Requirement of biphasic calcium release from the endoplasmic reticulum for Fas-mediated apoptosis

Wozniak, Ann L.; Wang, Xinmin; Stieren, Emily S.; Scarbrough, Shelby G.; Elferink, Cornelis; Boehning, Darren

doi:10.1083/jcb.200608035

Cited by 79 publications

(110 citation statements)

References 27 publications

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“…This is because HeLa cells, like most cells in culture, flatten out significantly when adherent obviating the need for confocal microscopy or other specialized equipment. We have found similar results in Jurkat cells adhered to poly-lysine coated coverslips 8 . In contrast to methodologies using dyes, it is also possible to non-invasively monitor mitochondrial calcium levels for hours using genetically encoded calcium indicators such as ratiometric pericam ( Figure 1E).…”

Section: Discussionsupporting

confidence: 79%

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Akimzhanov

Boehning

2011

JoVE

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Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis 1 . During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol 2 . It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. Highresolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology . Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 μM 4 . These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosisinducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

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Section: Discussionsupporting

confidence: 79%

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Akimzhanov

Boehning

2011

JoVE

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“…It interacts with Bcl-X L in the ER and regulates calcium signaling (29). Fas-mediated apoptosis shares some features with intrinsic death pathways, such as ER stress (43,44), and promotes a rise in intracellular calcium (45,46), which mediates some apoptotic features attributed to FasL-induced cell death (35). The cross-talk between the ER and mitochondria and the calcium efflux between them are important steps in Fas apoptotic signaling, at least in the immune system (47).…”

Section: Discussionmentioning

confidence: 99%

“…LFG Overexpression Down-regulates Calcium Release from the ER after FasL Stimulation-Calcium transfer from the ER to mitochondria is a crucial step in various forms of cell death (33,34), including FasL-induced apoptosis, as demonstrated in experiments in Jurkat cells (35). Bcl-X L exerts anti-apoptotic effects in the ER by blocking calcium release (36,37).…”

Section: Requirement Of Endogenous Bcl-x L For Lfg Anti-apoptotic Effmentioning

confidence: 99%

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Lifeguard Inhibits Fas Ligand-mediated Endoplasmic Reticulum-Calcium Release Mandatory for Apoptosis in Type II Apoptotic Cells

Urresti

Ruiz‐Meana

Coccia

et al. 2016

Journal of Biological Chemistry

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Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by coimmunoprecipitation experiments that LFG interacts with Bcl-X L and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-X L in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasLinduced death in a Bcl-X L dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG.

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“…37,41,42 To determine if this occurs in response to PCI-34051, the levels of cytosolic and mitochondrial cytochrome c were measured after fractionation at various timepoints. As shown in Figure 7, at 12 h in Jurkat cells, cytosolic cytochrome c is observed in both the PCI-34051 and FasL-treated samples, and this is increased at 24 h in both samples.…”

Section: Pci-34051 Induces Cytochrome C Release From Mitochondriamentioning

confidence: 99%

A novel histone deacetylase 8 (HDAC8)-specific inhibitor PCI-34051 induces apoptosis in T-cell lymphomas

et al. 2008

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We have developed a potent, histone deacetylase 8 (HDAC8)-specific inhibitor PCI-34051 with 4200-fold selectivity over the other HDAC isoforms. PCI-34051 induces caspase-dependent apoptosis in cell lines derived from T-cell lymphomas or leukemias, but not in other hematopoietic or solid tumor lines. Unlike broad-spectrum HDAC inhibitors, PCI-34051 does not cause detectable histone or tubulin acetylation. Cells defective in T-cell receptor signaling were still sensitive to PCI-34051-induced apoptosis, whereas a phospholipase C-c1 (PLCc1)-defective line was resistant. Jurkat cells showed a dosedependent decrease in PCI-34051-induced apoptosis upon treatment with a PLC inhibitor U73122, but not with an inactive analog. We found that rapid intracellular calcium mobilization from endoplasmic reticulum (ER) and later cytochrome c release from mitochondria are essential for the apoptotic mechanism. The rapid Ca 2 þ flux was dependent on PCI-34051 concentration, and was blocked by the PLC inhibitor U73122. Further, apoptosis was blocked by Ca 2 þ chelators (BAPTA) and enhanced by Ca 2 þ effectors (thapsigargin), supporting this model. These studies show that HDAC8-selective inhibitors have a unique mechanism of action involving PLCc1 activation and calcium-induced apoptosis, and could offer benefits including a greater therapeutic index for treating T-cell malignancies.

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Requirement of biphasic calcium release from the endoplasmic reticulum for Fas-mediated apoptosis

Cited by 79 publications

References 27 publications

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Lifeguard Inhibits Fas Ligand-mediated Endoplasmic Reticulum-Calcium Release Mandatory for Apoptosis in Type II Apoptotic Cells

A novel histone deacetylase 8 (HDAC8)-specific inhibitor PCI-34051 induces apoptosis in T-cell lymphomas

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