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Bruno Constantin - One of the best experts on this subject based on the ideXlab platform.
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Dystrophin/α1-syntrophin scaffold regulated PLC/PKC-dependent store-operated calcium entry in myotubes
Cell Calcium, 2012Co-Authors: Jessica Sabourin, Rania Harisseh, Thomas Harnois, Christophe Magaud, Nadine Déliot, Nicolas Bourmeyster, Bruno ConstantinAbstract:In skeletal muscles from patient suffering of Duchenne Muscular Dystrophy and from mdx mice, the absence of the Cytoskeleton Protein dystrophin has been shown to be essential for maintaining a normal calcium influx. We showed that a TRPC store-dependent cation influx is increased by loss of dystrophin or a scaffolding Protein α1-syntrophin, however the mechanisms of this calcium mishandling are incompletely understood. First of all, we confirmed that TRPC1 but also STIM1 and Orai1 are supporting the store-operated cation entry which is enhanced in dystrophin-deficient myotubes. Next, we demonstrated that inhibition of PLC or PKC in dystrophin-deficient myotubes restores elevated cation entry to normal levels similarly to enforced minidystrophin expression. In addition, silencing α1-syntrophin also increased cation influx in a PLC/PKC dependent pathway. We also showed that α1-syntrophin and PLCβ are part of a same Protein complex reinforcing the idea of their inter-relation in calcium influx regulation. This elevated cation entry was decreased to normal levels by chelating intracellular free calcium with BAPTA-AM. Double treatments with BAPTA-AM and PLC or PKC inhibitors suggested that the elevation of cation influx by PLC/PKC pathway is dependent on cytosolic calcium. All these results demonstrate an involvement in dystrophin-deficient myotubes of a specific calcium/PKC/PLC pathway in elevation of store-operated cation influx supported by the STIM1/Orai1/TRPC1 Proteins, which is normally regulated by the α1-syntrophin/dystrophin scaffold.
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Regulation of capacitative calcium entries by alpha1-syntrophin: association of TRPC1 with dystrophin complex and the PDZ domain of alpha1-syntrophin.
FASEB Journal, 2007Co-Authors: Aurélie Vandebrouck, Jessica Sabourin, Nicolas Bourmeyster, Jérôme Rivet, Haouria Balghi, Stéphane Sebille, Alain Kitzis, Guy Raymond, Christian Cognard, Bruno ConstantinAbstract:Calcium mishandling in Duchenne dystrophic muscle suggested that dystrophin, a membrane-associated Cytoskeleton Protein, might regulate calcium signaling cascade such as calcium influx pathway. It was previously shown that abnormal calcium entries involve uncontrolled stretch-activated currents and store-operated Ca2+ currents supported by TRPC1 channels. Moreover, our recent work demonstrated that reintroduction of minidystrophin in dystrophic myotubes restores normal capacitative calcium entries (CCEs). However, until now, no molecular link between the dystrophin complex and calcium entry channels has been described. This study is the first to show by coimmunoprecipitation assays the molecular association of TRPC1 with dystrophin and alpha1-syntrophin in muscle cells. TRPC1 was also associated with alpha1-syntrophin in dystrophic muscle cells independently of dystrophin. Furthermore, glutathione S-transferase (GST) pull-down assays showed that TRPC1 binds to the alpha1-syntrophin PDZ domain. Transfected recombinant alpha1-syntrophin formed a complex with TRPC1 channels and restored normal CCEs in dystrophic muscle cells. We suggest that normal regulation of CCEs in skeletal muscle depends on the association between TRPC1 channels and alpha1-syntrophin that may anchor the store-operated channels to the dystrophin-associated Protein complex (DAPC). The loss of this molecular association could participate in the calcium alterations observed in dystrophic muscle cells. This study provides a new model for the regulation of calcium influx by interaction with the scaffold of the DAPC in muscle cells.
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New insights in the regulation of calcium transfers by muscle dystrophin-based Cytoskeleton: implications in DMD.
Journal of Muscle Research and Cell Motility, 2006Co-Authors: Bruno Constantin, Stéphane Sebille, Christian CognardAbstract:Calcium mishandling in Duchenne muscular dystrophy (DMD) suggested that dystrophin, a membrane-associated Cytoskeleton Protein, may regulate calcium-signalling cascades such as calcium entries. Calcium overload in human DMD myotubes is dependent on their contractile activity suggesting the involvement of channels being activated during contraction and/or calcium release. Forced expression of mini-dystrophin in dystrophin-deficient myotubes, reactivates appropriate sarcolemmal expression of dystrophin-associated Proteins and restores normal calcium handling in the cytosol. Furthermore, the recombinant mini-dystrophin reduced the store-operated calcium influx across the sarcolemma, and the mitochondrial calcium uptake during this influx. A slow component of calcium release dependent on IP3R, as well as the production of IP3, were also reduced to normal levels by expression of mini-dystrophin. Our studies provide a new model for the convergent regulation of transmembrane calcium influx and IP3-dependent calcium release by the dystrophin-based Cytoskeleton (DBC). We also suggest molecular association of such channels with DBC which may provide the scaffold for assembling a multiProtein-signalling complex that modulates the channel activity. This suggests that the loss of this molecular association could participate in the alteration of calcium homeostasis observed in DMD muscle cells.
A Dagkesamanskaya - One of the best experts on this subject based on the ideXlab platform.
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Differential localisation of GFP fusions to Cytoskeleton-binding Proteins in animal, plant, and yeast cells. Green-fluorescent Protein.
Protoplasma, 2002Co-Authors: Antonius C J Timmers, Andreas Niebel, Christine Balagué, A DagkesamanskayaAbstract:The structure and functioning of the Cytoskeleton is controlled and regulated by Cytoskeleton-associated Proteins. Fused to the green-fluorescent Protein (GFP), these Proteins can be used as tools to monitor changes in the organisation of the Cytoskeleton in living cells and tissues in different organisms. Since the localisation of a specific Cytoskeleton Protein may indicate a particular function for the associated cytoskeletal element, studies of Cytoskeleton-binding Proteins fused to GFP may provide insight into the organisation and functioning of the Cytoskeleton. In this article, we focused on two animal Proteins, human T-plastin and bovine tau, and studied the distribution of their respective GFP fusions in animal COS cells, plant epidermal cells (Allium cepa), and yeast cells (Saccharomyces cerevisiae). Plastin-GFP localised preferentially to membrane ruffles, lamellipodia and focal adhesion points in COS cells, to the actin filament Cytoskeleton within cytoplasmic strands in onion epidermal cells, and to cortical actin patches in yeast cells. Thus, in these 3 very different types of cells plastin-GFP associated with mobile structures in which there are high rates of actin turnover. Chemical fixation was found to drastically alter the distribution of plastin-GFP. Tau-GFP bound to microtubules in COS cells and onion epidermal cells but failed to bind to yeast microtubules. Thus, animal and plant microtubules appear to have a common tau binding site which is absent in yeast. We conclude that the study of the distribution patterns of microtubule- and actin-filament-binding Proteins fused to GFP in heterologous systems should be a valuable tool in furthering our knowledge about Cytoskeleton function in eukaryotic cells.
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Differential localisation of GFP fusions to Cytoskeleton-binding Proteins in animal, plant, and yeast cells
Protoplasma, 2002Co-Authors: Antonius C J Timmers, Andreas Niebel, Christine Balagué, A DagkesamanskayaAbstract:The structure and functioning of the Cytoskeleton is controlled and regulated by Cytoskeleton-associated Proteins. Fused to the green-fluorescent Protein (GFP), these Proteins can be used as tools to monitor changes in the organisation of the Cytoskeleton in living cells and tissues in different organisms. Since the localisation of a specific Cytoskeleton Protein may indicate a particular function for the associated cytoskeletal element, studies of Cytoskeleton-binding Proteins fused to GFP may provide insight into the organisation and functioning of the Cytoskeleton. In this article, we focused on two animal Proteins, human T-plastin and bovine tau, and studied the distribution of their respective GFP fusions in animal COS cells, plant epidermal cells (Allium cepa), and yeast cells (Saccharomyces cerevisiae). Plastin-GFP localised preferentially to membrane ruffles, lamellipodia and focal adhesion points in COS cells, to the actin filament Cytoskeleton within cytoplasmic strands in onion epidermal cells, and to cortical actin patches in yeast cells. Thus, in these 3 very different types of cells plastin-GFP associated with mobile structures in which there are high rates of actin turnover. Chemical fixation was found to drastically alter the distribution of plastin-GFP. Tau-GFP bound to microtubules in COS cells and onion epidermal cells but failed to bind to yeast microtubules. Thus, animal and plant microtubules appear to have a common tau binding site which is absent in yeast. We conclude that the study of the distribution patterns of microtubule- and actin-filament-binding Proteins fused to GFP in heterologous systems should be a valuable tool in furthering our knowledge about Cytoskeleton function in eukaryotic cells.
Patrizia Lopresti - One of the best experts on this subject based on the ideXlab platform.
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Inducible Expression of a Truncated Form of Tau in Oligodendrocytes Elicits Gait Abnormalities and a Decrease in Myelin: Implications for Selective CNS Degenerative Diseases
Neurochemical Research, 2015Co-Authors: Patrizia LoprestiAbstract:The Cytoskeleton Protein Tau present in oligodendrocytes (OLGs) promotes cellular process outgrowth and myelination; whereas abnormally hyperphosphorylated Tau has been shown to be present in the most debilitating form of multiple sclerosis and in selective dementias. This research examined the functional consequences of expressing a truncated form of Tau in OLGs during the second postnatal life. In particular, this truncated form of Tau (∆Tau) retains the Fyn-binding domain but lacks the microtubule-binding domain. Similar to hyperphosphorylated Tau, ∆Tau cannot bind the Cytoskeleton and is missorted. The Cre/loxP recombination system was used to generate transgenic (TG) founder lines, which contain a Floxed LacZ-STOP cassette to prevent expression of enhanced green fluorescence Protein (EGFP)–∆Tau. The founder lines were then crossed with a Tamoxifen (TM)-inducible proteolipid Protein (PLP)-dependent Cre driver line. Myelin PLP is the major myelin Protein in the central nervous system (CNS). TM was given at postnatal day (p) 12 for 3 days, and CNS tissues were collected at p22. Only TG mice with both EGFP–∆Tau and Cre manifested an overt phenotype of loss of balance and stumbles starting around p18. CNS tissues obtained from TM-treated EGFP–∆Tau/Cre double transgenic mice had recombined PCR products, GFP, and diminished brain myelin. GFP was expressed in OLGs, but not in neurons or astrocytes. On the contrary, TM-treated TG mice with only one of the two transgenes, i.e., Cre or Tau, did not have recombinant PCR products, GFP, diminished myelin, or abnormal phenotype. Thus, this inducible model shows for the first time that a non-microtubule-associated Tau Protein in OLGs elicits both myelin decrease and gait abnormalities, similar to the occurrence in selective demyelinating and neurodegenerative diseases.
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Inducible Expression of a Truncated Form of Tau in Oligodendrocytes Elicits Gait Abnormalities and a Decrease in Myelin: Implications for Selective CNS Degenerative Diseases.
Neurochemical Research, 2015Co-Authors: Patrizia LoprestiAbstract:The Cytoskeleton Protein Tau present in oligodendrocytes (OLGs) promotes cellular process outgrowth and myelination; whereas abnormally hyperphosphorylated Tau has been shown to be present in the most debilitating form of multiple sclerosis and in selective dementias. This research examined the functional consequences of expressing a truncated form of Tau in OLGs during the second postnatal life. In particular, this truncated form of Tau (∆Tau) retains the Fyn-binding domain but lacks the microtubule-binding domain. Similar to hyperphosphorylated Tau, ∆Tau cannot bind the Cytoskeleton and is missorted. The Cre/loxP recombination system was used to generate transgenic (TG) founder lines, which contain a Floxed LacZ-STOP cassette to prevent expression of enhanced green fluorescence Protein (EGFP)–∆Tau. The founder lines were then crossed with a Tamoxifen (TM)-inducible proteolipid Protein (PLP)-dependent Cre driver line. Myelin PLP is the major myelin Protein in the central nervous system (CNS). TM was given at postnatal day (p) 12 for 3 days, and CNS tissues were collected at p22. Only TG mice with both EGFP–∆Tau and Cre manifested an overt phenotype of loss of balance and stumbles starting around p18. CNS tissues obtained from TM-treated EGFP–∆Tau/Cre double transgenic mice had recombined PCR products, GFP, and diminished brain myelin. GFP was expressed in OLGs, but not in neurons or astrocytes. On the contrary, TM-treated TG mice with only one of the two transgenes, i.e., Cre or Tau, did not have recombinant PCR products, GFP, diminished myelin, or abnormal phenotype. Thus, this inducible model shows for the first time that a non-microtubule-associated Tau Protein in OLGs elicits both myelin decrease and gait abnormalities, similar to the occurrence in selective demyelinating and neurodegenerative diseases.
Marina R Picciotto - One of the best experts on this subject based on the ideXlab platform.
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constitutive knockout of the membrane Cytoskeleton Protein beta adducin decreases mushroom spine density in the nucleus accumbens but does not prevent spine remodeling in response to cocaine
European Journal of Neuroscience, 2013Co-Authors: Yonwoo Jung, Patrick J Mulholland, Shari L Wiseman, Judson L Chandler, Marina R PicciottoAbstract:The adducin family of Proteins associates with the actin Cytoskeleton in a calcium-dependent manner. Beta adducin (βAdd) is involved in synaptic plasticity in the hippocampus; however, the role of βAdd in synaptic plasticity in other brain areas is unknown. Using diolistic labeling with the lipophilic dye DiI, we found that the density of mature mushroom-shaped spines was significantly decreased in the nucleus accumbens (NAc) in brain slices from βAdd KO mice as compared to their wildtype (WT) siblings. The effect of 10 days of daily cocaine (15 mg/kg) administration on NAc spine number and locomotor behavior was also measured in β Add WT and knockout (KO) mice. As expected, there was a significant increase in overall spine density in NAc slices from cocaine-treated WT mice at this time-point, however, there was a greater increase in the density of mushroom spines in β Add KO animals following chronic cocaine administration compared to WT. In addition, β Add KO mice showed elevated locomotor activity in response to cocaine treatment compared to WT siblings. These results indicate that β Add is required for stabilizing mature spines under basal conditions in the NAc, but that lack of this Protein does not prevent synaptic remodeling following repeated cocaine administration. In addition, these data are consistent with previous studies suggesting that β Add may normally be involved in stabilizing spines once drug- or experience-dependent remodeling has occurred.
Boris Zhivotovsky - One of the best experts on this subject based on the ideXlab platform.
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Caspase-2 promotes Cytoskeleton Protein degradation during apoptotic cell death
Cell Death & Disease, 2013Co-Authors: H Vakifahmetoglu-norberg, E Norberg, A B Perdomo, Fabiola Ciccosanti, Gian Maria Fimia, Maria Piacentini, M. Olsson, Sten Orrenius, Boris ZhivotovskyAbstract:The caspase family of proteases cleaves large number of Proteins resulting in major morphological and biochemical changes during apoptosis. Yet, only a few of these Proteins have been reported to selectively cleaved by caspase-2. Numerous observations link caspase-2 to the disruption of the Cytoskeleton, although it remains elusive whether any of the Cytoskeleton Proteins serve as bona fide substrates for caspase-2. Here, we undertook an unbiased proteomic approach to address this question. By differential proteome analysis using two-dimensional gel electrophoresis, we identified four Cytoskeleton Proteins that were degraded upon treatment with active recombinant caspase-2 in vitro. These Proteins were degraded in a caspase-2-dependent manner during apoptosis induced by DNA damage, Cytoskeleton disruption or endoplasmic reticulum stress. Hence, degradation of these Cytoskeleton Proteins was blunted by siRNA targeting of caspase-2 and when caspase-2 activity was pharmacologically inhibited. However, none of these Proteins was cleaved directly by caspase-2. Instead, we provide evidence that in cells exposed to apoptotic stimuli, caspase-2 probed these Proteins for proteasomal degradation. Taken together, our results depict a new role for caspase-2 in the regulation of the level of Cytoskeleton Proteins during apoptosis.
