Regulation of Endoplasmic Reticulum-Mitochondria Tethering and Ca2+ Fluxes by TDP-43 via GSK3ß.

Mitochondria-ER contacts (MERCs), tightly regulated by numerous tethering proteins that act as molecular and functional connections between the two organelles, are essential to maintain a variety of cellular functions. Such contacts are often compromised in the early stages of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). TDP-43, a nuclear protein mainly involved in RNA metabolism, has been repeatedly associated with ALS pathogenesis and other neurodegenerative diseases. Although TDP-43 neuropathological mechanisms are still unclear, the accumulation of the protein in cytoplasmic inclusions may underlie a protein loss-of-function effect. Accordingly, we investigated the impact of siRNA-mediated TDP-43 silencing on MERCs and the related cellular parameters in HeLa cells using GFP-based probes for MERCs quantification and aequorin-based probes for local Ca2+ measurements, combined with targeted protein and mRNA profiling. Our results demonstrated that TDP-43 down-regulation decreases MERCs density, thereby remarkably reducing mitochondria Ca2+ uptake after ER Ca2+ release. Thorough mRNA and protein analyses did not highlight altered expression of proteins involved in MERCs assembly or Ca2+-mediated ER-mitochondria cross-talk, nor alterations of mitochondrial density and morphology were observed by confocal microscopy. Further mechanistic inspections, however, suggested that the observed cellular alterations are correlated to increased expression/activity of GSK3ß, previously associated with MERCs disruption.

ER-Mitochondria Contact Sites Reporters: Strengths and Weaknesses of the Available Approaches.

Organelle intercommunication represents a wide area of interest. Over the last few decades, increasing evidence has highlighted the importance of organelle contact sites in many biological processes including Ca2+ signaling, lipid biosynthesis, apoptosis, and autophagy but also their involvement in pathological conditions. ER-mitochondria tethering is one of the most investigated inter-organelle communications and it is differently modulated in response to several cellular conditions including, but not limited to, starvation, Endoplasmic Reticulum (ER) stress, and mitochondrial shape modifications. Despite many studies aiming to understand their functions and how they are perturbed under different conditions, approaches to assess organelle proximity are still limited. Indeed, better visualization and characterization of contact sites remain a fascinating challenge. The aim of this review is to summarize strengths and weaknesses of the available methods to detect and quantify contact sites, with a main focus on ER-mitochondria tethering.

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

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.

Get Closer to the World of Contact Sites: A Beginner's Guide to Proximity-Driven Fluorescent Probes.

To maintain cellular homeostasis and to coordinate the proper response to a specific stimulus, information must be integrated throughout the cell in a well-organized network, in which organelles are the crucial nodes and membrane contact sites are the main edges. Membrane contact sites are the cellular subdomains where two or more organelles come into close apposition and interact with each other. Even though many inter-organelle contacts have been identified, most of them are still not fully characterized, therefore their study is an appealing and expanding field of research. Thanks to significant technological progress, many tools are now available or are in rapid development, making it difficult to choose which one is the most suitable for answering a specific biological question. Here we distinguish two different experimental approaches for studying inter-organelle contact sites. The first one aims to morphologically characterize the sites of membrane contact and to identify the molecular players involved, relying mainly on the application of biochemical and electron microscopy (EM)-related methods. The second approach aims to understand the functional importance of a specific contact, focusing on spatio-temporal details. For this purpose, proximity-driven fluorescent probes are the experimental tools of choice, since they allow the monitoring and quantification of membrane contact sites and their dynamics in living cells under different cellular conditions or upon different stimuli. In this review, we focus on these tools with the purpose of highlighting their great versatility and how they can be applied in the study of membrane contacts. We will extensively describe all the different types of proximity-driven fluorescent tools, discussing their benefits and drawbacks, ultimately providing some suggestions to choose and apply the appropriate methods on a case-to-case basis and to obtain the best experimental outcomes.

Stable Integration of Inducible SPLICS Reporters Enables Spatio-Temporal Analysis of Multiple Organelle Contact Sites upon Modulation of Cholesterol Traffic.

The study of organelle contact sites has received a great impulse due to increased interest in the understanding of their involvement in many disease conditions. Split-GFP-based contact sites (SPLICS) reporters emerged as essential tools to easily detect changes in a wide range of organelle contact sites in cultured cells and in vivo, e.g., in zebrafish larvae. We report here on the generation of a new vector library of SPLICS cloned into a piggyBac system for stable and inducible expression of the reporters in a cell line of interest to overcome any potential weakness due to variable protein expression in transient transfection studies. Stable HeLa cell lines expressing SPLICS between the endoplasmic reticulum (ER) and mitochondria (MT), the ER and plasma membrane (PM), peroxisomes (PO) and ER, and PO and MT, were generated and tested for their ability to express the reporters upon treatment with doxycycline. Moreover, to take advantage of these cellular models, we decided to follow the behavior of different membrane contact sites upon modulating cholesterol traffic. Interestingly, we found that the acute pharmacological inhibition of the intracellular cholesterol transporter 1 (NPC1) differently affects membrane contact sites, highlighting the importance of different interfaces for cholesterol sensing and distribution within the cell.

Split Green Fluorescent Protein-Based Contact Site Sensor (SPLICS ) for Heterotypic Organelle Juxtaposition as Applied to ER -Mitochondria Proximities.

In the last decades, membrane contact sites (MCSs) have been the object of intense investigation in different fields of cell physiology and pathology and their importance for the correct functioning of the cell is now widely recognized. MCS between any known intercellular organelles, including endoplasmic reticulum (ER), mitochondria, Golgi, endosomes, peroxisomes, lysosomes, lipid droplets, and the plasma membrane (PM), have been largely documented and in some cases the molecules responsible for the tethering also identified. They represent specific membrane hubs where a tightly coordinated exchange of ions, lipids, nutrients, and factors required to maintain proper cellular homeostasis takes place. Their delicate, dynamic, and sometimes elusive nature prevented and/or delayed the development of tools to easily image interorganelle proximity under physiological conditions and in living organisms. Nowadays, this aspect received great momentum due to the finding that MCSs' dysregulation is involved in several pathological conditions. We have recently developed modular, split-GFP-based contact site sensors (SPLICS) engineered to fluoresce when homo- and heterotypic juxtapositions between ER and mitochondria occur over a range of specific distances. Here we describe in detail, by highlighting strengths and weaknesses, the use and the application of these novel genetically encoded SPLICS sensors and how to properly quantify short- and long-range ER-mitochondria interactions.

Tau localises within mitochondrial sub-compartments and its caspase cleavage affects ER-mitochondria interactions and cellular Ca2+ handling.

Intracellular neurofibrillary tangles (NFT) composed by tau and extracellular amyloid beta (Aß) plaques accumulate in Alzheimer's disease (AD) and contribute to neuronal dysfunction. Mitochondrial dysfunction and neurodegeneration are increasingly considered two faces of the same coin and an early pathological event in AD. Compelling evidence indicates that tau and mitochondria are closely linked and suggests that tau-dependent modulation of mitochondrial functions might be a trigger for the neurodegeneration process; however, whether this occurs either directly or indirectly is not clear. Furthermore, whether tau influences cellular Ca2+ handling and ER-mitochondria cross-talk is yet to be explored. Here, by focusing on wt tau, either full-length (2N4R) or the caspase 3-cleaved form truncated at the C-terminus (2N4RΔC20), we examined the above-mentioned aspects. Using new genetically encoded split-GFP-based tools and organelle-targeted aequorin probes, we assessed: i) tau distribution within the mitochondrial sub-compartments; ii) the effect of tau on the short- (8-10 nm) and the long- (40-50 nm) range ER-mitochondria interactions; and iii) the effect of tau on cytosolic, ER and mitochondrial Ca2+ homeostasis. Our results indicate that a fraction of tau is found at the outer mitochondrial membrane (OMM) and within the inner mitochondrial space (IMS), suggesting a potential tau-dependent regulation of mitochondrial functions. The ER Ca2+ content and the short-range ER-mitochondria interactions were selectively affected by the expression of the caspase 3-cleaved 2N4RΔC20 tau, indicating that Ca2+ mis-handling and defects in the ER-mitochondria communications might be an important pathological event in tau-related dysfunction and thereby contributing to neurodegeneration. Finally, our data provide new insights into the molecular mechanisms underlying tauopathies.