Elisa Greotti

Mitofusin 2 ablation increases endoplasmic reticulum–mitochondria coupling

Significance The privileged interrelationship between mitochondria and the endoplasmic reticulum (ER) plays a key role in a variety of physiological functions, from lipid metabolism to Ca2+ signalling, and its modulation influences apoptotic susceptibility, mitophagy, and cellular bioenergetics. Among the several proteins known to influence ER–mitochondria interactions, mitofusin 2 (Mfn2) has been proposed to form a physical tether. In this study, we demonstrate that Mfn2 instead works as an ER–mitochondria tethering antagonist preventing an excessive, potentially toxic, proximity between the two organelles. Cells in which Mfn2 is ablated or reduced have an increased number of ER–mitochondria close contacts, potentiated Ca2+ transfer between the two organelles, and greater sensitivity to cell-death stimuli that implies mitochondria Ca2+ overload toxicity. The organization and mutual interactions between endoplasmic reticulum (ER) and mitochondria modulate key aspects of cell pathophysiology. Several proteins have been suggested to be involved in keeping ER and mitochondria at a correct distance. Among them, in mammalian cells, mitofusin 2 (Mfn2), located on both the outer mitochondrial membrane and the ER surface, has been proposed to be a physical tether between the two organelles, forming homotypic interactions and heterocomplexes with its homolog Mfn1. Recently, this widely accepted model has been challenged using quantitative EM analysis. Using a multiplicity of morphological, biochemical, functional, and genetic approaches, we demonstrate that Mfn2 ablation increases the structural and functional ER–mitochondria coupling. In particular, we show that in different cell types Mfn2 ablation or silencing increases the close contacts between the two organelles and strengthens the efficacy of inositol trisphosphate (IP3)-induced Ca2+ transfer from the ER to mitochondria, sensitizing cells to a mitochondrial Ca2+ overload-dependent death. We also show that the previously reported discrepancy between electron and fluorescence microscopy data on ER–mitochondria proximity in Mfn2-ablated cells is only apparent. By using a different type of morphological analysis of fluorescent images that takes into account (and corrects for) the gross modifications in mitochondrial shape resulting from Mfn2 ablation, we demonstrate that an increased proximity between the organelles is also observed by confocal microscopy when Mfn2 levels are reduced. Based on these results, we propose a new model for ER–mitochondria juxtaposition in which Mfn2 works as a tethering antagonist preventing an excessive, potentially toxic, proximity between the two organelles.

Presenilin 2 Modulates Endoplasmic Reticulum-Mitochondria Coupling by Tuning the Antagonistic Effect of Mitofusin 2

Communication between organelles plays key roles in cell biology. In particular, physical and functional coupling of the endoplasmic reticulum (ER) and mitochondria is crucial for regulation of various physiological and pathophysiological processes. Here, we demonstrate that Presenilin 2 (PS2), mutations in which underlie familial Alzheimers disease (FAD), promotes ER-mitochondria coupling only in the presence of mitofusin 2 (Mfn2). PS2 is not necessary for the antagonistic effect of Mfn2 on organelle coupling, although its abundance can tune it. The two proteins physically interact, whereas their homologues Mfn1 and PS1 are dispensable for this interplay. Moreover, PS2 mutants associated with FAD are more effective than the wild-type form in modulating ER-mitochondria tethering because their binding to Mfn2 in mitochondria-associated membranes is favored. We propose a revised model for ER-mitochondria interaction to account for these findings and discuss possible implications for FAD pathogenesis.

Ca2+ and cAMP cross‐talk in mitochondria

While mitochondrial Ca2+ homeostasis has been studied for several decades and many of the functional roles of Ca2+ accumulation within the matrix have been at least partially clarified, the possibility of modulation of the organelle functions by cAMP remains largely unknown. In this contribution we briefly summarize the key aspects of Ca2+ and cAMP signalling pathways in mitochondria. In particular, we focus on recent findings concerning the discovery of an autonomous cAMP toolkit within the mitochondrial matrix, its crossroad with mitochondrial Ca2+ signalling and its role in controlling ATP synthesis. The discovery of a cAMP signalling, and the demonstration of a cross‐talk between cAMP and Ca2+ inside mitochondria, open the way to a re‐evaluation of these organelles as integrators of multiple second messengers. A description of the main methods presently available to measure Ca2+ and cAMP in mitochondria of living cells with genetically encoded probes is also presented.

On the role of Mitofusin 2 in endoplasmic reticulum–mitochondria tethering

The recent paper by Naon et al. (1) claims that their new data “definitively” prove the role of Mitofusin 2 (Mfn2) as an endoplasmic reticulum (ER)–mitochondria tether, supporting their original proposal (2) and arguing against evidence presented by ourselves and others (3⇓⇓–6) suggesting that Mfn2 is a negative regulator of tethering. A careful reading of the paper highlights that Naon et al.’s (1) claims are not supported by the data presented. Below are some pivotal examples. First, the key parameter (number of ER–mitochondria contacts) that both we and others (3, 4) found doubled in Mfn2 −/− cells is simply not addressed. Second, the ER–mitochondria distance was determined by electron microscopy to be 2 nm larger in Mfn2 −/− cells, compared with wild-type; using the … [↵][1]1To whom correspondence may be addressed. Email: tullio.pozzan{at}unipd.it or paola.pizzo{at}unipd.it. [1]: #xref-corresp-1-1

Exploring cells with targeted biosensors

Cellular signaling networks are composed of multiple pathways, often interconnected, that form complex networks with great potential for cross-talk. Signal decoding depends on the nature of the message as well as its amplitude, temporal pattern, and spatial distribution. In addition, the existence of membrane-bound organelles, which are both targets and generators of messages, add further complexity to the system. The availability of sensors that can localize to specific compartments in live cells and monitor their targets with high spatial and temporal resolution is thus crucial for a better understanding of cell pathophysiology. For this reason, over the last four decades, a variety of strategies have been developed, not only to generate novel and more sensitive probes for ions, metabolites, and enzymatic activity, but also to selectively deliver these sensors to specific intracellular compartments. In this review, we summarize the principles that have been used to target organic or protein sensors to different cellular compartments and their application to cellular signaling.

Spying on organelle Ca2+ in living cells: the mitochondrial point of view

Over the past years, the use of genetically encoded Ca2+ indicators (GECIs), derived from aequorin and green fluorescent protein, has profoundly transformed the study of Ca2+ homeostasis in living cells leading to novel insights into functional aspects of Ca2+ signalling. Particularly relevant for a deeper understanding of these key aspects of cell pathophysiology has been the possibility of imaging changes in Ca2+ concentration not only in the cytoplasm, but also inside organelles. In this review, we will provide an overview of the ongoing developments in the use of GECIs, with particular focus on mitochondrially targeted probes. Indeed, due to recent advances in organelle Ca2+ imaging with GECIs, mitochondria are now at the centre of renewed interest: they play key roles both in the physiology of the cell and in multiple pathological conditions relevant to human health.

Optogenetic control of mitochondrial metabolism and Ca2+ signaling by mitochondria-targeted opsins

Significance Mitochondrial functions depend on the steep H+ electrochemical gradient (ΔμH+) across their inner membrane. The available tools for controlling this gradient are essentially limited to inhibitors of the respiratory chain or of the H+ ATPase or to uncouplers, poisons plagued by important side effects and that lack both cell and spatial specificity. We show here that, by transfecting cells with the cDNA encoding channelrhodopsins specifically targeted to the inner mitochondrial membrane, we can obtain an accurate and spatially confined, light-dependent control of mitochondrial membrane potential and, as a consequence, of a series of mitochondrial activities ranging from electron transport to ATP synthesis and Ca2+ signaling. Key mitochondrial functions such as ATP production, Ca2+ uptake and release, and substrate accumulation depend on the proton electrochemical gradient (ΔμH+) across the inner membrane. Although several drugs can modulate ΔμH+, their effects are hardly reversible, and lack cellular specificity and spatial resolution. Although channelrhodopsins are widely used to modulate the plasma membrane potential of excitable cells, mitochondria have thus far eluded optogenetic control. Here we describe a toolkit of optometabolic constructs based on selective targeting of channelrhodopsins with distinct functional properties to the inner mitochondrial membrane of intact cells. We show that our strategy enables a light-dependent control of the mitochondrial membrane potential (Δψm) and coupled mitochondrial functions such as ATP synthesis by oxidative phosphorylation, Ca2+ dynamics, and respiratory metabolism. By directly modulating Δψm, the mitochondria-targeted opsins were used to control complex physiological processes such as spontaneous beats in cardiac myocytes and glucose-dependent ATP increase in pancreatic β-cells. Furthermore, our optometabolic tools allow modulation of mitochondrial functions in single cells and defined cell regions.

Characterization of the ER-Targeted Low Affinity Ca2+ Probe D4ER

Calcium ion (Ca2+) is a ubiquitous intracellular messenger and changes in its concentration impact on nearly every aspect of cell life. Endoplasmic reticulum (ER) represents the major intracellular Ca2+ store and the free Ca2+ concentration ([Ca2+]) within its lumen ([Ca2+]ER) can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca2+ sensors have been developed over the last years. However, most of them are non-ratiometric and, thus, their signal is difficult to calibrate in live cells and is affected by shifts in the focal plane and artifactual movements of the sample. On the other hand, existing ratiometric Ca2+ probes are plagued by different drawbacks, such as a double dissociation constant (Kd) for Ca2+, low dynamic range, and an affinity for the cation that is too high for the levels of [Ca2+] in the ER lumen. Here, we report the characterization of a recently generated ER-targeted, Förster resonance energy transfer (FRET)-based, Cameleon probe, named D4ER, characterized by suitable Ca2+ affinity and dynamic range for monitoring [Ca2+] variations within the ER. As an example, resting [Ca2+]ER have been evaluated in a known paradigm of altered ER Ca2+ homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer’s Disease-linked protein Presenilin 2 (PS2). The lower Ca2+ affinity of the D4ER probe, compared to that of the previously generated D1ER, allowed the detection of a conspicuous, more clear-cut, reduction in ER Ca2+ content in cells expressing mutated PS2, compared to controls.

The ER-mitochondria connection: New players in Alzheimer's disease

(APP) by b-secretase (mainly BACE1) and g-secretase; a multi-protein complex containing proteolytically active Presenilin-1 (PS1) or Presenilin-2 (PS2). Most mutations in PS1, PS2 or APp, known to cause early-onset AD, predominantly result in an absolute or relative increase in Ab 42 by altered g-secretase cleavage. Since altered g-secretase function is fundamental to the AD process, we hypothesised that proteins or drugs that interact with the secretases may regulate their function and therefore represent potential therapeutic targets. Methods: We have used systems biology technology to search the human proteome for proteins that are known to interact with our seeds BACE1, PS1 or PS2.We then searched these proteins at topological distance 1 (D1) for further interactions at topological distance 2 (D2), focusing on proteins that are expressed in the brain. In addition, we performed a search of D1 and D2 proteins for targets of currently approved drugs.Results:We found 36 (BACE1), 176 (PS1) and 64 (PS2) D1 proteins. Two of the PS1 interactors are known targets for approved drugs (with unknown pharmacological function). In our D2 search, 2 potential g-secretase modulators were highlighted based on their interactions with both PS1 and nicastrin. We also found 5 drug targets (with known pharmacological action) at D2 from PS1 and 3 from BACE1.Conclusions: Our comprehensive search has revealed 15 proteins that interact with PS1 that have not been studied in the field of AD. We are currently verifying these interactions by co-immunoprecipitation and co-localisation in human cell lines. The confirmed interactions will then be investigated for potential modulation of g-secretase activity in vitro. We will also test the effect of the 5 drugs on the interactions of D2 targets to evaluate any potential modulation of Ab production.

Highlighting the endoplasmic reticulum-mitochondria connection: Focus on Mitofusin 2

&NA; The endoplasmic reticulum (ER) and the mitochondrial network are two highly interconnected cellular structures. By proteinaceous tethers, specialized membrane domains of the ER are tightly associated with the outer membrane of mitochondria, allowing the assembly of signaling platforms where different cell functions take place or are modulated, such as lipid biosynthesis, Ca2+ homeostasis, inflammation, autophagy and apoptosis. The ER‐mitochondria coupling is highly dynamic and contacts between the two organelles can be modified in their number, extension and thickness by different stimuli. Importantly, several pathological conditions, such as cancer, neurodegenerative diseases and metabolic syndromes show alterations in this feature, underlining the key role of ER‐mitochondria crosstalk in cell physiology. In this contribution, we will focus on one of the major modulator of ER‐mitochondria apposition, Mitofusin 2, discussing the structure of the protein and its debated role on organelles tethering. Moreover, we will critically describe different techniques commonly used to investigate this crucial issue, highlighting their advantages, drawbacks and limits. Graphical abstract Figure. No caption available.