György Csordás

Structural and functional features and significance of the physical linkage between ER and mitochondria

The role of mitochondria in cell metabolism and survival is controlled by calcium signals that are commonly transmitted at the close associations between mitochondria and endoplasmic reticulum (ER). However, the physical linkage of the ER–mitochondria interface and its relevance for cell function remains elusive. We show by electron tomography that ER and mitochondria are adjoined by tethers that are ∼10 nm at the smooth ER and ∼25 nm at the rough ER. Limited proteolysis separates ER from mitochondria, whereas expression of a short “synthetic linker” (<5 nm) leads to tightening of the associations. Although normal connections are necessary and sufficient for proper propagation of ER-derived calcium signals to the mitochondria, tightened connections, synthetic or naturally observed under apoptosis-inducing conditions, make mitochondria prone to Ca2+ overloading and ensuing permeability transition. These results reveal an unexpected dependence of cell function and survival on the maintenance of proper spacing between the ER and mitochondria.

Quasi-synaptic calcium signal transmission between endoplasmic reticulum and mitochondria.

Transmission of cytosolic [Ca2+] ([Ca2+]c) oscillations into the mitochondrial matrix is thought to be supported by local calcium control between IP3 receptor Ca2+ channels (IP3R) and mitochondria, but study of the coupling mechanisms has been difficult. We established a permeabilized cell model in which the Ca2+ coupling between endoplasmic reticulum (ER) and mitochondria is retained, and mitochondrial [Ca2+] ([Ca2+]m) can be monitored by fluorescence imaging. We demonstrate that maximal activation of mitochondrial Ca2+ uptake is evoked by IP3‐induced perimitochondrial [Ca2+] elevations, which appear to reach values >20‐fold higher than the global increases of [Ca2+]c. Incremental doses of IP3 elicited [Ca2+]m elevations that followed the quantal pattern of Ca2+ mobilization, even at the level of individual mitochondria. In contrast, gradual increases of IP3 evoked relatively small [Ca2+]m responses despite eliciting similar [Ca2+]c increases. We conclude that each mitochondrial Ca2+ uptake site faces multiple IP3R, a concurrent activation of which is required for optimal activation of mitochondrial Ca2+ uptake. This architecture explains why calcium oscillations evoked by synchronized periodic activation of IP3R are particularly effective in establishing dynamic control over mitochondrial metabolism. Furthermore, our data reveal fundamental functional similarities between ER–mitochondrial Ca2+ coupling and synaptic transmission.

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.

SR/ER-mitochondrial local communication: Calcium and ROS

Mitochondria form junctions with the sarco/endoplasmic reticulum (SR/ER), which support signal transduction and biosynthetic pathways and affect organellar distribution. Recently, these junctions have received attention because of their pivotal role in mediating calcium signal propagation to the mitochondria, which is important for both ATP production and mitochondrial cell death. Many of the SR/ER-mitochondrial calcium transporters and signaling proteins are sensitive to redox regulation and are directly exposed to the reactive oxygen species (ROS) produced in the mitochondria and SR/ER. Although ROS has been emerging as a novel signaling entity, the redox signaling of the SR/ER-mitochondrial interface is yet to be elucidated. We describe here possible mechanisms of the mutual interaction between local Ca(2+) and ROS signaling in the control of SR/ER-mitochondrial function.

The machinery of local Ca2+ signalling between sarco-endoplasmic reticulum and mitochondria.

Growing evidence suggests that propagation of cytosolic [Ca2+] ([Ca2+]c) spikes and oscillations to the mitochondria is important for the control of fundamental cellular functions. Delivery of [Ca2+]c spikes to the mitochondria may utilize activation of the mitochondrial Ca2+ uptake sites by the large local [Ca2+]c rise occurring in the vicinity of activated sarco‐endoplasmic reticulum (SR/ER) Ca2+ release channels. Although direct measurement of the local [Ca2+]c sensed by the mitochondria has been difficult, recent studies shed some light onto the molecular mechanism of local Ca2+ communication between SR/ER and mitochondria. Subdomains of the SR/ER are in close contact with mitochondria and display a concentration of Ca2+ release sites, providing the conditions for an effective delivery of released Ca2+ to the mitochondrial targets. Furthermore, many functional properties of the signalling between SR/ER Ca2+ release sites and mitochondrial Ca2+ uptake sites, including transient microdomains of high [Ca2+], saturation of mitochondrial Ca2+ uptake sites by released Ca2+, connection of multiple release sites to each uptake site and quantal transmission, are analogous to the features of the coupling between neurotransmitter release sites and postsynaptic receptors in synaptic transmission. As such, Ca2+ signal transmission between SR/ER and mitochondria may utilize discrete communication sites and a closely related functional architecture to that used for synaptic signal propagation between cells.

Mitofusin 2-Containing Mitochondrial-Reticular Microdomains Direct Rapid Cardiomyocyte Bioenergetic Responses Via Interorganelle Ca 2! Crosstalk

Rationale: Mitochondrial Ca2+ uptake is essential for the bioenergetic feedback response through stimulation of Krebs cycle dehydrogenases. Close association of mitochondria to the sarcoplasmic reticulum (SR) may explain efficient mitochondrial Ca2+ uptake despite low Ca2+ affinity of the mitochondrial Ca2+ uniporter. However, the existence of such mitochondrial Ca2+ microdomains and their functional role are presently unresolved. Mitofusin (Mfn) 1 and 2 mediate mitochondrial outer membrane fusion, whereas Mfn2 but not Mfn1 tethers endoplasmic reticulum to mitochondria in noncardiac cells. Objective: To elucidate roles for Mfn1 and 2 in SR-mitochondrial tethering, Ca2+ signaling, and bioenergetic regulation in cardiac myocytes. Methods and Results: Fruit fly heart tubes deficient of the Drosophila Mfn ortholog MARF had increased contraction-associated and caffeine-sensitive Ca2+ release, suggesting a role for Mfn in SR Ca2+ handling. Whereas cardiac-specific Mfn1 ablation had no effects on murine heart function or Ca2+ cycling, Mfn2 deficiency decreased cardiomyocyte SR-mitochondrial contact length by 30% and reduced the content of SR-associated proteins in mitochondria-associated membranes. This was associated with decreased mitochondrial Ca2+ uptake (despite unchanged mitochondrial membrane potential) but increased steady-state and caffeine-induced SR Ca2+ release. Accordingly, Ca2+-induced stimulation of Krebs cycle dehydrogenases during &bgr;-adrenergic stimulation was hampered in Mfn2-KO but not Mfn1-KO myocytes, evidenced by oxidation of the redox states of NAD(P)H/NAD(P)+ and FADH2/FAD. Conclusions: Physical tethering of SR and mitochondria via Mfn2 is essential for normal interorganelle Ca2+ signaling in the myocardium, consistent with a requirement for SR-mitochondrial Ca2+ signaling through microdomains in the cardiomyocyte bioenergetic feedback response to physiological stress.

Ca2+-dependent Control of the Permeability Properties of the Mitochondrial Outer Membrane and Voltage-dependent Anion-selective Channel (VDAC)

Cell function depends on the distribution of cytosolic and mitochondrial factors across the outer mitochondrial membrane (OMM). Passage of metabolites through the OMM has been attributed to the voltage-dependent anion-selective channel (VDAC), which can form a large conductance and permanently open a channel in lipid bilayers. However, recent data indicate that the transport of metabolites through the OMM is controlled in the cells. Recognizing that the bilayer studies had been commonly conducted at supraphysiological [Ca2+] and [K+], we determined the effect of Ca2+ on VDAC activity. In liposomes, the purified VDAC displays Ca2+-dependent control of the molecular cut-off size and shows Ca2+-regulated Ca2+ permeability in the physiological [Ca2+] range. In bilayer experiments, at submicromolar [Ca2+], the purified VDAC or isolated OMM does not show sustained large conductance but rather exhibits gating between a nonconducting state and various subconductance states. Ca2+ addition causes a reversible increase in the conductance and may evoke channel opening to full conductance. Furthermore, single cell imaging data indicate that Ca2+ may facilitate the cation and ATP transport across the OMM. Thus, the VDAC gating is dependent on the physiological concentrations of cations, allowing the OMM to control the passage of ions and some small molecules. The OMM barrier is likely to decrease during the calcium signal.

Old players in a new role: mitochondria-associated membranes, VDAC, and ryanodine receptors as contributors to calcium signal propagation from endoplasmic reticulum to the mitochondria.

In many cell types, IP(3) and ryanodine receptor (IP(3)R/RyR)-mediated Ca(2+) mobilization from the sarcoendoplasmic reticulum (ER/SR) results in an elevation of mitochondrial matrix [Ca(2+)]. Although delivery of the released Ca(2+) to the mitochondria has been established as a fundamental signaling process, the molecular mechanism underlying mitochondrial Ca(2+) uptake remains a challenge for future studies. The Ca(2+) uptake can be divided into the following three steps: (1) Ca(2+) movement from the IP(3)R/RyR to the outer mitochondrial membrane (OMM); (2) Ca(2+) transport through the OMM; and (3) Ca(2+) transport through the inner mitochondrial membrane (IMM). Evidence has been presented that Ca(2+) delivery to the OMM is facilitated by a local coupling between closely apposed regions of the ER/SR and mitochondria. Recent studies of the dynamic changes in mitochondrial morphology and visualization of the subcellular pattern of the calcium signal provide important clues to the organization of the ER/SR-mitochondrial interface. Interestingly, key steps of phospholipid synthesis and transfer to the mitochondria have also been confined to subdomains of the ER tightly associated with the mitochondria, referred as mitochondria-associated membranes (MAMs). Through the OMM, the voltage-dependent anion channels (VDAC, porin) have been thought to permit free passage of ions and other small molecules. However, recent studies suggest that the VDAC may represent a regulated step in Ca(2+) transport from IP(3)R/RyR to the IMM. A novel proposal regarding the IMM Ca(2+) uptake site is a mitochondrial RyR that would mediate rapid Ca(2+) uptake by mitochondria in excitable cells. An overview of the progress in these directions is described in the present paper.