Diego De Stefani

A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter

Mitochondrial Ca2+ homeostasis has a key role in the regulation of aerobic metabolism and cell survival, but the molecular identity of the Ca2+ channel, the mitochondrial calcium uniporter, is still unknown. Here we have identified in silico a protein (named MCU) that shares tissue distribution with MICU1 (also known as CBARA1), a recently characterized uniporter regulator, is present in organisms in which mitochondrial Ca2+ uptake was demonstrated and whose sequence includes two transmembrane domains. Short interfering RNA (siRNA) silencing of MCU in HeLa cells markedly reduced mitochondrial Ca2+ uptake. MCU overexpression doubled the matrix Ca2+ concentration increase evoked by inositol 1,4,5-trisphosphate-generating agonists, thus significantly buffering the cytosolic elevation. The purified MCU protein showed channel activity in planar lipid bilayers, with electrophysiological properties and inhibitor sensitivity of the uniporter. A mutant MCU, in which two negatively charged residues of the putative pore-forming region were replaced, had no channel activity and reduced agonist-dependent matrix Ca2+ concentration transients when overexpressed in HeLa cells. Overall, these data demonstrate that the 40-kDa protein identified is the channel responsible for ruthenium-red-sensitive mitochondrial Ca2+ uptake, thus providing a molecular basis for this process of utmost physiological and pathological relevance.

Mitochondria as sensors and regulators of calcium signalling

During the past two decades calcium (Ca2+) accumulation in energized mitochondria has emerged as a biological process of utmost physiological relevance. Mitochondrial Ca2+ uptake was shown to control intracellular Ca2+ signalling, cell metabolism, cell survival and other cell-type specific functions by buffering cytosolic Ca2+ levels and regulating mitochondrial effectors. Recently, the identity of mitochondrial Ca2+ transporters has been revealed, opening new perspectives for investigation and molecular intervention.

Ca2+ transfer from the ER to mitochondria: when, how and why

The heterogenous subcellular distribution of a wide array of channels, pumps and exchangers allows extracellular stimuli to induce increases in cytoplasmic Ca(2+) concentration ([Ca(2+)]c) with highly defined spatial and temporal patterns, that in turn induce specific cellular responses (e.g. contraction, secretion, proliferation or cell death). In this extreme complexity, the role of mitochondria was considered marginal, till the direct measurement with targeted indicators allowed to appreciate that rapid and large increases of the [Ca(2+)] in the mitochondrial matrix ([Ca(2+)]m) invariably follow the cytosolic rises. Given the low affinity of the mitochondrial Ca(2+) transporters, the close proximity to the endoplasmic reticulum (ER) Ca(2+)-releasing channels was shown to be responsible for the prompt responsiveness of mitochondria. In this review, we will summarize the current knowledge of: i) the mitochondrial and ER Ca(2+) channels mediating the ion transfer, ii) the structural and molecular foundations of the signaling contacts between the two organelles, iii) the functional consequences of the [Ca(2+)]m increases, and iv) the effects of oncogene-mediated signals on mitochondrial Ca(2+) homeostasis. Despite the rapid progress carried out in the latest years, a deeper molecular understanding is still needed to unlock the secrets of Ca(2+) signaling machinery.

Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB

The view of the lysosome as the terminal end of cellular catabolic pathways has been challenged by recent studies showing a central role of this organelle in the control of cell function. Here we show that a lysosomal Ca2+ signalling mechanism controls the activities of the phosphatase calcineurin and of its substrate TFEB, a master transcriptional regulator of lysosomal biogenesis and autophagy. Lysosomal Ca2+ release through mucolipin 1 (MCOLN1) activates calcineurin, which binds and dephosphorylates TFEB, thus promoting its nuclear translocation. Genetic and pharmacological inhibition of calcineurin suppressed TFEB activity during starvation and physical exercise, while calcineurin overexpression and constitutive activation had the opposite effect. Induction of autophagy and lysosomal biogenesis through TFEB required MCOLN1-mediated calcineurin activation. These data link lysosomal calcium signalling to both calcineurin regulation and autophagy induction and identify the lysosome as a hub for the signalling pathways that regulate cellular homeostasis.

The mitochondrial calcium uniporter is a multimer that can include a dominant‐negative pore‐forming subunit

Mitochondrial calcium uniporter (MCU) channel is responsible for Ruthenium Red‐sensitive mitochondrial calcium uptake. Here, we demonstrate MCU oligomerization by immunoprecipitation and Förster resonance energy transfer (FRET) and characterize a novel protein (MCUb) with two predicted transmembrane domains, 50% sequence similarity and a different expression profile from MCU. Based on computational modelling, MCUb includes critical amino‐acid substitutions in the pore region and indeed MCUb does not form a calcium‐permeable channel in planar lipid bilayers. In HeLa cells, MCUb is inserted into the oligomer and exerts a dominant‐negative effect, reducing the [Ca2+]mt increases evoked by agonist stimulation. Accordingly, in vitro co‐expression of MCUb with MCU drastically reduces the probability of observing channel activity in planar lipid bilayer experiments. These data unveil the structural complexity of MCU and demonstrate a novel regulatory mechanism, based on the inclusion of dominant‐negative subunits in a multimeric channel, that underlies the fine control of the physiologically and pathologically relevant process of mitochondrial calcium homeostasis.

MICU1 and MICU2 Finely Tune the Mitochondrial Ca2+ Uniporter by Exerting Opposite Effects on MCU Activity

Summary Mitochondrial calcium accumulation was recently shown to depend on a complex composed of an inner-membrane channel (MCU and MCUb) and regulatory subunits (MICU1, MCUR1, and EMRE). A fundamental property of MCU is low activity at resting cytosolic Ca2+ concentrations, preventing deleterious Ca2+ cycling and organelle overload. Here we demonstrate that these properties are ensured by a regulatory heterodimer composed of two proteins with opposite effects, MICU1 and MICU2, which, both in purified lipid bilayers and in intact cells, stimulate and inhibit MCU activity, respectively. Both MICU1 and MICU2 are regulated by calcium through their EF-hand domains, thus accounting for the sigmoidal response of MCU to [Ca2+] in situ and allowing tight physiological control. At low [Ca2+], the dominant effect of MICU2 largely shuts down MCU activity; at higher [Ca2+], the stimulatory effect of MICU1 allows the prompt response of mitochondria to Ca2+ signals generated in the cytoplasm.

Loss-of-function mutations in MICU1 cause a brain and muscle disorder linked to primary alterations in mitochondrial calcium signaling

Mitochondrial Ca2+ uptake has key roles in cell life and death. Physiological Ca2+ signaling regulates aerobic metabolism, whereas pathological Ca2+ overload triggers cell death. Mitochondrial Ca2+ uptake is mediated by the Ca2+ uniporter complex in the inner mitochondrial membrane, which comprises MCU, a Ca2+-selective ion channel, and its regulator, MICU1. Here we report mutations of MICU1 in individuals with a disease phenotype characterized by proximal myopathy, learning difficulties and a progressive extrapyramidal movement disorder. In fibroblasts from subjects with MICU1 mutations, agonist-induced mitochondrial Ca2+ uptake at low cytosolic Ca2+ concentrations was increased, and cytosolic Ca2+ signals were reduced. Although resting mitochondrial membrane potential was unchanged in MICU1-deficient cells, the mitochondrial network was severely fragmented. Whereas the pathophysiology of muscular dystrophy and the core myopathies involves abnormal mitochondrial Ca2+ handling, the phenotype associated with MICU1 deficiency is caused by a primary defect in mitochondrial Ca2+ signaling, demonstrating the crucial role of mitochondrial Ca2+ uptake in humans.

Mitochondrial Ca2+ uptake contributes to buffering cytoplasmic Ca2+ peaks in cardiomyocytes

Mitochondrial ability of shaping Ca2+ signals has been demonstrated in a large number of cell types, but it is still debated in heart cells. Here, we take advantage of the molecular identification of the mitochondrial Ca2+ uniporter (MCU) and of unique targeted Ca2+ probes to directly address this issue. We demonstrate that, during spontaneous Ca2+ pacing, Ca2+ peaks on the outer mitochondrial membrane (OMM) are much greater than in the cytoplasm because of a large number of Ca2+ hot spots generated on the OMM surface. Cytoplasmic Ca2+ peaks are reduced or enhanced by MCU overexpression and siRNA silencing, respectively; the opposite occurs within the mitochondrial matrix. Accordingly, the extent of contraction is reduced by overexpression of MCU and augmented by its down-regulation. Modulation of MCU levels does not affect the ATP content of the cardiomyocytes. Thus, in neonatal cardiac myocytes, mitochondria significantly contribute to buffering the amplitude of systolic Ca2+ rises.

Critical reappraisal confirms that Mitofusin 2 is an endoplasmic reticulum–mitochondria tether

Significance Organelles engage in heterotypic interactions crucial for metabolic and signaling cascades. The best-studied case of this heterotypic interaction is that between the mitochondria and endoplasmic reticulum (ER), crucial for transfer of lipids and especially Ca2+ between the two organelles. The original discovery that the mitochondria-shaping protein Mitofusin 2 (Mfn2) physically tethers the ER to mitochondria was recently challenged. Here, electron microscopy and fluorescent probes of organelle proximity provide definitive evidence that constitutive or acute Mfn2 ablation increases the distance between the ER and mitochondria. Functionally, this process reduces mitochondrial Ca2+ uptake without altering the mitochondrial Ca2+ uniporter complex in multiple tissues. Thus, the discoveries of the role of ER–mitochondria juxtaposition in cell biology based on Mfn2 as a tool remain unchallenged. The discovery of the multiple roles of mitochondria–endoplasmic reticulum (ER) juxtaposition in cell biology often relied upon the exploitation of Mitofusin (Mfn) 2 as an ER–mitochondria tether. However, this established Mfn2 function was recently questioned, calling for a critical re-evaluation of Mfn2’s role in ER–mitochondria cross-talk. Electron microscopy and fluorescence-based probes of organelle proximity confirmed that ER–mitochondria juxtaposition was reduced by constitutive or acute Mfn2 deletion. Functionally, mitochondrial uptake of Ca2+ released from the ER was reduced following acute Mfn2 ablation, as well as in Mfn2−/− cells overexpressing the mitochondrial calcium uniporter. Mitochondrial Ca2+ uptake rate and extent were normal in isolated Mfn2−/− liver mitochondria, consistent with the finding that acute or chronic Mfn2 ablation or overexpression did not alter mitochondrial calcium uniporter complex component levels. Hence, Mfn2 stands as a bona fide ER–mitochondria tether whose ablation decreases interorganellar juxtaposition and communication.

Enjoy the Trip: Calcium in Mitochondria Back and Forth

In the last 5 years, most of the molecules that control mitochondrial Ca(2+) homeostasis have been finally identified. Mitochondrial Ca(2+) uptake is mediated by the Mitochondrial Calcium Uniporter (MCU) complex, a macromolecular structure that guarantees Ca(2+) accumulation inside mitochondrial matrix upon increases in cytosolic Ca(2+). Conversely, Ca(2+) release is under the control of the Na(+)/Ca(2+) exchanger, encoded by the NCLX gene, and of a H(+)/Ca(2+) antiporter, whose identity is still debated. The low affinity of the MCU complex, coupled to the activity of the efflux systems, protects cells from continuous futile cycles of Ca(2+) across the inner mitochondrial membrane and consequent massive energy dissipation. In this review, we discuss the basic principles that govern mitochondrial Ca(2+) homeostasis and the methods used to investigate the dynamics of Ca(2+) concentration within the organelles. We discuss the functional and structural role of the different molecules involved in mitochondrial Ca(2+) handling and their pathophysiological role.