Browse
Submit Data
Databases
API
Help

Dataset Information

33 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

BOK controls apoptosis by Ca²⁺ transfer through ER-mitochondrial contact sites.

ABSTRACT: Calcium transfer from the endoplasmic reticulum (ER) to mitochondria is a critical contributor to apoptosis. B cell lymphoma 2 (BCL-2) ovarian killer (BOK) localizes to the ER and binds the inositol 1,4,5-trisphosophate receptor (IP3R). Here, we show that BOK is necessary for baseline mitochondrial calcium levels and stimulus-induced calcium transfer from the ER to the mitochondria. Murine embryonic fibroblasts deficient for BOK have decreased proximity of the ER to the mitochondria and altered protein composition of mitochondria-associated membranes (MAMs), which form essential calcium microdomains. Rescue of the ER-mitochondrial juxtaposition with drug-inducible interorganelle linkers reveals a kinetic disruption, which when overcome in Bok^-/- cells is still insufficient to rescue thapsigargin-induced calcium transfer and apoptosis. Likewise, a BOK mutant unable to interact with IP3R restores ER-mitochondrial proximity, but not ER-mitochondrial calcium transfer, MAM protein composition, or apoptosis. This work identifies the dynamic coordination of ER-mitochondrial contact by BOK as an important control point for apoptosis.

SUBMITTER: Carpio MA

PROVIDER: S-EPMC7995216 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Reduced ER-mitochondrial contact sites and mitochondrial Ca2+ flux in PRKN-mutant patient tyrosine hydroxylase reporter iPSC lines.

Project description:Endoplasmic reticulum-mitochondrial contact sites (ERMCS) play an important role in mitochondrial dynamics, calcium signaling, and autophagy. Disruption of the ERMCS has been linked to several neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). However, the etiological role of ERMCS in these diseases remains unclear. We previously established tyrosine hydroxylase reporter (TH-GFP) iPSC lines from a PD patient with a PRKN mutation to perform correlative light-electron microscopy (CLEM) analysis and live cell imaging in GFP-expressing dopaminergic neurons. Here, we analyzed ERMCS in GFP-expressing PRKN-mutant dopaminergic neurons from patients using CLEM and a proximity ligation assay (PLA). The PLA showed that the ERMCS were significantly reduced in PRKN-mutant patient dopaminergic neurons compared to the control under normal conditions. The reduction of the ERMCS in PRKN-mutant patient dopaminergic neurons was further enhanced by treatment with a mitochondrial uncoupler. In addition, mitochondrial calcium imaging showed that mitochondrial Ca2+ flux was significantly reduced in PRKN-mutant patient dopaminergic neurons compared to the control. These results suggest a defect in calcium flux from ER to mitochondria is due to the decreased ERMCS in PRKN-mutant patient dopaminergic neurons. Our study of ERMCS using TH-GFP iPSC lines would contribute to further understanding of the mechanisms of dopaminergic neuron degeneration in patients with PRKN mutations.

| S-EPMC10514478 | biostudies-literature

ORP3 phosphorylation regulates phosphatidylinositol 4-phosphate and Ca2+ dynamics at plasma membrane-ER contact sites.

Project description:Oxysterol-binding protein (OSBP)-related proteins (ORPs) mediate non-vesicular lipid transfer between intracellular membranes. Phosphoinositide (PI) gradients play important roles in the ability of OSBP and some ORPs to transfer cholesterol and phosphatidylserine between the endoplasmic reticulum (ER) and other organelle membranes. Here, we show that plasma membrane (PM) association of ORP3 (also known as OSBPL3), a poorly characterized ORP family member, is triggered by protein kinase C (PKC) activation, especially when combined with Ca2+ increases, and is determined by both PI(4,5)P 2 and PI4P After activation, ORP3 efficiently extracts PI4P and to a lesser extent phosphatidic acid from the PM, and slightly increases PM cholesterol levels. Full activation of ORP3 resulted in decreased PM PI4P levels and inhibited Ca2+ entry via the store-operated Ca2+ entry pathway. The C-terminal region of ORP3 that follows the strictly defined lipid transfer domain was found to be critical for the proper localization and function of the protein.

| S-EPMC7097422 | biostudies-literature

PERK is required at the ER-mitochondrial contact sites to convey apoptosis after ROS-based ER stress.

Project description:Endoplasmic reticulum stress is emerging as an important modulator of different pathologies and as a mechanism contributing to cancer cell death in response to therapeutic agents. In several instances, oxidative stress and the onset of endoplasmic reticulum (ER) stress occur together; yet, the molecular events linking reactive oxygen species (ROS) to ER stress-mediated apoptosis are currently unknown. Here, we show that PERK (RNA-dependent protein kinase (PKR)-like ER kinase), a key ER stress sensor of the unfolded protein response, is uniquely enriched at the mitochondria-associated ER membranes (MAMs). PERK(-/-) cells display disturbed ER morphology and Ca(2+) signaling as well as significantly weaker ER-mitochondria contact sites. Re-expression of a kinase-dead PERK mutant but not the cytoplasmic deletion mutant of PERK in PERK(-/-) cells re-establishes ER-mitochondria juxtapositions and mitochondrial sensitization to ROS-mediated stress. In contrast to the canonical ER stressor thapsigargin, during ROS-mediated ER stress, PERK contributes to apoptosis twofold by sustaining the levels of pro-apoptotic C/EBP homologous protein (CHOP) and by facilitating the propagation of ROS signals between the ER and mitochondria through its tethering function. Hence, this study reveals an unprecedented role of PERK as a MAMs component required to maintain the ER-mitochondria juxtapositions and propel ROS-mediated mitochondrial apoptosis. Furthermore, it suggests that loss of PERK may cause defects in cell death sensitivity in pathological conditions linked to ROS-mediated ER stress.

| S-EPMC3469056 | biostudies-literature

Remodeling of ER-plasma membrane contact sites but not STIM1 phosphorylation inhibits Ca2+ influx in mitosis.

Project description:Store-operated Ca2+ entry (SOCE), mediated by the endoplasmic reticulum (ER) Ca2+ sensor stromal interaction molecule 1 (STIM1) and the plasma membrane (PM) channel Orai1, is inhibited during mitosis. STIM1 phosphorylation has been suggested to mediate this inhibition, but it is unclear whether additional pathways are involved. Here, we demonstrate using various approaches, including a nonphosphorylatable STIM1 knock-in mouse, that STIM1 phosphorylation is not required for SOCE inhibition in mitosis. Rather, multiple pathways converge to inhibit Ca2+ influx in mitosis. STIM1 interacts with the cochaperone BAG3 and localizes to autophagosomes in mitosis, and STIM1 protein levels are reduced. The density of ER-PM contact sites (CSs) is also dramatically reduced in mitosis, thus physically preventing STIM1 and Orai1 from interacting to activate SOCE. Our findings provide insights into ER-PM CS remodeling during mitosis and a mechanistic explanation of the inhibition of Ca2+ influx that is required for cell cycle progression.

| S-EPMC6535005 | biostudies-literature

BOK controls ER proteostasis and physiological ER stress responses in neurons.

Project description:The Bcl-2 family proteins BAK and BAX control the crucial step of pore formation in the mitochondrial outer membrane during intrinsic apoptosis. Bcl-2-related ovarian killer (BOK) is a Bcl-2 family protein with a high sequence similarity to BAK and BAX. However, intrinsic apoptosis can proceed in the absence of BOK. Unlike BAK and BAX, BOK is primarily located on the endoplasmic reticulum (ER) and Golgi membranes, suggesting a role for BOK in regulating ER homeostasis. In this study, we report that BOK is required for a full ER stress response. Employing previously characterized fluorescent protein-based ER stress reporter cell systems, we show that BOK-deficient cells have an attenuated response to ER stress in all three signaling branches of the unfolded protein response. Fluo-4-based confocal Ca2+ imaging revealed that disruption of ER proteostasis in BOK-deficient cells was not linked to altered ER Ca2+ levels. Fluorescence recovery after photobleaching (FRAP) experiments using GRP78/BiP-eGFP demonstrated that GRP78 motility was significantly lower in BOK-deficient cells. This implied that less intraluminal GRP78 was freely available and more of the ER chaperone bound to unfolded proteins. Collectively, these experiments suggest a new role for BOK in the protection of ER proteostasis and cellular responses to ER stress.

| S-EPMC9434404 | biostudies-literature

IP3 Receptors Preferentially Associate with ER-Lysosome Contact Sites and Selectively Deliver Ca2+ to Lysosomes.

Project description:Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) allow extracellular stimuli to redistribute Ca2+ from the ER to cytosol or other organelles. We show, using small interfering RNA (siRNA) and vacuolar H+-ATPase (V-ATPase) inhibitors, that lysosomes sequester Ca2+ released by all IP3R subtypes, but not Ca2+ entering cells through store-operated Ca2+ entry (SOCE). A low-affinity Ca2+ sensor targeted to lysosomal membranes reports large, local increases in cytosolic [Ca2+] during IP3-evoked Ca2+ release, but not during SOCE. Most lysosomes associate with endoplasmic reticulum (ER) and dwell at regions populated by IP3R clusters, but IP3Rs do not assemble ER-lysosome contacts. Increasing lysosomal pH does not immediately prevent Ca2+ uptake, but it causes lysosomes to slowly redistribute and enlarge, reduces their association with IP3Rs, and disrupts Ca2+ exchange with ER. In a "piston-like" fashion, ER concentrates cytosolic Ca2+ and delivers it, through large-conductance IP3Rs, to a low-affinity lysosomal uptake system. The involvement of IP3Rs allows extracellular stimuli to regulate Ca2+ exchange between the ER and lysosomes.

| S-EPMC6302550 | biostudies-literature

Sar1, a Novel Regulator of ER-Mitochondrial Contact Sites.

Project description:Endoplasmic reticulum (ER)-mitochondrial contact sites play a pivotal role in exchange of lipids and ions between the two organelles. How size and function of these contact sites are regulated remains elusive. Here we report a previously unanticipated, but conserved role of the small GTPase Sar1 in the regulation of ER-mitochondrial contact site size. Activated Sar1 introduces membrane curvature through its N-terminal amphiphatic helix at the ER-mitochondria interphase and thereby reducing contact size. Conversely, the S. cerevisiae N3-Sar1 mutant, in which curvature induction is decreased, caused an increase in ER-mitochondrial contacts. As a consequence, ER tubules are no longer able to mark the prospective scission site on mitochondria, thereby impairing mitochondrial dynamics. Consistently, blocking mitochondrial fusion partially rescued, whereas deletion of the dynamin-like protein enhanced the phenotype in the sar1D32G mutant. We conclude that Sar1 regulates the size of ER-mitochondria contact sites through its effects on membrane curvature.

| S-EPMC4839682 | biostudies-literature

E-syt1 Re-arranges STIM1 Clusters to Stabilize Ring-shaped ER-PM Contact Sites and Accelerate Ca2+ Store Replenishment.

Project description:In many non-excitable cells, the depletion of endoplasmic reticulum (ER) Ca2+ stores leads to the dynamic formation of membrane contact sites (MCSs) between the ER and the plasma membrane (PM), which activates the store-operated Ca2+ entry (SOCE) to refill the ER store. Two different Ca2+-sensitive proteins, STIM1 and extended synaptotagmin-1 (E-syt1), are activated during this process. Due to the lack of live cell super-resolution imaging, how MCSs are dynamically regulated by STIM1 and E-syt1 coordinately during ER Ca2+ store depletion and replenishment remain unknown. With home-built super-resolution microscopes that provide superior axial and lateral resolution in live cells, we revealed that extracellular Ca2+ influx via SOCE activated E-syt1s to move towards the PM by ~12 nm. Unexpectedly, activated E-syt1s did not constitute the MCSs per se, but re-arranged neighboring ER structures into ring-shaped MCSs (230~280 nm in diameter) enclosing E-syt1 puncta, which helped to stabilize MCSs and accelerate local ER Ca2+ replenishment. Overall, we have demonstrated different roles of STIM1 and E-syt1 in MCS formation regulation, SOCE activation and ER Ca2+ store replenishment.

| S-EPMC6408583 | biostudies-literature

An Endosomal NAADP-Sensitive Two-Pore Ca2+ Channel Regulates ER-Endosome Membrane Contact Sites to Control Growth Factor Signaling.

Project description:Membrane contact sites are regions of close apposition between organelles that facilitate information transfer. Here, we reveal an essential role for Ca2+ derived from the endo-lysosomal system in maintaining contact between endosomes and the endoplasmic reticulum (ER). Antagonizing action of the Ca2+-mobilizing messenger NAADP, inhibiting its target endo-lysosomal ion channel, TPC1, and buffering local Ca2+ fluxes all clustered and enlarged late endosomes/lysosomes. We show that TPC1 localizes to ER-endosome contact sites and is required for their formation. Reducing NAADP-dependent contacts delayed EGF receptor de-phosphorylation consistent with close apposition of endocytosed receptors with the ER-localized phosphatase PTP1B. In accord, downstream MAP kinase activation and mobilization of ER Ca2+ stores by EGF were exaggerated upon NAADP blockade. Membrane contact sites between endosomes and the ER thus emerge as Ca2+-dependent hubs for signaling.

| S-EPMC5318655 | biostudies-literature

MITOL prevents ER stress-induced apoptosis by IRE1α ubiquitylation at ER-mitochondria contact sites.

Project description:Unresolved endoplasmic reticulum (ER) stress shifts the unfolded protein response signaling from cell survival to cell death, although the switching mechanism remains unclear. Here, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) inhibits ER stress-induced apoptosis through ubiquitylation of IRE1α at the mitochondria-associated ER membrane (MAM). MITOL promotes K63-linked chain ubiquitination of IRE1α at lysine 481 (K481), thereby preventing hyper-oligomerization of IRE1α and regulated IRE1α-dependent decay (RIDD). Therefore, under ER stress, MITOL depletion or the IRE1α mutant (K481R) allows for IRE1α hyper-oligomerization and enhances RIDD activity, resulting in apoptosis. Similarly, in the spinal cord of MITOL-deficient mice, ER stress enhances RIDD activity and subsequent apoptosis. Notably, unresolved ER stress attenuates IRE1α ubiquitylation, suggesting that this directs the apoptotic switch of IRE1α signaling. Our findings suggest that mitochondria regulate cell fate under ER stress through IRE1α ubiquitylation by MITOL at the MAM.

| S-EPMC6669929 | biostudies-literature