Structural and functional features and significance of the physical linkage between ER and mitochondria (original) (raw)

Ca(2+) transfer from the ER to mitochondria: when, how and why

Biochimica et biophysica acta, 2009

The heterogenous subcellular distribution of a wide array of channels, pumps and exchangers allows extracellular stimuli to induce increases in cytoplasmic Ca2+ concentration ([Ca2+]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 [Ca2+] in the mitochondrial matrix ([Ca2+]m) invariably follow the cytosolic rises. Given the low affinity of the mitochondrial Ca2+ transporters, the close proximity to the endoplasmic reticulum (ER) Ca2+-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 Ca2+ 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 [Ca2+]m increases, and iv) the effects of oncogene-mediated signals on mitochondrial Ca2+ homeostasis. Despite the rapid progress carried out in the latest years, a deeper molecular understanding is still needed to unlock the secrets of Ca2+ signaling machinery.

The Machinery of Local Ca2+ Signalling Between Sarco-Endoplasmic Reticulum and Mitochondria

The Journal of …, 2000

Although the large capacity of isolated mitochondria to take up Ca¥ has been well known since the 1960's, its relevance under physiological conditions remained subject to controversy until recently. Mitochondria were believed to be relatively insensitive to physiological [Ca¥]c increases since the rise of global [Ca¥]c to 500 nÒ-1 ìÒ during IP× receptor (IP×R)-or ryanodine receptor (RyR)-driven [Ca¥]c spiking is probably not sufficient to activate the low affinity mitochondrial Ca¥ uptake mechanisms. However, a different picture emerged from experiments that utilized novel approaches to directly measure mitochondrial matrix [Ca¥] ([Ca¥]m) in living cells. Experiments using Ca¥-sensitive photoproteins targeted to the mitochondria, or fluorescent Ca¥ tracers loaded into the mitochondria, demonstrated increases of [Ca¥]m that occurred simultaneously with [Ca¥]c spikes and oscillations (Rizzuto et al. 1993, 1994; Hajn oczky et al. 1995). These results have been explained by a close coupling of IP×R-and RyR-mediated Ca¥ release to mitochondrial Ca¥ uptake, allowing mitochondrial uptake sites to sense the high local [Ca¥]c adjacent to the activated release sites. The obligatory components of the local Ca¥ transfer are the SRÏER Ca¥ release sites (RyRÏIP×R) and the mitochondrial Ca¥ uptake sites (Ca¥ uniporter), but the SRÏER Ca¥ uptake sites, the mitochondrial Ca¥ release sites and Ca¥ binding proteins are also important Ca¥-handling elements of the SRÏER-mitochondrial communication. The complex regulation of RyR by Ca¥, and IP×R by IP× and Ca¥, allows these Ca¥ channels to exhibit rapid and concerted activation and inactivation, giving rise to bursts of Ca¥ release from the high [Ca¥] SRÏER lumen to the low [Ca¥] cytosol. At low [Ca¥]c levels, the membrane potential (ÄØm)-driven Ca¥ uniporter-mediated mitochondrial Ca¥ influx is balanced by Ca¥ efflux, but large or sustained elevations of [Ca¥]c effectively activate the mitochondrial Ca¥ uniporter and may result in robust [Ca¥]m signals. For a comprehensive analysis of Ca¥ transport properties of SRÏER and mitochondria we refer

Structural and functional link between the mitochondrial network and the endoplasmic reticulum

The international journal of biochemistry & cell biology, 2009

Mitochondrial and endoplasmic reticulum (ER) networks are fundamental for the maintenance of cellular homeostasis and for determination of cell fate under stress conditions. Recent structural and functional studies revealed the interaction of these networks. These zones of close contact between ER and mitochondria called MAM (mitochondria associated membranes) support communication between the two organelles including bioenergetics and cell survival. The existence of macromolecular complexes in these contact sites has also been revealed. In this contribution, we will review: i) the ER and mitochondria structure and their dynamics, ii) the basic principles of ER mitochondrial Ca 2+ transport, (iii) the physiological/pathological role of this crosstalk.

IP3 receptor isoforms differently regulate ER-mitochondrial contacts and local calcium transfer

Nature Communications, 2019

Contact sites of endoplasmic reticulum (ER) and mitochondria locally convey calcium signals between the IP3 receptors (IP3R) and the mitochondrial calcium uniporter, and are central to cell survival. It remains unclear whether IP3Rs also have a structural role in contact formation and whether the different IP3R isoforms have redundant functions. Using an IP3R-deficient cell model rescued with each of the three IP3R isoforms and an array of super-resolution and ultrastructural approaches we demonstrate that IP3Rs are required for maintaining ER-mitochondrial contacts. This role is independent of calcium fluxes. We also show that, while each isoform can support contacts, type 2 IP3R is the most effective in delivering calcium to the mitochondria. Thus, these studies reveal a non-canonical, structural role for the IP3Rs and direct attention towards the type 2 IP3R that was previously neglected in the context of ER-mitochondrial calcium signaling.

Mitochondria and Ca2+ in cell physiology and pathophysiology

Cell Calcium, 2000

There is now a consensus that mitochondria take up and accumulate Ca 2; during physiological [Ca 2; ] c signalling. This contribution will consider some of the functional consequences of mitochondrial Ca 2; uptake for cell physiology and pathophysiology. The ability to remove Ca 2; from local cytosol enables mitochondria to regulate the [Ca 2; ] in microdomains close to IP3-sensitive Ca 2; -release channels. The [Ca 2; ] sensitivity of these channels means that, by regulating local [Ca 2; ] c , mitochondrial Ca 2; uptake modulates the rate and extent of propagation of [Ca 2; ] c waves in a variety of cell types. The coincidence of mitochondrial Ca 2; uptake with oxidative stress may open the mitochondrial permeability transition pore (mPTP). This is a catastrophic event for the cell that will initiate pathways to cell death either by necrotic or apoptotic pathways. A model is presented in which illumination of an intramitochondrial fluorophore is used to generate oxygen radical species within mitochondria. This causes mitochondrial Ca 2; loading from SR and triggers mPTP opening. In cardiomyocytes, mPTP opening leads to ATP consumption by the mitochondrial ATPase and so results in ATP depletion, rigor and necrotic cell death. In central mammalian neurons exposed to glutamate, a cellular Ca 2; overload coincident with NO production also causes loss of mitochondrial potential and cell death, but mPTP involvement has proven more difficult to demonstrate unequivocally. A cartoon to illustrate the features of the mitochondrial permeability transition pore and its pharmacology. The mPTP seems to consist of an association between the adenine nucleotide translocase (ANT), the voltage dependent anion channel (VDAC) of the outer mitochondrial membrane and cyclophilin D (Cyp D). Opening of the pore is modulated primarily by [Ca 2; ] m , ROS, NO, many sulphydryl reagents and by bongrekate or atractyloside, amongst many agents and conditions (from Duchen, 2000, with permission).

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

Nature, 2011

Mitochondrial Ca 2+ homeostasis plays a key role in the regulation of aerobic metabolism and cell survival 1 , but the molecular identity of the Ca 2+ channel, the mitochondrial calcium uniporter 2 , was still unknown. We have identified in silico a protein (denominated MCU) that shares tissue distribution with MICU1, a recently characterized uniporter regulator 3 , coexists with uniporter activity in phylogeny and includes two trasmembrane domains in the sequence. siRNA silencing of MCU in HeLa cells drastically reduced mitochondrial Ca 2+ uptake. MCU overexpression doubled the [Ca 2+ ] mt rise evoked by IP 3 -generating agonists, thus significantly buffering the cytosolic elevation. The purified MCU protein exhibited 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 [Ca 2+ ] mt transients when overexpressed in HeLa cells. Overall, these data demonstrate that the identified 40 kDa protein is the channel responsible for Ruthenium Red-sensitive mitochondrial Ca 2+ uptake, thus providing molecular basis for this process of utmost physiological and pathological relevance.

Mitochondria-Associated Membranes (MAMs) as Hotspot Ca2+ Signaling Units

Advances in Experimental Medicine and Biology, 2012

The tight interplay between endoplasmic reticulum (ER) and mitochondria is a key determinant of cell function and survival through the control of intracellular calcium (Ca 2+ ) signaling. The specifi c sites of physical association between ER and mitochondria are known as mitochondria-associated membranes (MAMs). It has recently become clear that MAMs are crucial for highly effi cient transmission of Ca 2+ from the ER to mitochondria, thus controlling fundamental processes involved in energy production and also determining cell fate by triggering or preventing apoptosis. In this contribution, we summarize the main features of the Ca 2+ -signaling toolkit, covering also the latest breakthroughs in the fi eld, such as the identifi cation of novel candidate proteins implicated in mitochondrial Ca 2+ transport and the recent A. Bononi et al. direct characterization of the high-Ca 2+ microdomains between ER and mitochondria. We review the main functions of these two organelles, with special emphasis on Ca 2+ handling and on the structural and molecular foundations of the signaling contacts between them. Additionally, we provide important examples of the physiopathological role of this cross-talk, briefl y describing the key role played by MAMs proteins in many diseases, and shedding light on the essential role of mitochondria-ER interactions in the maintenance of cellular homeostasis and the determination of cell fate.

The Role of Mitochondria for Ca2+ Refilling of the Endoplasmic Reticulum

Journal of Biological Chemistry, 2005

Endoplasmic reticulum (ER) Ca 2؉ refilling is an active process to ensure an appropriate ER Ca 2؉ content under basal conditions and to maintain or restore ER Ca 2؉ concentration during/after cell stimulation. The mechanisms to achieve successful ER Ca 2؉ refilling are multiple and built on a concerted action of processes that provide a suitable reservoir for Ca 2؉ sequestration into the ER. Despite mitochondria having been found to play an essential role in the maintenance of capacitative Ca 2؉ entry by buffering subplasmalemmal Ca 2؉ , their contribution to ER Ca 2؉ refilling was not subjected to detailed analysis so far. Thus, this study was designed to elucidate the involvement of mitochondria in Ca 2؉ store refilling during and after cell stimulation. ER Ca 2؉ refilling was found to be accomplished even during continuous inositol 1,4,5-trisphosphate (IP 3)-triggered ER Ca 2؉ release by an agonist. Basically, ER Ca 2؉ refilling depended on the presence of extracellular Ca 2؉ as the source and sarcoplasmic/endoplasmic reticulum Ca 2؉ ATPase (SERCA) activity. Interestingly, in the presence of an IP 3-generating agonist, ER Ca 2؉ refilling was prevented by the inhibition of trans-mitochondrial Ca 2؉ flux by CGP 37157 (7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one) that precludes the mitochondrial Na ؉ /Ca 2؉ exchanger as well as by mitochondrial depolarization using a mixture of oligomycin and antimycin A. In contrast, after the removal of the agonist, ER refilling was found to be largely independent of trans-mitochondrial Ca 2؉ flux. Under these conditions, ER Ca 2؉ refilling took place even without an associated Ca 2؉ elevation in the deeper cytosol, thus, indicating that superficial ER domains mimic mitochondrial Ca 2؉ buffering and efficiently sequester subplasmalemmal Ca 2؉ and consequently facilitate capacitative Ca 2؉ entry. Hence, these data point to different contribution of mitochondria in the process of ER Ca 2؉ refilling based on the presence or absence of IP 3 , which represents the turning point for the dependence or autonomy of ER Ca 2؉ refilling from trans-mitochondrial Ca 2؉ flux.

Mitochondrial localization as a determinant of capacitative Ca2+ entry in HeLa cells

Cell Calcium, 2004

Whether different subsets of mitochondria play distinct roles in shaping intracellular Ca 2+ signals is presently unresolved. Here, we determine the role of mitochondria located beneath the plasma membrane in controlling (a) Ca 2+ release from the endoplasmic reticulum (ER) and (b) capacitative Ca 2+ entry. By over-expression of the dynactin subunit dynamitin, and consequent inhibition of the fission factor, dynamin-related protein (Drp-1), mitochondria were relocalised from the plasma membrane towards the nuclear periphery in HeLa cells. The impact of these changes on free calcium concentration in the cytosol ([Ca 2+ ] c ), mitochondria ([Ca 2+ ] m ) and ER ([Ca 2+ ] ER ) was then monitored with specifically-targeted aequorins. Whilst dynamitin over-expression increased the number of close contacts between the ER and mitochondria by >2.5-fold, assessed using organelle-targeted GFP variants, histamine-induced changes in organellar [Ca 2+ ] were unaffected. By contrast, Ca 2+ influx elicited significantly smaller increases in [Ca 2+ ] c and [Ca 2+ ] m in dynamitin-expressing than in control cells. These data suggest that the strategic localisation of a subset of mitochondria beneath the plasma membrane is required for normal Ca 2+ influx, but that the transfer of Ca 2+ ions between the ER and mitochondria is relatively insensitive to gross changes in the spatial relationship between these two organelles.

Subplasmalemmal Mitochondria Modulate the Activity of Plasma Membrane Ca2+-ATPases

Journal of Biological Chemistry, 2005

Mitochondria are dynamic organelles that modulate cellular Ca 2؉ signals by interacting with Ca 2؉ transporters on the plasma membrane or the endoplasmic reticulum (ER). To study how mitochondria dynamics affects cell Ca 2؉ homeostasis, we overexpressed two mitochondrial fission proteins, hFis1 and Drp1, and measured Ca 2؉ changes within the cytosol and the ER in HeLa cells. Both proteins fragmented mitochondria, decreased their total volume by 25-40%, and reduced the fraction of subplasmalemmal mitochondria by 4-fold. The cytosolic Ca 2؉ signals elicited by histamine were unaltered in cells lacking subplasmalemmal mitochondria as long as Ca 2؉ was present in the medium, but the signals were significantly blunted when Ca 2؉ was removed. Upon Ca 2؉ withdrawal, the free ER Ca 2؉ concentration decreased rapidly, and hFis1 cells were unable to respond to repetitive histamine stimulations. . 2 The abbreviations used are: [Ca 2ϩ ] cyt , cytosolic [Ca 2ϩ ]; [Ca 2ϩ ] ER , endoplasmic reticulum [Ca 2ϩ ]; [Ca 2ϩ ] mit , mitochondrial [Ca 2ϩ ]; CGP 37157, (7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one); ER, endoplasmic reticulum; GFP, green fluorescent protein; PMCA, plasma membrane Ca 2ϩ -ATPase; RFP mit , mitochondrial red fluorescent protein; SERCA, sarco/endoplasmic reticulum Ca 2ϩ ATPase; YC4.1 ER , yellow cameleon targeted to the ER; YC3.6 pm , yellow cameleon targeted to the plasma membrane.