Limits...
Mitochondrial regulation of store-operated calcium signaling in T lymphocytes.

Hoth M, Fanger CM, Lewis RS - J. Cell Biol. (1997)

Bottom Line: Ca2+ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca2+), rapid (detectable within 8 s), and does not readily saturate.Under these conditions, the rate of Ca2+ influx in single cells undergoes abrupt transitions from a high influx to a low influx state.These results demonstrate that mitochondria not only buffer the Ca2+ that enters T cells via store-operated Ca2+ channels, but also play an active role in modulating the rate of capacitative Ca2+ entry.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, California 94305-5426, USA. mhoth@leland.stanford.edu

ABSTRACT
Mitochondria act as potent buffers of intracellular Ca2+ in many cells, but a more active role in modulating the generation of Ca2+ signals is not well established. We have investigated the ability of mitochondria to modulate store-operated or "capacitative" Ca2+ entry in Jurkat leukemic T cells and human T lymphocytes using fluorescence imaging techniques. Depletion of the ER Ca2+ store with thapsigargin (TG) activates Ca2+ release-activated Ca2+ (CRAC) channels in T cells, and the ensuing influx of Ca2+ loads a TG-insensitive intracellular store that by several criteria appears to be mitochondria. Loading of this store is prevented by carbonyl cyanide m-chlorophenylhydrazone or by antimycin A1 + oligomycin, agents that are known to inhibit mitochondrial Ca2+ import by dissipating the mitochondrial membrane potential. Conversely, intracellular Na+ depletion, which inhibits Na+-dependent Ca2+ export from mitochondria, enhances store loading. In addition, we find that rhod-2 labels mitochondria in T cells, and it reports changes in Ca2+ levels that are consistent with its localization in the TG-insensitive store. Ca2+ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca2+), rapid (detectable within 8 s), and does not readily saturate. The rate of mitochondrial Ca2+ uptake is sensitive to extracellular [Ca2+], indicating that mitochondria sense Ca2+ gradients near CRAC channels. Remarkably, mitochondrial uncouplers or Na+ depletion prevent the ability of T cells to maintain a high rate of capacitative Ca2+ entry over prolonged periods of >10 min. Under these conditions, the rate of Ca2+ influx in single cells undergoes abrupt transitions from a high influx to a low influx state. These results demonstrate that mitochondria not only buffer the Ca2+ that enters T cells via store-operated Ca2+ channels, but also play an active role in modulating the rate of capacitative Ca2+ entry.

Show MeSH

Related in: MedlinePlus

Rhod-2/AM labels mitochondria and reports changes in intramitochondrial [Ca2+]. (A) Confocal micrographs of Jurkat cells  after incubation with rhod-2/AM (left) or MitoTracker™ Green FM, a mitochondrial marker (center). Rhod-2 labeling of mitochondria  (arrowhead) is easily distinguished from labeling of the nucleolus (arrow). An overlay of the two images demonstrates specific colocalization of the two dyes in the mitochondria (right). (B) Mitochondrial Ca2+ imaging with rhod-2. In these pseudocolored video microscopic images of a single TG-treated cell, rhod-2 fluorescence in the mitochondria increases within 3 min after readdition of 2 mM Ca2+  (top). 1 μM CCCP prevents the fluorescence increase in another cell (bottom). Warmer colors indicate higher rhod-2 fluorescence. (C)  Quantification of rhod-2 fluorescence in “hot spots” in cells of the experiment shown in B. Hot spot fluorescence was measured before  and 3 min after readdition of 2 mM Ca2+. The fluorescence intensity before readdition was normalized to one. 35 cells (control) and 28  cells (+CCCP) were analyzed. Error bars reflect standard deviations. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139882&req=5

Figure 5: Rhod-2/AM labels mitochondria and reports changes in intramitochondrial [Ca2+]. (A) Confocal micrographs of Jurkat cells after incubation with rhod-2/AM (left) or MitoTracker™ Green FM, a mitochondrial marker (center). Rhod-2 labeling of mitochondria (arrowhead) is easily distinguished from labeling of the nucleolus (arrow). An overlay of the two images demonstrates specific colocalization of the two dyes in the mitochondria (right). (B) Mitochondrial Ca2+ imaging with rhod-2. In these pseudocolored video microscopic images of a single TG-treated cell, rhod-2 fluorescence in the mitochondria increases within 3 min after readdition of 2 mM Ca2+ (top). 1 μM CCCP prevents the fluorescence increase in another cell (bottom). Warmer colors indicate higher rhod-2 fluorescence. (C) Quantification of rhod-2 fluorescence in “hot spots” in cells of the experiment shown in B. Hot spot fluorescence was measured before and 3 min after readdition of 2 mM Ca2+. The fluorescence intensity before readdition was normalized to one. 35 cells (control) and 28 cells (+CCCP) were analyzed. Error bars reflect standard deviations. Bar, 5 μm.

Mentions: For qualitative measurements of mitochondrial Ca2+, cells were loaded with rhod-2/AM (Molecular Probes) in Ringer's solution at a concentration of 4–8 μM (2 mM stock in DMSO) for 30–40 min at 20°–25°C. Cells were illuminated at 540 ± 12 nm (bandpass filter; Chroma Technology Corp.), and the fluorescence emission at 605 ± 28 nm (bandpass filter; Chroma Technology Corp.) was captured and analyzed as described above for fura-2. For these experiments, a ×63 Zeiss Neofluar objective (NA 1.25) was used. Movement of mitochondria posed a serious problem for time-lapse imaging. To minimize movement artifacts, we first flattened the cells by attaching them to poly-ornithine–coated coverslips followed by incubation for 30 min with 10 μg/ml phytohemagglutinin in Ringer's solution. In control experiments using fura-2–loaded cells, phytohemagglutinin did not appear to affect Ca2+ plateaus or responses to mitochondrial inhibitors, implying that it does not alter mitochondrial Ca2+ uptake. The microscope was focused on the mitochondria immediately before collecting each set of data (see Fig. 5, B and C). Rhod-2 fluorescence was not calibrated in terms of [Ca2+]m but is expressed relative to the initial fluorescence intensity.


Mitochondrial regulation of store-operated calcium signaling in T lymphocytes.

Hoth M, Fanger CM, Lewis RS - J. Cell Biol. (1997)

Rhod-2/AM labels mitochondria and reports changes in intramitochondrial [Ca2+]. (A) Confocal micrographs of Jurkat cells  after incubation with rhod-2/AM (left) or MitoTracker™ Green FM, a mitochondrial marker (center). Rhod-2 labeling of mitochondria  (arrowhead) is easily distinguished from labeling of the nucleolus (arrow). An overlay of the two images demonstrates specific colocalization of the two dyes in the mitochondria (right). (B) Mitochondrial Ca2+ imaging with rhod-2. In these pseudocolored video microscopic images of a single TG-treated cell, rhod-2 fluorescence in the mitochondria increases within 3 min after readdition of 2 mM Ca2+  (top). 1 μM CCCP prevents the fluorescence increase in another cell (bottom). Warmer colors indicate higher rhod-2 fluorescence. (C)  Quantification of rhod-2 fluorescence in “hot spots” in cells of the experiment shown in B. Hot spot fluorescence was measured before  and 3 min after readdition of 2 mM Ca2+. The fluorescence intensity before readdition was normalized to one. 35 cells (control) and 28  cells (+CCCP) were analyzed. Error bars reflect standard deviations. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139882&req=5

Figure 5: Rhod-2/AM labels mitochondria and reports changes in intramitochondrial [Ca2+]. (A) Confocal micrographs of Jurkat cells after incubation with rhod-2/AM (left) or MitoTracker™ Green FM, a mitochondrial marker (center). Rhod-2 labeling of mitochondria (arrowhead) is easily distinguished from labeling of the nucleolus (arrow). An overlay of the two images demonstrates specific colocalization of the two dyes in the mitochondria (right). (B) Mitochondrial Ca2+ imaging with rhod-2. In these pseudocolored video microscopic images of a single TG-treated cell, rhod-2 fluorescence in the mitochondria increases within 3 min after readdition of 2 mM Ca2+ (top). 1 μM CCCP prevents the fluorescence increase in another cell (bottom). Warmer colors indicate higher rhod-2 fluorescence. (C) Quantification of rhod-2 fluorescence in “hot spots” in cells of the experiment shown in B. Hot spot fluorescence was measured before and 3 min after readdition of 2 mM Ca2+. The fluorescence intensity before readdition was normalized to one. 35 cells (control) and 28 cells (+CCCP) were analyzed. Error bars reflect standard deviations. Bar, 5 μm.
Mentions: For qualitative measurements of mitochondrial Ca2+, cells were loaded with rhod-2/AM (Molecular Probes) in Ringer's solution at a concentration of 4–8 μM (2 mM stock in DMSO) for 30–40 min at 20°–25°C. Cells were illuminated at 540 ± 12 nm (bandpass filter; Chroma Technology Corp.), and the fluorescence emission at 605 ± 28 nm (bandpass filter; Chroma Technology Corp.) was captured and analyzed as described above for fura-2. For these experiments, a ×63 Zeiss Neofluar objective (NA 1.25) was used. Movement of mitochondria posed a serious problem for time-lapse imaging. To minimize movement artifacts, we first flattened the cells by attaching them to poly-ornithine–coated coverslips followed by incubation for 30 min with 10 μg/ml phytohemagglutinin in Ringer's solution. In control experiments using fura-2–loaded cells, phytohemagglutinin did not appear to affect Ca2+ plateaus or responses to mitochondrial inhibitors, implying that it does not alter mitochondrial Ca2+ uptake. The microscope was focused on the mitochondria immediately before collecting each set of data (see Fig. 5, B and C). Rhod-2 fluorescence was not calibrated in terms of [Ca2+]m but is expressed relative to the initial fluorescence intensity.

Bottom Line: Ca2+ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca2+), rapid (detectable within 8 s), and does not readily saturate.Under these conditions, the rate of Ca2+ influx in single cells undergoes abrupt transitions from a high influx to a low influx state.These results demonstrate that mitochondria not only buffer the Ca2+ that enters T cells via store-operated Ca2+ channels, but also play an active role in modulating the rate of capacitative Ca2+ entry.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, California 94305-5426, USA. mhoth@leland.stanford.edu

ABSTRACT
Mitochondria act as potent buffers of intracellular Ca2+ in many cells, but a more active role in modulating the generation of Ca2+ signals is not well established. We have investigated the ability of mitochondria to modulate store-operated or "capacitative" Ca2+ entry in Jurkat leukemic T cells and human T lymphocytes using fluorescence imaging techniques. Depletion of the ER Ca2+ store with thapsigargin (TG) activates Ca2+ release-activated Ca2+ (CRAC) channels in T cells, and the ensuing influx of Ca2+ loads a TG-insensitive intracellular store that by several criteria appears to be mitochondria. Loading of this store is prevented by carbonyl cyanide m-chlorophenylhydrazone or by antimycin A1 + oligomycin, agents that are known to inhibit mitochondrial Ca2+ import by dissipating the mitochondrial membrane potential. Conversely, intracellular Na+ depletion, which inhibits Na+-dependent Ca2+ export from mitochondria, enhances store loading. In addition, we find that rhod-2 labels mitochondria in T cells, and it reports changes in Ca2+ levels that are consistent with its localization in the TG-insensitive store. Ca2+ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca2+), rapid (detectable within 8 s), and does not readily saturate. The rate of mitochondrial Ca2+ uptake is sensitive to extracellular [Ca2+], indicating that mitochondria sense Ca2+ gradients near CRAC channels. Remarkably, mitochondrial uncouplers or Na+ depletion prevent the ability of T cells to maintain a high rate of capacitative Ca2+ entry over prolonged periods of >10 min. Under these conditions, the rate of Ca2+ influx in single cells undergoes abrupt transitions from a high influx to a low influx state. These results demonstrate that mitochondria not only buffer the Ca2+ that enters T cells via store-operated Ca2+ channels, but also play an active role in modulating the rate of capacitative Ca2+ entry.

Show MeSH
Related in: MedlinePlus