The spatial relation between mitochondria and endoplasmic reticulum (ER) in living HeLa cells was analyzed at high resolution in three dimensions with two differently colored, specifically targeted green fluorescent proteins. Numerous close contacts were observed between these organelles, and mitochondria in situ formed a largely interconnected, dynamic network. A Ca ²⁺ -sensitive photoprotein targeted to the outer face of the inner mitochondrial membrane showed that, upon opening of the inositol 1,4,5-triphosphate (IP ₃ )–gated channels of the ER, the mitochondrial surface was exposed to a higher concentration of Ca ²⁺ than was the bulk cytosol. These results emphasize the importance of cell architecture and the distribution of organelles in regulation of Ca ²⁺ signaling.

Central to the role of the mitochondrion in cellular metabolism is its ability to control the fluxes of the key signalling ion, Ca2+. This is done by a highly selective ion channel known as the mitochondrial calcium uniporter. The molecular nature of this channel has remained elusive, but now two groups report the identification of a 40-kilodalton protein in the inner membrane of mitochondria as the active channel of the uniporter. This protein contains two transmembrane domains and exhibits calcium-channel activity in vitro and in vivo. Mitochondrial Ca2+ homeostasis has a key role in the regulation of aerobic metabolism and cell survival1, but the molecular identity of the Ca2+ channel, the mitochondrial calcium uniporter2, 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 regulator3, 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.

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 voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane mediates metabolic flow, Ca2+, and cell death signaling between the endoplasmic reticulum (ER) and mitochondrial networks. We demonstrate that VDAC1 is physically linked to the endoplasmic reticulum Ca2+-release channel inositol 1,4,5-trisphosphate receptor (IP3R) through the molecular chaperone glucose-regulated protein 75 (grp75). Functional interaction between the channels was shown by the recombinant expression of the ligand-binding domain of the IP3R on the ER or mitochondrial surface, which directly enhanced Ca2+ accumulation in mitochondria. Knockdown of grp75 abolished the stimulatory effect, highlighting chaperone-mediated conformational coupling between the IP3R and the mitochondrial Ca2+ uptake machinery. Because organelle Ca2+ homeostasis influences fundamentally cellular functions and death signaling, the central location of grp75 may represent an important control point of cell fate and pathogenesis.

There is a growing consensus that the various forms of cell death (necrosis, apoptosis and autophagy) are not separated by strict boundaries, but rather share molecular effectors and signaling routes. Among the latter, a clear role is played by calcium (Ca²⁺), the ubiquitous second messenger involved in the control of a broad variety of physiological events. Fine tuning of intracellular Ca²⁺ homeostasis by anti- and proapoptotic proteins shapes the Ca²⁺ signal to which mitochondria and other cellular effectors are exposed, and hence the efficiency of various cell death inducers. Here, we will review: (i) the evidence linking calcium homeostasis to the regulation of apoptotic, and more recently autophagic cell death, (ii) the discussion of mitochondria as a critical, although not unique checkpoint and (iii) the molecular and functional elucidation of ER/mitochondria contacts, corresponding to the mitochondria-associated membrane (MAM) subfraction and proposed to be a specialized signaling microdomain.