Tünde Golenár

Imaging Interorganelle Contacts and Local Calcium Dynamics at the ER-Mitochondrial Interface

The ER-mitochondrial junction provides a local calcium signaling domain that is critical for both matching energy production with demand and the control of apoptosis. Here, we visualize ER-mitochondrial contact sites and monitor the localized [Ca(2+)] changes ([Ca(2+)](ER-mt)) using drug-inducible fluorescent interorganelle linkers. We show that all mitochondria have contacts with the ER, but plasma membrane (PM)-mitochondrial contacts are less frequent because of interleaving ER stacks in both RBL-2H3 and H9c2 cells. Single mitochondria display discrete patches of ER contacts and show heterogeneity in the ER-mitochondrial Ca(2+) transfer. Pericam-tagged linkers revealed IP(3)-induced [Ca(2+)](ER-mt) signals that exceeded 9 microM and endured buffering bulk cytoplasmic [Ca(2+)] increases. Altering linker length to modify the space available for the Ca(2+) transfer machinery had a biphasic effect on [Ca(2+)](ER-mt) signals. These studies provide direct evidence for the existence of high-Ca(2+) microdomains between the ER and mitochondria and suggest an optimal gap width for efficient Ca(2+) transfer.

MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism

The mitochondrial calcium uniporter (MCU) mediates calcium uptake by mitochondria and thus regulates cellular bioenergetics, but how MCU activity is modulated is not fully understood. Madesh, Foskett and colleagues report that the integral mitochondrial membrane protein MCUR1 (mitochondrial calcium uniporter regulator 1) binds to the MCU and promotes MCU-dependent calcium uptake to control ATP production and autophagy.

MICU1 controls both the threshold and cooperative activation of the mitochondrial Ca2+ uniporter

Mitochondrial Ca(2+) uptake via the uniporter is central to cell metabolism, signaling, and survival. Recent studies identified MCU as the uniporters likely pore and MICU1, an EF-hand protein, as its critical regulator. How this complex decodes dynamic cytoplasmic [Ca(2+)] ([Ca(2+)]c) signals, to tune out small [Ca(2+)]c increases yet permit pulse transmission, remains unknown. We report that loss of MICU1 in mouse liver and cultured cells causes mitochondrial Ca(2+) accumulation during small [Ca(2+)]c elevations but an attenuated response to agonist-induced [Ca(2+)]c pulses. The latter reflects loss of positive cooperativity, likely via the EF-hands. MICU1 faces the intermembrane space and responds to [Ca(2+)]c changes. Prolonged MICU1 loss leads to an adaptive increase in matrix Ca(2+) binding, yet cells show impaired oxidative metabolism and sensitization to Ca(2+) overload. Collectively, the data indicate that MICU1 senses the [Ca(2+)]c to establish the uniporters threshold and gain, thereby allowing mitochondria to properly decode different inputs.

Isoform- and Species-specific Control of Inositol 1,4,5-Trisphosphate (IP3) Receptors by Reactive Oxygen Species

Background: Reactive oxygen species (ROS) affect cytoplasmic calcium signaling. Results: Superoxide anion causes oxidation of the IP3 receptor and sensitization of calcium release to promote cytoplasmic calcium oscillations and mitochondrial calcium uptake. Conclusion: Physiologically relevant ROS controls cytoplasmic and mitochondrial calcium transport through IP3 receptors. Significance: Mechanisms of calcium and ROS interactions are relevant for both physiological and pathophysiological signaling. Reactive oxygen species (ROS) stimulate cytoplasmic [Ca2+] ([Ca2+]c) signaling, but the exact role of the IP3 receptors (IP3R) in this process remains unclear. IP3Rs serve as a potential target of ROS produced by both ER and mitochondrial enzymes, which might locally expose IP3Rs at the ER-mitochondrial associations. Also, IP3Rs contain multiple reactive thiols, common molecular targets of ROS. Therefore, we have examined the effect of superoxide anion (O2⨪) on IP3R-mediated Ca2+ signaling. In human HepG2, rat RBL-2H3, and chicken DT40 cells, we observed [Ca2+]c spikes and frequency-modulated oscillations evoked by a O2⨪ donor, xanthine (X) + xanthine oxidase (XO), dose-dependently. The [Ca2+]c signal was mediated by ER Ca2+ mobilization. X+XO added to permeabilized cells promoted the [Ca2+]c rise evoked by submaximal doses of IP3, indicating that O2⨪ directly sensitizes IP3R-mediated Ca2+ release. In response to X+XO, DT40 cells lacking two of three IP3R isoforms (DKO) expressing either type 1 (DKO1) or type 2 IP3Rs (DKO2) showed a [Ca2+]c signal, whereas DKO expressing type 3 IP3R (DKO3) did not. By contrast, IgM that stimulates IP3 formation, elicited a [Ca2+]c signal in every DKO. X+XO also facilitated the Ca2+ release evoked by submaximal IP3 in permeabilized DKO1 and DKO2 but was ineffective in DKO3 or in DT40 lacking every IP3R (TKO). However, X+XO could also facilitate the effect of suboptimal IP3 in TKO transfected with rat IP3R3. Although in silico studies failed to identify a thiol missing in the chicken IP3R3, an X+XO-induced redox change was documented only in the rat IP3R3. Thus, ROS seem to specifically sensitize IP3Rs through a thiol group(s) within the IP3R, which is probably inaccessible in the chicken IP3R3.

Calcium transport across the inner mitochondrial membrane: Molecular mechanisms and pharmacology

Growing evidence supports that mitochondrial calcium uptake is important for cell metabolism, signaling and survival. However, both the molecular nature of the mitochondrial Ca(2+) transport sites and the calcium signals they respond to remained elusive. Recent RNA interference studies have identified new candidate proteins for Ca(2+) transport across the inner mitochondrial membrane, including LETM1, MCU, MICU1 and NCLX. The sensitivity of these factors to several drugs has been tested and in parallel, some new inhibitors of mitochondrial Ca(2+) uptake have been described. This paper provides an update on the pharmacological aspects of the molecular mechanisms of the inner mitochondrial membrane Ca(2+) transport.

Reliance of ER-mitochondrial calcium signaling on mitochondrial EF-hand Ca2+ binding proteins. Miros, MICUs, LETM1 and solute carriers

Endoplasmic reticulum (ER) and mitochondria are functionally distinct with regard to membrane protein biogenesis and oxidative energy production, respectively, but cooperate in several essential cell functions, including lipid biosynthesis, cell signaling and organelle dynamics. The interorganellar cooperation requires local communication that can occur at the strategically positioned and dynamic associations between ER and mitochondria. Calcium is locally transferred from ER to mitochondria at the associations and exerts regulatory effects on numerous proteins. A common Ca(2+) sensing mechanism is the EF-hand Ca(2+) binding domain, many of which can be found in proteins of the mitochondria, including Miro1&2, MICU1,2&3, LETM1 and mitochondrial solute carriers. Recently, these proteins have triggered much interest and were described in reports with diverging conclusions. The present essay focuses on their shared features and established specific functions.

Tissue-Specific Mitochondrial Decoding of Cytoplasmic Ca2+ Signals is Controlled by the Stoichiometry of MICU1/2 and MCU

SUMMARY Mitochondrial Ca2+ uptake through the Ca2+ uniporter supports cell functions, including oxidative metabolism, while meeting tissue-specific calcium signaling patterns and energy needs. The molecular mechanisms underlying tissue-specific control of the uniporter are unknown. Here, we investigated a possible role for tissue-specific stoichiometry between the Ca2+-sensing regulators (MICUs) and pore unit (MCU) of the uniporter. Low MICU1:MCU protein ratio lowered the [Ca2+] threshold for Ca2+ uptake and activation of oxidative metabolism but decreased the cooperativity of uniporter activation in heart and skeletal muscle compared to liver. In MICU1-overexpressing cells, MICU1 was pulled down by MCU proportionally to MICU1 overexpression, suggesting that MICU1:MCU protein ratio directly reflected their association. Overexpressing MICU1 in the heart increased MICU1:MCU ratio, leading to liver-like mitochondrial Ca2+ uptake phenotype and cardiac contractile dysfunction. Thus, the proportion of MICU1-free and MICU1-associated MCU controls these tissue-specific uniporter phenotypes and downstream Ca2+ tuning of oxidative metabolism.

Natural and Induced Mitochondrial Phosphate Carrier Loss DIFFERENTIAL DEPENDENCE OF MITOCHONDRIAL METABOLISM AND DYNAMICS AND CELL SURVIVAL ON THE EXTENT OF DEPLETION

The relevance of mitochondrial phosphate carrier (PiC), encoded by SLC25A3, in bioenergetics is well accepted. However, little is known about the mechanisms mediating the cellular impairments induced by pathological SLC25A3 variants. To this end, we investigated the pathogenicity of a novel compound heterozygous mutation in SLC25A3. First, each variant was modeled in yeast, revealing that substituting GSSAS for QIP within the fifth matrix loop is incompatible with survival on non-fermentable substrate, whereas the L200W variant is functionally neutral. Next, using skin fibroblasts from an individual expressing these variants and HeLa cells with varying degrees of PiC depletion, PiC loss of ∼60% was still compatible with uncompromised maximal oxidative phosphorylation (oxphos), whereas lower maximal oxphos was evident at ∼85% PiC depletion. Furthermore, intact mutant fibroblasts displayed suppressed mitochondrial bioenergetics consistent with a lower substrate availability rather than phosphate limitation. This was accompanied by slowed proliferation in glucose-replete medium; however, proliferation ceased when only mitochondrial substrate was provided. Both mutant fibroblasts and HeLa cells with 60% PiC loss showed a less interconnected mitochondrial network and a mitochondrial fusion defect that is not explained by altered abundance of OPA1 or MFN1/2 or relative amount of different OPA1 forms. Altogether these results indicate that PiC depletion may need to be profound (>85%) to substantially affect maximal oxphos and that pathogenesis associated with PiC depletion or loss of function may be independent of phosphate limitation when ATP requirements are not high.

Mitochondrial Ca2+ Uptake; Regulation by Ca2+, Inhibition by Minocyclin

Mitochondrial Ca2+ uptake is mediated by the low-affinity Ca2+ uniporter (MCU) that is controlled allosterically by Ca2+. The Ca2+ effect might be mediated by MICU1, a recently identified obligatory component of MCU. Ca2+ induces sensitization of MCU but some reports claimed Ca2+-induced desensitization. Here, we compared recovery kinetics of cytosolic [Ca2+] ([Ca2+]c) elevations following addition of varying Ca2+ doses in suspensions of permeabilized RBL-2H3 cells. The recovery rates (reflecting mitochondrial Ca2+ uptake) progressively increased with the Ca2+ dose. The [Ca2+]c recovery steady state was similar after 7.5-50uM CaCl2 pulses, but was reached faster after the larger Ca2+ doses. Thus, a pre-exposure to high [Ca2+]c enhanced the permeability of MCU in a prolonged manner resulting in accelerated mitochondrial Ca2+ uptake even at low [Ca2+]c. We are currently investigating the molecular mechanism underlying the effect of Ca2+.The most specific pharmacological inhibitor of MCU is Ru360 that has limited plasma membrane permeability. Minocyclin, an anti-inflammatory drug has been suggested to exert its cytoprotective and anti-apoptotic effect via interfering with mitochondrial Ca2+ uptake, based on studies of isolated mitochondria. However, minocyclin has also been shown to act as a Ca2+-dependent protonophore. We found that minocyclin inhibits the rapid phase of IP3-induced mitochondrial matrix [Ca2+] response at concentrations of 80-120uM in permeabilized RBL-2H3 cells almost as effectively as Ru360 (80% vs. 92% inhibition, respectively). In minocyclin-pretreated cells, the IP3-induced Ca2+ release caused only slow and small mitochondrial depolarization, while bulk [Ca2+]c elevation by addition of large amounts of Ca2+ elicited almost complete depolarization. These data suggest that the protonophore action of minocyclin requires relatively large [Ca2+]c. Thus, minocyclin seems to be an effective inhibitor of mitochondrial Ca2+ uptake sites during physiological [Ca2+]c signals and exerts its protonophore effect under conditions of large global Ca2+ exposure.