C. Castelbou - Academia.edu (original) (raw)

Papers by C. Castelbou

Research paper thumbnail of Ca2+ Homeostasis during Mitochondrial Fragmentation and Perinuclear Clustering Induced by hFis1

Journal of Biological Chemistry, 2004

Mitochondria modulate Ca 2؉ signals by taking up, buffering, and releasing Ca 2؉ at key locations... more Mitochondria modulate Ca 2؉ signals by taking up, buffering, and releasing Ca 2؉ at key locations near Ca 2؉ release or influx channels. The role of such local interactions between channels and organelles is difficult to establish in living cells because mitochondria form an interconnected network constantly remodeled by coordinated fusion and fission reactions. To study the effect of a controlled disruption of the mitochondrial network on Ca 2؉ homeostasis, we took advantage of hFis1, a protein that promotes mitochondrial fission by recruiting the dynamin-related protein, Drp1. hFis1 expression in HeLa cells induced a rapid and complete fragmentation of mitochondria, which redistributed away from the plasma membrane and clustered around the nucleus. Despite the dramatic morphological alteration, hFis1-fragmented mitochondria maintained a normal transmembrane potential and pH and took up normally the Ca 2؉ released from intracellular stores upon agonist stimulation, as measured with a targeted ratiometric pericam probe. In contrast, hFis1fragmented mitochondria took up more slowly the Ca 2؉ entering across plasma membrane channels, because the Ca 2؉ ions reaching mitochondria propagated faster and in a more coordinated manner in interconnected than in fragmented mitochondria. In parallel cytosolic fura-2 measurements, the capacitative Ca 2؉ entry (CCE) elicited by store depletion was only marginally reduced by hFis1 expression. Regardless of mitochondria shape and location, disruption of mitochondrial potential with uncouplers or oligomycin/ rotenone reduced CCE by ϳ35%. These observations indicate that close contact to Ca 2؉ influx channels is not required for CCE modulation and that the formation of a mitochondrial network facilitates Ca 2؉ propagation within interconnected mitochondria.

Research paper thumbnail of Homeostasis during mitochondrial fragmentation and perinuclear clustering induced by hfis1

Research paper thumbnail of NCLX Protein, but Not LETM1, Mediates Mitochondrial Ca2+ Extrusion, Thereby Limiting Ca2+-induced NAD(P)H Production and Modulating Matrix Redox State

Journal of Biological Chemistry, 2014

Background: Whether mitochondrial Ca 2ϩ extrusion is mediated by NCLX (mitochondrial sodium/calci... more Background: Whether mitochondrial Ca 2ϩ extrusion is mediated by NCLX (mitochondrial sodium/calcium exchanger) or LETM1 (leucine zipper-EF-hand-containing transmembrane protein 1) and controls matrix redox state is unknown. Results: NCLX, but not LETM1, increases Ca 2ϩ extrusion, limits NAD(P)H production, and promotes matrix oxidation. Conclusion: NCLX controls the duration of matrix Ca 2ϩ elevations and their impact on redox signaling. Significance: NCLX is a potential target for the treatment of redox-dependent diseases.

Research paper thumbnail of Hv1 proton channels differentially regulate the pH of neutrophil and macrophage phagosomes by sustaining the production of phagosomal ROS that inhibit the delivery of vacuolar ATPases

Journal of Leukocyte Biology, 2014

Production of ROS and maintenance of an appropriate pH within the lumen of neutrophil and macroph... more Production of ROS and maintenance of an appropriate pH within the lumen of neutrophil and macrophage phagosomes are important for an effective immune response. Hv1 proton channels sustain ROS production at the plasma membrane, but their role in phagosomes is not known. Here, we tested whether Hv1 channels regulate the pH p and sustain phagosomal ROS production in neutrophils and macrophages. The presence of Hv1 channels on phagosomes of human neutrophils and mouse macrophages was confirmed by Western blot and immunostaining. Phagosomal ROS production, measured with OxyBurst-coupled targets, was reduced in neutrophils and macrophages isolated from Hv1-deficient mice. Ratiometric imaging of FITC-coupled targets showed that phagosomes acidified more slowly in Hv1-deficient macrophages and transiently alkalinized when the V-ATPase was inhibited. In WT neutrophils, 97% of phagosomes remained neutral 30 min after particle ingestion, whereas 37% of Hv1-deficient phagosomes were alkaline (pHϾ8.3) and 14% acidic (pHϽ6.3). The subpopulation of acidic phagosomes was eliminated by V-ATPase inhibition, whereas NOX inhibition caused a rapid acidification, independently of Hv1 expression. Finally, V-ATPase accumulation on phagosomes was inversely correlated to intraphagosomal ROS production in neutrophils. These data indicate that Hvcn1 ablation deregulates neutrophil pH p , leading to alkalinization in phagosomes with residual ROS production or to the early accumulation of V-ATPase on phagosomes that fail to mount an oxidative response. Hv1 channels therefore differentially regulate the pH p in neutrophils and macrophages, sustaining rapid acidification in macrophage phagosomes and maintaining a neutral pH in neutrophil phagosomes. J. Leukoc. Biol. 95: 827-839; 2014.

Research paper thumbnail of Calreticulin Differentially Modulates Calcium Uptake and Release in the Endoplasmic Reticulum and Mitochondria

Journal of Biological Chemistry, 2002

To study the role of calreticulin in Ca(2+) homeostasis and apoptosis, we generated cells inducib... more To study the role of calreticulin in Ca(2+) homeostasis and apoptosis, we generated cells inducible for full-length or truncated calreticulin and measured Ca(2+) signals within the cytosol, the endoplasmic reticulum (ER), and mitochondria with "cameleon" indicators. Induction of calreticulin increased the free Ca(2+) concentration within the ER lumen, [Ca(2+)](ER), from 306 +/- 31 to 595 +/- 53 microm, and doubled the rate of ER refilling. [Ca(2+)](ER) remained elevated in the presence of thapsigargin, an inhibitor of SERCA-type Ca(2+) ATPases. Under these conditions, store-operated Ca(2+) influx appeared inhibited but could be reactivated by decreasing [Ca(2+)](ER) with the low affinity Ca(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. In contrast, [Ca(2+)](ER) decreased much faster during stimulation with carbachol. The larger ER release was associated with a larger cytosolic Ca(2+) response and, surprisingly, with a shorter mitochondrial Ca(2+) response. The reduced mitochondrial signal was not associated with visible morphological alterations of mitochondria or with disruption of the contacts between mitochondria and the ER but correlated with a reduced mitochondrial membrane potential. Altered ER and mitochondrial Ca(2+) responses were also observed in cells expressing an N-truncated calreticulin but not in cells overexpressing calnexin, a P-domain containing chaperone, indicating that the effects were mediated by the unique C-domain of calreticulin. In conclusion, calreticulin overexpression increases Ca(2+) fluxes across the ER but decreases mitochondrial Ca(2+) and membrane potential. The increased Ca(2+) turnover between the two organelles might damage mitochondria, accounting for the increased susceptibility of cells expressing high levels of calreticulin to apoptotic stimuli.

Research paper thumbnail of Uncoupling Protein 3 (UCP3) Modulates the Activity of Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) by Decreasing Mitochondrial ATP Production

Journal of Biological Chemistry, 2011

Research paper thumbnail of Subplasmalemmal Mitochondria Modulate the Activity of Plasma Membrane Ca2+-ATPases

Journal of Biological Chemistry, 2005

Mitochondria are dynamic organelles that modulate cellular Ca 2؉ signals by interacting with Ca 2... more Mitochondria are dynamic organelles that modulate cellular Ca 2؉ signals by interacting with Ca 2؉ transporters on the plasma membrane or the endoplasmic reticulum (ER). To study how mitochondria dynamics affects cell Ca 2؉ homeostasis, we overexpressed two mitochondrial fission proteins, hFis1 and Drp1, and measured Ca 2؉ changes within the cytosol and the ER in HeLa cells. Both proteins fragmented mitochondria, decreased their total volume by 25-40%, and reduced the fraction of subplasmalemmal mitochondria by 4-fold. The cytosolic Ca 2؉ signals elicited by histamine were unaltered in cells lacking subplasmalemmal mitochondria as long as Ca 2؉ was present in the medium, but the signals were significantly blunted when Ca 2؉ was removed. Upon Ca 2؉ withdrawal, the free ER Ca 2؉ concentration decreased rapidly, and hFis1 cells were unable to respond to repetitive histamine stimulations. . 2 The abbreviations used are: [Ca 2ϩ ] cyt , cytosolic [Ca 2ϩ ]; [Ca 2ϩ ] ER , endoplasmic reticulum [Ca 2ϩ ]; [Ca 2ϩ ] mit , mitochondrial [Ca 2ϩ ]; CGP 37157, (7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one); ER, endoplasmic reticulum; GFP, green fluorescent protein; PMCA, plasma membrane Ca 2ϩ -ATPase; RFP mit , mitochondrial red fluorescent protein; SERCA, sarco/endoplasmic reticulum Ca 2ϩ ATPase; YC4.1 ER , yellow cameleon targeted to the ER; YC3.6 pm , yellow cameleon targeted to the plasma membrane.

Research paper thumbnail of Ca2+ Homeostasis during Mitochondrial Fragmentation and Perinuclear Clustering Induced by hFis1

Journal of Biological Chemistry, 2004

Mitochondria modulate Ca 2؉ signals by taking up, buffering, and releasing Ca 2؉ at key locations... more Mitochondria modulate Ca 2؉ signals by taking up, buffering, and releasing Ca 2؉ at key locations near Ca 2؉ release or influx channels. The role of such local interactions between channels and organelles is difficult to establish in living cells because mitochondria form an interconnected network constantly remodeled by coordinated fusion and fission reactions. To study the effect of a controlled disruption of the mitochondrial network on Ca 2؉ homeostasis, we took advantage of hFis1, a protein that promotes mitochondrial fission by recruiting the dynamin-related protein, Drp1. hFis1 expression in HeLa cells induced a rapid and complete fragmentation of mitochondria, which redistributed away from the plasma membrane and clustered around the nucleus. Despite the dramatic morphological alteration, hFis1-fragmented mitochondria maintained a normal transmembrane potential and pH and took up normally the Ca 2؉ released from intracellular stores upon agonist stimulation, as measured with a targeted ratiometric pericam probe. In contrast, hFis1fragmented mitochondria took up more slowly the Ca 2؉ entering across plasma membrane channels, because the Ca 2؉ ions reaching mitochondria propagated faster and in a more coordinated manner in interconnected than in fragmented mitochondria. In parallel cytosolic fura-2 measurements, the capacitative Ca 2؉ entry (CCE) elicited by store depletion was only marginally reduced by hFis1 expression. Regardless of mitochondria shape and location, disruption of mitochondrial potential with uncouplers or oligomycin/ rotenone reduced CCE by ϳ35%. These observations indicate that close contact to Ca 2؉ influx channels is not required for CCE modulation and that the formation of a mitochondrial network facilitates Ca 2؉ propagation within interconnected mitochondria. . 1 The abbreviations and trivial names used are: ER, endoplasmic reticulum; CCE, capacitative Ca 2ϩ entry; CCCP, carbonylcyanide mchlorophenylhydrazone; [Ca 2ϩ ] mit , mitochondrial [Ca 2ϩ ]; [Ca 2ϩ ] cyt , cytosolic [Ca 2ϩ ]; RP3.1 mit , ratiometric pericam targeted to the mitochondrial matrix; SERCA, sarco/endoplasmic reticulum Ca 2ϩ ATPase; I CRAC , Ca 2ϩ release-activated Ca 2ϩ current; ⌬ m , mitochondrial membrane potential; TMRM, tetramethylrhodamine methyl ester; GFP, green fluorescent protein.

Research paper thumbnail of Ca2+ Homeostasis during Mitochondrial Fragmentation and Perinuclear Clustering Induced by hFis1

Journal of Biological Chemistry, 2004

Mitochondria modulate Ca 2؉ signals by taking up, buffering, and releasing Ca 2؉ at key locations... more Mitochondria modulate Ca 2؉ signals by taking up, buffering, and releasing Ca 2؉ at key locations near Ca 2؉ release or influx channels. The role of such local interactions between channels and organelles is difficult to establish in living cells because mitochondria form an interconnected network constantly remodeled by coordinated fusion and fission reactions. To study the effect of a controlled disruption of the mitochondrial network on Ca 2؉ homeostasis, we took advantage of hFis1, a protein that promotes mitochondrial fission by recruiting the dynamin-related protein, Drp1. hFis1 expression in HeLa cells induced a rapid and complete fragmentation of mitochondria, which redistributed away from the plasma membrane and clustered around the nucleus. Despite the dramatic morphological alteration, hFis1-fragmented mitochondria maintained a normal transmembrane potential and pH and took up normally the Ca 2؉ released from intracellular stores upon agonist stimulation, as measured with a targeted ratiometric pericam probe. In contrast, hFis1fragmented mitochondria took up more slowly the Ca 2؉ entering across plasma membrane channels, because the Ca 2؉ ions reaching mitochondria propagated faster and in a more coordinated manner in interconnected than in fragmented mitochondria. In parallel cytosolic fura-2 measurements, the capacitative Ca 2؉ entry (CCE) elicited by store depletion was only marginally reduced by hFis1 expression. Regardless of mitochondria shape and location, disruption of mitochondrial potential with uncouplers or oligomycin/ rotenone reduced CCE by ϳ35%. These observations indicate that close contact to Ca 2؉ influx channels is not required for CCE modulation and that the formation of a mitochondrial network facilitates Ca 2؉ propagation within interconnected mitochondria.

Research paper thumbnail of Homeostasis during mitochondrial fragmentation and perinuclear clustering induced by hfis1

Research paper thumbnail of NCLX Protein, but Not LETM1, Mediates Mitochondrial Ca2+ Extrusion, Thereby Limiting Ca2+-induced NAD(P)H Production and Modulating Matrix Redox State

Journal of Biological Chemistry, 2014

Background: Whether mitochondrial Ca 2ϩ extrusion is mediated by NCLX (mitochondrial sodium/calci... more Background: Whether mitochondrial Ca 2ϩ extrusion is mediated by NCLX (mitochondrial sodium/calcium exchanger) or LETM1 (leucine zipper-EF-hand-containing transmembrane protein 1) and controls matrix redox state is unknown. Results: NCLX, but not LETM1, increases Ca 2ϩ extrusion, limits NAD(P)H production, and promotes matrix oxidation. Conclusion: NCLX controls the duration of matrix Ca 2ϩ elevations and their impact on redox signaling. Significance: NCLX is a potential target for the treatment of redox-dependent diseases.

Research paper thumbnail of Hv1 proton channels differentially regulate the pH of neutrophil and macrophage phagosomes by sustaining the production of phagosomal ROS that inhibit the delivery of vacuolar ATPases

Journal of Leukocyte Biology, 2014

Production of ROS and maintenance of an appropriate pH within the lumen of neutrophil and macroph... more Production of ROS and maintenance of an appropriate pH within the lumen of neutrophil and macrophage phagosomes are important for an effective immune response. Hv1 proton channels sustain ROS production at the plasma membrane, but their role in phagosomes is not known. Here, we tested whether Hv1 channels regulate the pH p and sustain phagosomal ROS production in neutrophils and macrophages. The presence of Hv1 channels on phagosomes of human neutrophils and mouse macrophages was confirmed by Western blot and immunostaining. Phagosomal ROS production, measured with OxyBurst-coupled targets, was reduced in neutrophils and macrophages isolated from Hv1-deficient mice. Ratiometric imaging of FITC-coupled targets showed that phagosomes acidified more slowly in Hv1-deficient macrophages and transiently alkalinized when the V-ATPase was inhibited. In WT neutrophils, 97% of phagosomes remained neutral 30 min after particle ingestion, whereas 37% of Hv1-deficient phagosomes were alkaline (pHϾ8.3) and 14% acidic (pHϽ6.3). The subpopulation of acidic phagosomes was eliminated by V-ATPase inhibition, whereas NOX inhibition caused a rapid acidification, independently of Hv1 expression. Finally, V-ATPase accumulation on phagosomes was inversely correlated to intraphagosomal ROS production in neutrophils. These data indicate that Hvcn1 ablation deregulates neutrophil pH p , leading to alkalinization in phagosomes with residual ROS production or to the early accumulation of V-ATPase on phagosomes that fail to mount an oxidative response. Hv1 channels therefore differentially regulate the pH p in neutrophils and macrophages, sustaining rapid acidification in macrophage phagosomes and maintaining a neutral pH in neutrophil phagosomes. J. Leukoc. Biol. 95: 827-839; 2014.

Research paper thumbnail of Calreticulin Differentially Modulates Calcium Uptake and Release in the Endoplasmic Reticulum and Mitochondria

Journal of Biological Chemistry, 2002

To study the role of calreticulin in Ca(2+) homeostasis and apoptosis, we generated cells inducib... more To study the role of calreticulin in Ca(2+) homeostasis and apoptosis, we generated cells inducible for full-length or truncated calreticulin and measured Ca(2+) signals within the cytosol, the endoplasmic reticulum (ER), and mitochondria with "cameleon" indicators. Induction of calreticulin increased the free Ca(2+) concentration within the ER lumen, [Ca(2+)](ER), from 306 +/- 31 to 595 +/- 53 microm, and doubled the rate of ER refilling. [Ca(2+)](ER) remained elevated in the presence of thapsigargin, an inhibitor of SERCA-type Ca(2+) ATPases. Under these conditions, store-operated Ca(2+) influx appeared inhibited but could be reactivated by decreasing [Ca(2+)](ER) with the low affinity Ca(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. In contrast, [Ca(2+)](ER) decreased much faster during stimulation with carbachol. The larger ER release was associated with a larger cytosolic Ca(2+) response and, surprisingly, with a shorter mitochondrial Ca(2+) response. The reduced mitochondrial signal was not associated with visible morphological alterations of mitochondria or with disruption of the contacts between mitochondria and the ER but correlated with a reduced mitochondrial membrane potential. Altered ER and mitochondrial Ca(2+) responses were also observed in cells expressing an N-truncated calreticulin but not in cells overexpressing calnexin, a P-domain containing chaperone, indicating that the effects were mediated by the unique C-domain of calreticulin. In conclusion, calreticulin overexpression increases Ca(2+) fluxes across the ER but decreases mitochondrial Ca(2+) and membrane potential. The increased Ca(2+) turnover between the two organelles might damage mitochondria, accounting for the increased susceptibility of cells expressing high levels of calreticulin to apoptotic stimuli.

Research paper thumbnail of Uncoupling Protein 3 (UCP3) Modulates the Activity of Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) by Decreasing Mitochondrial ATP Production

Journal of Biological Chemistry, 2011

Research paper thumbnail of Subplasmalemmal Mitochondria Modulate the Activity of Plasma Membrane Ca2+-ATPases

Journal of Biological Chemistry, 2005

Mitochondria are dynamic organelles that modulate cellular Ca 2؉ signals by interacting with Ca 2... more Mitochondria are dynamic organelles that modulate cellular Ca 2؉ signals by interacting with Ca 2؉ transporters on the plasma membrane or the endoplasmic reticulum (ER). To study how mitochondria dynamics affects cell Ca 2؉ homeostasis, we overexpressed two mitochondrial fission proteins, hFis1 and Drp1, and measured Ca 2؉ changes within the cytosol and the ER in HeLa cells. Both proteins fragmented mitochondria, decreased their total volume by 25-40%, and reduced the fraction of subplasmalemmal mitochondria by 4-fold. The cytosolic Ca 2؉ signals elicited by histamine were unaltered in cells lacking subplasmalemmal mitochondria as long as Ca 2؉ was present in the medium, but the signals were significantly blunted when Ca 2؉ was removed. Upon Ca 2؉ withdrawal, the free ER Ca 2؉ concentration decreased rapidly, and hFis1 cells were unable to respond to repetitive histamine stimulations. . 2 The abbreviations used are: [Ca 2ϩ ] cyt , cytosolic [Ca 2ϩ ]; [Ca 2ϩ ] ER , endoplasmic reticulum [Ca 2ϩ ]; [Ca 2ϩ ] mit , mitochondrial [Ca 2ϩ ]; CGP 37157, (7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one); ER, endoplasmic reticulum; GFP, green fluorescent protein; PMCA, plasma membrane Ca 2ϩ -ATPase; RFP mit , mitochondrial red fluorescent protein; SERCA, sarco/endoplasmic reticulum Ca 2ϩ ATPase; YC4.1 ER , yellow cameleon targeted to the ER; YC3.6 pm , yellow cameleon targeted to the plasma membrane.

Research paper thumbnail of Ca2+ Homeostasis during Mitochondrial Fragmentation and Perinuclear Clustering Induced by hFis1

Journal of Biological Chemistry, 2004

Mitochondria modulate Ca 2؉ signals by taking up, buffering, and releasing Ca 2؉ at key locations... more Mitochondria modulate Ca 2؉ signals by taking up, buffering, and releasing Ca 2؉ at key locations near Ca 2؉ release or influx channels. The role of such local interactions between channels and organelles is difficult to establish in living cells because mitochondria form an interconnected network constantly remodeled by coordinated fusion and fission reactions. To study the effect of a controlled disruption of the mitochondrial network on Ca 2؉ homeostasis, we took advantage of hFis1, a protein that promotes mitochondrial fission by recruiting the dynamin-related protein, Drp1. hFis1 expression in HeLa cells induced a rapid and complete fragmentation of mitochondria, which redistributed away from the plasma membrane and clustered around the nucleus. Despite the dramatic morphological alteration, hFis1-fragmented mitochondria maintained a normal transmembrane potential and pH and took up normally the Ca 2؉ released from intracellular stores upon agonist stimulation, as measured with a targeted ratiometric pericam probe. In contrast, hFis1fragmented mitochondria took up more slowly the Ca 2؉ entering across plasma membrane channels, because the Ca 2؉ ions reaching mitochondria propagated faster and in a more coordinated manner in interconnected than in fragmented mitochondria. In parallel cytosolic fura-2 measurements, the capacitative Ca 2؉ entry (CCE) elicited by store depletion was only marginally reduced by hFis1 expression. Regardless of mitochondria shape and location, disruption of mitochondrial potential with uncouplers or oligomycin/ rotenone reduced CCE by ϳ35%. These observations indicate that close contact to Ca 2؉ influx channels is not required for CCE modulation and that the formation of a mitochondrial network facilitates Ca 2؉ propagation within interconnected mitochondria. . 1 The abbreviations and trivial names used are: ER, endoplasmic reticulum; CCE, capacitative Ca 2ϩ entry; CCCP, carbonylcyanide mchlorophenylhydrazone; [Ca 2ϩ ] mit , mitochondrial [Ca 2ϩ ]; [Ca 2ϩ ] cyt , cytosolic [Ca 2ϩ ]; RP3.1 mit , ratiometric pericam targeted to the mitochondrial matrix; SERCA, sarco/endoplasmic reticulum Ca 2ϩ ATPase; I CRAC , Ca 2ϩ release-activated Ca 2ϩ current; ⌬ m , mitochondrial membrane potential; TMRM, tetramethylrhodamine methyl ester; GFP, green fluorescent protein.