International Journal of Molecular Sciences Review ER–Mitochondria Contact Sites Reporters: Strengths and Weaknesses of the Available Approaches Flavia Giamogante 1 , Lucia Barazzuol 1, Marisa Brini 2,* and Tito Calì 1,3,* 1 Department of Biomedical Sciences, University of Padova, 35131 Padova, Italy; fl[email protected] (F.G.); [email protected] (L.B.) 2 Department of Biology, University of Padova, 35131 Padova, Italy 3 Padova Neuroscience Center (PNC), University of Padova, 35131 Padova, Italy * Correspondence: [email protected] (M.B.); [email protected] (T.C.) Received: 22 September 2020; Accepted: 30 October 2020; Published: 31 October 2020 Abstract: 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. Keywords: ER–mitochondria tethering; split-GFP; SPLICS; organelle contact sites 1. Introduction In eukaryotes, organelles are delimited by a membrane that enables them to perform specific and unique tasks, whose characterization has been the goal of researchers for many decades. In the last years, however, the inter-organelle communication received momentum and represents an emerging aspect in cell biology. It is now well established that organelles are not isolated, but they are interconnected, thereby forming a communicating network [1]. Proteins identified at the interface between organelles seem to act as tethers that physically bridge two opposing membranes and join them to each other [2,3]; others have a specific role that occurs at the contact sites, such as the ability to transfer small molecules in a non-vesicular manner [4,5]. The interplay between the endoplasmic reticulum (ER) and mitochondria has been one of the first identified and, to date, still represents one of the best characterized forms. ER–mitochondria juxtaposition has been firstly observed by electron microscopy (EM) in the late 1950s in rat tissue; however, at that time, it was considered an artifact due to fixation [6]. Conversely, later approaches compatible with living cells analysis have validated their existence [7]. Now, it is well established that ER–mitochondrial tethering plays a crucial role in several cellular pathways, such as Ca2+ homeostasis [8,9], lipid transfer [10], mitochondrial dynamics [11], apoptosis, and mitophagy [12,13]. Many shreds of evidence report that its alteration is linked to the development of different disorders including diabetes [14], cancer [15], and neurodegenerative diseases (e.g., Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS) [16]). Int. J. Mol. Sci. 2020, 21, 8157; doi:10.3390/ijms21218157 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 8157 2 of 18 Int. J. Mol. Sci. 2020, 21, 8157 2 of 18 However, most of the research aimed at characterizing ER–mitochondria contact-site componentsHowever, and most their of thefunctions research face aimed the atcomplex characterizing issue of ER–mitochondria unequivocally identifying contact-site componentsthe contact, definingand their its functions function, face and the monitoring complex issue its changes of unequivocally upon perturbations. identifying the To contact,date, several defining methods its function, need toand be monitoring combined itsto changesextract all upon the perturbations.mentioned information To date, several since an methods optimized need approach to be combined that can to provideextract all them the mentionedat once is informationstill missing. since Indeed, an optimized the transient approach nature that and can variable provide abundance them at once of is ER– still mitochondriamissing. 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Proteins Proteins Involved Involved in ER–Mitochondria Interplay Among the organelles interacting with the ER, ER, wh whichich include include the the Golgi Golgi apparatus, apparatus, mitochondria, mitochondria, peroxisomes, andand the the plasma plasma membrane, membrane, mitochondria mitochondria establish establish one of theone most of andthe well-characterizedmost and well- characterizedconnection [6]. connection Electron tomography [6]. Electron was tomography used to measure was used ER–mitochondria to measure ER–mitochondria proximity and itproximity revealed andthat it the revealed distance that between the distance the two between organelles the istwo approximately organelles is of approximately 10–30 nm [3,8 ].of This10–30 distance nm [3,8]. is closeThis distanceenough to is proposeclose enough that the to ERpropose and the that outer the mitochondria ER and the outer membrane mitochondria (OMM) canmembrane be tethered (OMM) together can beby proteinstethered locatedtogether on by the proteins opposing located membranes—indeed, on the opposing mitochondria-associated membranes—indeed, ERmitochondria- membranes associated(MAMs) can ER be membranes physically separated(MAMs) can from be thephysical otherly organelle separated membranes from the other [17–20 organelle]. We also membranes know that [17–20].their functional We also interplayknow that [ their21] is functional guaranteed interplay by specific, [21] is stabilized guaranteed contacts by specific, [22] that stabilized are able contacts to stay [22]tethered that toare each able other to stay even tethered when the to organelles each other are even moving when along the the organelles cytoskeleton are [23moving]. Many along proteins the cytoskeletonhave been identified [23]. Many in the proteins MAMs but,have for been many iden of them,tified thein the biological MAMs role but, is notfor yetmany fully of understood them, the biological(Figure1). role is not yet fully understood (Figure 1). Figure 1. 1. SchematicSchematic representation representation of the of thetethering tethering complexes complexes at the at endoplasmic the endoplasmic reticulum reticulum (ER)– mitochondria(ER)–mitochondria interface. interface. In yeast cells, the ER–mitochondria connection is ensured by a multiprotein complex called the ER–mitochondria encounter structure (ERMES), containing Mdm12, Mdm34, Mdm10, and Mmm1 proteins [[24].24]. This physical tethering allows efficient efficient lipid transport by soluble lipid-carrier proteins such as ceramide-transfer protein (CERT) and oxysterol-binding protein (OSBP) [[25].25]. In mammalian cells, the the ER–mitochondria ER–mitochondria interface interface is is more more complex. complex. Indeed, Indeed, a plethora a plethora of ofproteins proteins are are involved involved in maintaining and modulating this interaction. Mitofusin 2 (MFN2) is one of such players. It is a Int. J. Mol. Sci. 2020, 21, 8157 3 of 18 in maintaining and modulating this interaction. Mitofusin 2 (MFN2) is one of such players. It is a mitochondrial fusion guanosine-50-triphosphate (GTP)ases, localized at the outer membrane of mitochondria (OMM) and found in MAMs [26–29]. MFN2 plays a crucial role in mitochondrial fusion together with Optic Atrophy 1 OPA1, another mitochondrial fusion GTPase located on the inner membrane of mitochondria [30]. It has been reported that during the mitochondrial fusion process, mitochondrial MFN2 assembles homo- or heterodimer complexes with ER-residing MFN2 [28,31]. The interaction between the protein tyrosine phosphatase-interacting protein-51 (PTPIP51), located on the OMM, and an ER-resident protein, the vesicle-associated membrane protein B (VAPB),