György Szabadkai

Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels

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.

Bidirectional Ca2+-dependent control of mitochondrial dynamics by the Miro GTPase

Calcium oscillations suppress mitochondrial movements along the microtubules to support on-demand distribution of mitochondria. To activate this mechanism, Ca2+ targets a yet unidentified cytoplasmic factor that does not seem to be a microtubular motor or a kinase/phosphatase. Here, we have studied the dependence of mitochondrial dynamics on the Miro GTPases that reside in the mitochondria and contain two EF-hand Ca2+-binding domains, in H9c2 cells and primary neurons. At resting cytoplasmic [Ca2+] ([Ca2+]c), movements of the mitochondria were enhanced by Miro overexpression irrespective of the presence of the EF-hands. The Ca2+-induced arrest of mitochondrial motility was also promoted by Miro overexpression and was suppressed when either the Miro were depleted or their EF-hand was mutated. Miro also enhanced the fusion state of the mitochondria at resting [Ca2+]c but promoted mitochondrial fragmentation at high [Ca2+]c. These effects of Miro on mitochondrial morphology seem to involve Drp1 suppression and activation, respectively. In primary neurons, Miro also caused an increase in dendritic mitochondrial mass and enhanced mitochondrial calcium signaling. Thus, Miro proteins serve as a [Ca2+]c-sensitive switch and bifunctional regulator for both the motility and fusion-fission dynamics of the mitochondria.

Recombinant expression of the voltage-dependent anion channel enhances the transfer of Ca2+ microdomains to mitochondria

Although the physiological relevance of mitochondrial Ca2+ homeostasis is widely accepted, no information is yet available on the molecular identity of the proteins involved in this process. Here we analyzed the role of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane in the transmission of Ca2+ signals between the ER and mitochondria by measuring cytosolic and organelle [Ca2+] with targeted aequorins and Ca2+-sensitive GFPs. In HeLa cells and skeletal myotubes, the transient expression of VDAC enhanced the amplitude of the agonist-dependent increases in mitochondrial matrix Ca2+ concentration by allowing the fast diffusion of Ca2+ from ER release sites to the inner mitochondrial membrane. Indeed, high speed imaging of mitochondrial and cytosolic [Ca2+] changes showed that the delay between the rises occurring in the two compartments is significantly shorter in VDAC-overexpressing cells. As to the functional consequences, VDAC-overexpressing cells are more susceptible to ceramide-induced cell death, thus confirming that mitochondrial Ca2+ uptake plays a key role in the process of apoptosis. These results reveal a novel function for the widely expressed VDAC channel, identifying it as a molecular component of the routes for Ca2+ transport across the mitochondrial membranes.

Participation of endoplasmic reticulum and mitochondrial calcium handling in apoptosis: more than just neighborhood?

Over the past few years, extensive progress has been made in elucidating the role of calcium in the signaling of apoptosis. This has led to the characterization of calciums role in the induction of apoptosis and in the regulation of effector proteases. In this review, we attempt to summarize the current knowledge regarding a segment of these studies, the interaction between the endoplasmic reticulum (ER) and mitochondria. This interface has been shown to play a crucial role in transferring agonist induced Ca2+ signals to mitochondria during physiological processes. Recent evidence, however, extended the role of this Ca2+ transfer to apoptotic pathways, showing that modulation of mitochondrial Ca2+ uptake from the ER side has a prominent role in modulating cellular fate.

Endoplasmic reticulum, Bcl-2 and Ca2+ handling in apoptosis.

In the complex signalling interplay that allows extracellular signals to be decoded into activation of apoptotic cell death, Ca(2+) plays a significant role. This is supported not only by evidence linking alterations in Ca(2+) homeostasis to the triggering of apoptotic (and in some cases necrotic) cell death, but also by recent data indicating that a key anti-apoptotic protein, Bcl-2, has a direct effect on ER Ca(2+) handling. We will briefly summarise the first aspect, and describe in more detail these new data, demonstrating that (i) Bcl-2 reduces the state of filling of the ER Ca(2+) store and (ii) this Ca(2+) signalling alteration renders the cells less sensitive to apoptotic stimuli. Overall, these results suggest that calcium homeostasis may represent a pharmacological target in the fundamental pathological process of apoptosis.

Novel role for polycystin-1 in modulating cell proliferation through calcium oscillations in kidney cells

Abstract.u2003 Objectives: Polycystin‐1 (PC1), a signalling receptor regulating Ca2+‐permeable cation channels, is mutated in autosomal dominant polycystic kidney disease, which is typically characterized by increased cell proliferation. However, the precise mechanisms by which PC1 functions on Ca2+ homeostasis, signalling and cell proliferation remain unclear. Here, we investigated the possible role of PC1 as a modulator of non‐capacitative Ca2+ entry (NCCE) and Ca2+ oscillations, with downstream effects on cell proliferation. Results and discussion: By employing RNA interference, we show that depletion of endogenous PC1 in HEK293 cells leads to an increase in serum‐induced Ca2+ oscillations, triggering nuclear factor of activated T cell activation and leading to cell cycle progression. Consistently, Ca2+ oscillations and cell proliferation are increased in PC1‐mutated kidney cystic cell lines, but both abnormal features are reduced in cells that exogenously express PC1. Notably, blockers of the NCCE pathway, but not of the CCE, blunt abnormal oscillation and cell proliferation. Our study therefore provides the first demonstration that PC1 modulates Ca2+ oscillations and a molecular mechanism to explain the association between abnormal Ca2+ homeostasis and cell proliferation in autosomal dominant polycystic kidney disease.

Chaperones as parts of organelle networks.

The efficiency, divergence, and specificity of virtually all intracellular metabolic and signalling pathways largely depend on their compartmentalized organization. A corollary of the requirement of compartmentalization is the dynamic structural partition of the intracellular space by endomembrane systems. A branch of these membranes communicate with the extracellular space through the endo- and exocytotic processes. Others, like the mitochondrial and endoplasmic reticulum networks accomplish a further role, being fundamental for the maintenance of cellular energy balance and for determination of cell fate under stress conditions. Recent structural and functional studies revealed that the interaction of these networks and the connectivity state of mitochondria controls metabolic flow, protein transport, intracellular Ca2+ signalling, and cell death. Moreover, reflecting the fact that the above processes are accomplished in a microdomain between collaborating organelle membranes, the existence of macromolecular complexes at their contact sites have also been revealed. Being not only assistants of nascent protein folding, chaperones are proposed to participate in assembling and maintaining the function of the above complexes. In this chapter we discuss recently found examples of such an assembly of protein interactions driven by chaperone proteins, and their role in regulating physiological and pathological processes.

p66Shc aging protein in control of fibroblasts cell fate.

Reactive oxygen species (ROS) are wieldy accepted as one of the main factors of the aging process. These highly reactive compounds modify nucleic acids, proteins and lipids and affect the functionality of mitochondria in the first case and ultimately of the cell. Any agent or genetic modification that affects ROS production and detoxification can be expected to influence longevity. On the other hand, genetic manipulations leading to increased longevity can be expected to involve cellular changes that affect ROS metabolism. The 66-kDa isoform of the growth factor adaptor Shc (p66Shc) has been recognized as a relevant factor to the oxygen radical theory of aging. The most recent data indicate that p66Shc protein regulates life span in mammals and its phosphorylation on serine 36 is important for the initiation of cell death upon oxidative stress. Moreover, there is strong evidence that apart from aging, p66Shc may be implicated in many oxidative stress-associated pathologies, such as diabetes, mitochondrial and neurodegenerative disorders and tumorigenesis. This article summarizes recent knowledge about the role of p66Shc in aging and senescence and how this protein can influence ROS production and detoxification, focusing on studies performed on skin and skin fibroblasts.

Modulation of Calcium Homeostasis by the Endoplasmic Reticulum in Health and Disease

The endoplasmic reticulum (ER) is the main intracellular agonist-sensitive Ca2+ store, and is involved in the regulation of a wide range of cellular functions depending on cytosolic Ca2+. In addition, it has recently been recognized that Ca2+ regulates also processes occurring in the ER lumen, such as protein synthesis and trafficking, and cellular responses to stress. Accordingly, perturbation of ER Ca2+ homeostasis appears to be a key component in the development of several pathological situations. In this chapter, after providing an overview of the Ca2+ signaling components of the ER, we briefly summarize their role in basic pathophysiological processes and specific diseases.