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Biomedical subjects

Serge Arnaudeau

Publications and source records attributed to Serge Arnaudeau.

10 recordsLinked to original sources
PubMed2006

Calcium sources used by post-natal human myoblasts during initial differentiation.

Increases in cytoplasmic Ca(2+) are crucial for inducing the initial steps of myoblast differentiation that ultimately lead to fusion; yet the mechanisms that produce this elevated Ca(2+) have not been fully resolved. For example, it is still unclear whether the increase comes exclusively from membrane Ca(2+) influx or also from Ca(2+) release from internal stores. To address this, we investigated early differentiation of myoblast clones each derived from single post-natal human satellite cells. Initial differentiation was assayed by immunostaining myonuclei for the transcription factor MEF2. When Ca(2+) influx was eliminated by using low external Ca(2+) media, we found that approximately half the clones could still differentiate. Of the clones that required influx of external Ca(2+), most clones used T-type Ca(2+) channels, but others used store-operated channels as influx-generating mechanisms. On the other hand, clones that differentiated in low external Ca(2+) relied on Ca(2+) release from internal stores through IP(3) receptors. Interestingly, by following clones over time, we observed that some switched their preferred Ca(2+) source: clones that initially used calcium release from internal stores to differentiate later required Ca(2+) influx and inversely. In conclusion, we show that human myoblasts can use three alternative mechanisms to increase cytoplasmic Ca(2+) at the onset of the differentiation process: influx through T-types Ca(2+) channels, influx through store operated channels and release from internal stores through IP(3) receptors. In addition, we suggest that, probably because Ca(2+) elevation is essential during initial differentiation, myoblasts may be able to select between these alternate Ca(2+) pathways.

Calcium↗
PubMed2006

Pax6-induced alteration of cell fate: shape changes, expression of neuronal alpha tubulin, postmitotic phenotype, and cell migration.

The transcription factor Pax6 plays an important role in the development of the central nervous system. To understand its mechanism of action, we transduced HeLa cells with a Pax6-expressing lentiviral vector. Upon transduction, HeLa cells markedly changed shape and formed neuritelike extensions. Pax6-transduced HeLa cells expressed high levels of neuronal alpha3 tubulin, demonstrating a partial transdifferentiation towards a neuronal phenotype. Neurons are postmitotic cells. Pax6-transduced HeLa cells became postmitotic through mechanisms involving up-regulation of p53 and cyclin-dependent kinase inhibitor p21. One of the most striking effects of Pax6 was observed by time-lapse videomicroscopy: cells started to dissociate from cell clusters and displayed intense migratory activity. Migration was accompanied by dynamic and reversible shape changes. Our results identified three elements of Pax6 action: (i) expression of neuron-specific genes; (ii) establishment of a postmitotic phenotype; and (iii) involvement in the regulation of cell shape and cell migration.

Animals↗
PubMed2005

Subplasmalemmal mitochondria modulate the activity of plasma membrane Ca2+-ATPases.

Mitochondria are dynamic organelles that modulate cellular Ca2+ signals by interacting with Ca2+ transporters on the plasma membrane or the endoplasmic reticulum (ER). To study how mitochondria dynamics affects cell Ca2+ homeostasis, we overexpressed two mitochondrial fission proteins, hFis1 and Drp1, and measured Ca2+ 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 Ca2+ signals elicited by histamine were unaltered in cells lacking subplasmalemmal mitochondria as long as Ca2+ was present in the medium, but the signals were significantly blunted when Ca2+ was removed. Upon Ca2+ withdrawal, the free ER Ca2+ concentration decreased rapidly, and hFis1 cells were unable to respond to repetitive histamine stimulations. The loss of stored Ca2+ was due to an increased activity of plasma membrane Ca2+-ATPase (PMCA) pumps and was associated with an increased influx of Ca2+ and Mn2+ across store-operated Ca2+ channels. The increased Ca2+ influx compensated for the loss of stored Ca2+, and brief Ca2+ additions between successive agonist stimulations fully corrected subsequent histamine responses. We propose that the lack of subplasmalemmal mitochondria disrupts the transfer of Ca2+ from plasma membrane channels to the ER and that the resulting increase in subplasmalemmal [Ca2+] up-regulates the activity of PMCA. The increased Ca2+ extrusion promotes ER depletion and the subsequent activation of store-operated Ca2+ channels. Cells thus adapt to the lack of subplasmalemmal mitochondria by relying on external rather than on internal Ca2+ for signaling.

Calcium↗
PubMed2005

Gelsolin mediates calcium-dependent disassembly of Listeria actin tails.

The role of intracellular Ca2+ in the regulation of actin filament assembly and disassembly has not been clearly defined. We show that reduction of intracellular free Ca2+ concentration ([Ca2+]i) to <40 nM in Listeria monocytogenes-infected, EGFP-actin-transfected Madin-Darby canine kidney cells results in a 3-fold lengthening of actin filament tails. This increase in tail length is the consequence of marked slowing of the actin filament disassembly rate, without a significant change in assembly rate. The Ca2+-sensitive actin-severing protein gelsolin concentrates in the Listeria rocket tails at normal resting [Ca2+]i and disassociates from the tails when [Ca2+]i is lowered. Reduction in [Ca2+]i also blocks the severing activity of gelsolin, but not actin-depolymerizing factor (ADF)/cofilin microinjected into Listeria-infected cells. In Xenopus extracts, Listeria tail lengths are also calcium-sensitive, markedly shortening on addition of calcium. Immunodepletion of gelsolin, but not Xenopus ADF/cofilin, eliminates calcium-sensitive actin-filament shortening. Listeria tail length is also calcium-insensitive in gelsolin-null mouse embryo fibroblasts. We conclude that gelsolin is the primary Ca2+-sensitive actin filament recycling protein in the cell and is capable of enhancing Listeria actin tail disassembly at normal resting [Ca2+]i (145 nM). These experiments illustrate the unique and complementary functions of gelsolin and ADF/cofilin in the recycling of actin filaments.

Actin Depolymerizing Factors↗
PubMed2004

Membrane hyperpolarization triggers myogenin and myocyte enhancer factor-2 expression during human myoblast differentiation.

It is widely thought that myogenin is one of the earliest detectable markers of skeletal muscle differentiation. Here we show that, during human myoblast differentiation, an inward rectifier K(+) channel (Kir2.1) and its associated hyperpolarization trigger expression and activity of the myogenic transcription factors, myogenin and myocyte enhancer factor-2 (MEF2). Furthermore, Kir2.1 current precedes and is required for the developmental increase in expression/activity of myogenin and MEF2. Drugs or antisense reducing Kir2.1 current diminished or suppressed fusion as well as expression/activity of myogenin and MEF2. In contrast, LY294002, an inhibitor of phosphatidylinositol 3-kinase (a pathway controlling initiation of the myogenic program) that inhibited both myogenin/MEF2 expression and fusion, did not affect Kir2.1 current. This non-blockade by LY294002 indicates that Kir2.1 acts upstream of myogenin and MEF2. We propose that Kir2.1 channel activation is a required key early event that initiates myogenesis by turning on myogenin and MEF2 transcription factors via a hyperpolarization-activated Ca(2+)-dependent pathway.

Blotting, Western↗
PubMed2004

Extracellular hypotonicity increases Na,K-ATPase cell surface expression via enhanced Na+ influx in cultured renal collecting duct cells.

In the renal collecting duct (CD), the Na,K-ATPase, which provides the driving force for Na+ absorption, is under tight multifactorial control. Because CD cells are physiologically exposed to variations of interstitial and tubular fluid osmolarities, the effects of extracellular anisotonicity on Na,K-ATPase cell surface expression were studied. Results show that hypotonic conditions increased, whereas hypertonic conditions had no effect on Na,K-ATPase cell surface expression in confluent mpkCCDcl4 cells. Incubating cells with amphotericin B, which increases [Na+]i, under isotonic or anisotonic conditions, revealed that Na,K-ATPase recruitment to the cell surface was not directly related to variations of cell volume and osmolarity. The effects of amphotericin B and extracellular hypotonicity were not additive, and both were prevented by protein kinase A and proteasome inhibitors, suggesting a common mechanism of action. In line with this hypothesis, extracellular hypotonicity induced a sustained stimulation of the amiloride-sensitive short-circuit current, indicating increased Na+ influx through the apical epithelial Na+ channel. Moreover, inhibiting apical Na+ entry by amiloride, a blocker of epithelial Na+ channel, or incubating cells in Na+ -free medium prevented the cell surface recruitment of Na,K-ATPase in response to extracellular hypotonicity. Altogether, these findings strongly suggest that extracellular hypotonicity stimulates apical Na+ influx leading to increased [Na+]i, protein kinase A activation, and recruitment of Na,K-ATPase units to the cell surface of mpkCCDcl4 cells.

Adenosine Triphosphatases↗
PubMed2003

Intracellular transport of calcium from plasma membrane to mitochondria in adrenal H295R cells: implication for steroidogenesis.

Angiotensin II and extracellular potassium stimulate aldosterone production in adrenal glomerulosa cells by mobilizing the calcium messenger system. This response requires calcium influx across the plasma membrane, followed by calcium uptake into the mitochondria. It has been proposed that calcium is transported to the mitochondria via the lumen of the endoplasmic reticulum, acting as a kind of intracellular calcium pipeline. This hypothesis has been tested in the present study by measuring intramitochondrial calcium variations in H295R cells with a new fluorescent calcium probe, ratiometric pericam. Calyculin A, a protein phosphatase inhibitor, induced the formation of a large cortical layer of actin filaments, removing the peripheral endoplasmic reticulum away from the plasma membrane and thereby physically uncoupling the calcium channels from the pipeline. The mitochondrial calcium response to potassium was markedly reduced after calyculin treatment, but that of AngII was unaffected. Under the same conditions, potassium-stimulated pregnenolone and aldosterone production was significantly reduced, whereas the steroidogenic response to AngII remained unchanged. The inhibitory action of calyculin A on the responses to potassium was not mediated by a modification of the calcium channel activity and was not accompanied by a reduction of the cytosolic calcium response. It therefore appears that, in H295R cells, the organization of the actin cytoskeleton at the cell periphery influences the steroidogenic action of potassium, but not the response to angiotensin II. The response to potassium is proposed to be dependent on the endoplasmic reticulum-mediated transfer of calcium entering through plasma membrane calcium channels to the mitochondria.

Actins↗
PubMed2003

Acceleration of human myoblast fusion by depolarization: graded Ca2+ signals involved.

We have previously shown that human myoblasts do not fuse when their voltage fails to reach the domain of a window T-type Ca(2+) current. We demonstrate, by changing the voltage in the window domain, that the Ca(2+) signal initiating fusion is not of the all-or-none type, but can be graded and is interpreted as such by the differentiation program. This was carried out by exploiting the properties of human ether-à-go-go related gene K(+) channels that we found to be expressed in human myoblasts. Methanesulfonanilide class III antiarrhythmic agents or antisense-RNA vectors were used to suppress completely ether-à-go-go related gene current. Both procedures induced a reproducible depolarization from -74 to -64 mV, precisely in the window domain where the T-type Ca(2+) current increases with voltage. This 10 mV depolarization raised the cytoplasmic free Ca(2+) concentration, and triggered a tenfold acceleration of myoblast fusion. Our results suggest that any mechanism able to modulate intracellular Ca(2+) concentration could affect the rate of myoblast fusion.

Calcium↗
PubMed2002

Calreticulin differentially modulates calcium uptake and release in the endoplasmic reticulum and mitochondria.

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.

Apoptosis↗