditions and in the presence of SNP. The resting [Ca 2+ ] elevation due to SNP was accompanied by a depression of the peak calcium release elicited by pulses to +10 mV. The effects of SNP could be reproduced by the chemically distinct NO donor NOC-12. They could be reversed upon exposure of the fibres to the thiol reducing agent dithiothreitol. Results suggest that large levels of NO produce a redox-sensitive continuous leak of Ca 2+ from the SR, through a limited number of release channels that do not close once they are activated by membrane depolarization. This SR Ca 2+ leak and the resulting increase in resting [Ca 2+ ] may be important in mediating the effects of excess NO on voltage-activated calcium release.