Diana Pendin

Balancing ER dynamics: Shaping, bending, severing, and mending membranes

The endoplasmic reticulum is a multifunctional organelle composed of functionally and morphologically distinct domains. These include the relatively planar nuclear envelope and the peripheral ER, a network of sheet-like cisternae interconnected with tubules that spread throughout the cytoplasm. The ER is highly dynamic and the shape of its domains as well as their relative content are in constant flux. The multiple forces driving these morphological changes depend on the interaction between the ER and microtubules, membrane fusion and fission events and the action of proteins capable of actively shaping membranes. The interplay between these forces is ultimately responsible for the dynamic morphology of the ER, which in turn is crucial for properly executing the varied functions of this organelle.

Reduction of endoplasmic reticulum stress attenuates the defects caused by Drosophila mitofusin depletion

The developmental and motor defects evident in flies depleted of the mitofusin Marf can be ameliorated by treatments that reduce ER stress, confirming an active role for ER stress in the observed phenotypes.

GTP-dependent packing of a three-helix bundle is required for atlastin-mediated fusion

The mechanisms governing atlastin-mediated membrane fusion are unknown. Here we demonstrate that a three-helix bundle (3HB) within the middle domain is required for oligomerization. Mutation of core hydrophobic residues within these helices inactivates atlastin function by preventing membrane tethering and the subsequent fusion. GTP binding induces a conformational change that reorients the GTPase domain relative to the 3HB to permit self-association, but the ability to hydrolyze GTP is required for full fusion, indicating that nucleotide binding and hydrolysis play distinct roles. Oligomerization of atlastin stimulates its ability to hydrolyze GTP, and the energy released drives lipid bilayer merger. Mutations that prevent atlastin self-association also abolish oligomerization-dependent stimulation of GTPase activity. Furthermore, increasing the distance of atlastin complex formation from the membrane inhibits fusion, suggesting that this distance is crucial for atlastin to promote fusion.

The effects of ER morphology on synaptic structure and function in Drosophila melanogaster

ABSTRACT Hereditary spastic paraplegia (HSP) is a set of genetic diseases caused by mutations in one of 72 genes that results in age-dependent corticospinal axon degeneration accompanied by spasticity and paralysis. Two genes implicated in HSPs encode proteins that regulate endoplasmic reticulum (ER) morphology. Atlastin 1 (ATL1, also known as SPG3A) encodes an ER membrane fusion GTPase and reticulon 2 (RTN2, also known as SPG12) helps shape ER tube formation. Here, we use a new fluorescent ER marker to show that the ER within wild-type Drosophila motor nerve terminals forms a network of tubules that is fragmented and made diffuse upon loss of the atlastin 1 ortholog atl. atl or Rtnl1 loss decreases evoked transmitter release and increases arborization. Similar to other HSP proteins, Atl inhibits bone morphogenetic protein (BMP) signaling, and loss of atl causes age-dependent locomotor deficits in adults. These results demonstrate a crucial role for ER in neuronal function, and identify mechanistic links between ER morphology, neuronal function, BMP signaling and adult behavior. Highlighted Article: The ER within Drosophila motor nerve terminals forms a network of tubules. Loss of the fusion GTPase atlastin fragments synaptic ER. Alteration of ER structure decreases evoked neurotransmitter release.

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.

The Concerted Action of Mitochondrial Dynamics and Positioning: New Characters in Cancer Onset and Progression.

Mitochondria are dynamic organelles whose morphology and activity are extremely variable, depending on the metabolic state of the cell. In particular, their shape and movements within the cell are finely regulated by an increasing number of proteins, which take part in the process of mitochondrial fission/fusion and connect the organelles to the cytoskeleton. As to their activities, mitochondria are considered to be at the crossroad between cell life and death since, on the one hand, they are essential in ATP production and in multiple metabolic pathways but, on the other, they are involved in the intrinsic apoptotic cascade, triggered by different stress conditions. Importantly, the process of mitochondrial Ca2+ uptake, as well as the morphology and the dynamics of these organelles, is known to deeply impact on both pro-survival and pro-death mitochondrial activities. Recently, increasing evidence has accrued on a central role of deregulated mitochondrial functionalities in the onset and progression of different pathologies, ranging from neurodegenerative diseases to cancer. In this contribution, we will present the latest findings connecting alterations in the machineries that control mitochondrial dynamics and localization to specific cancer hallmarks, highlighting the importance of mitochondria for the viability of cancer cells and discussing their role as promising targets for the development of novel anticancer therapies.

Mitofusin 2: from functions to disease

Mitochondria are highly dynamic organelles whose functions are essential for cell viability. Within the cell, the mitochondrial network is continuously remodeled through the balance between fusion and fission events. Moreover, it dynamically contacts other organelles, particularly the endoplasmic reticulum, with which it enterprises an important functional relationship able to modulate several cellular pathways. Being mitochondria key bioenergetics organelles, they have to be transported to all the specific high-energy demanding sites within the cell and, when damaged, they have to be efficiently removed. Among other proteins, Mitofusin 2 represents a key player in all these mitochondrial activities (fusion, trafficking, turnover, contacts with other organelles), the balance of which results in the appropriate mitochondrial shape, function, and distribution within the cell. Here we review the structural and functional properties of Mitofusin 2, highlighting its crucial role in several cell pathways, as well as in the pathogenesis of neurodegenerative diseases, metabolic disorders, cardiomyopathies, and cancer.

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.