Critical reappraisal confirms that Mitofusin 2 is an endoplasmic reticulum–mitochondria tether

Abstract

The discovery of the multiple roles of mitochondria-endoplasmic reticulum (ER) juxtaposition in cell biology often relied upon the exploitation of Mitofusin (Mfn) 2 as an ER-mitochondria tether. However, this established Mfn2 function was recently questioned, calling for a critical re-evaluation of Mfn2's role in ER-mitochondria cross-talk. Electron microscopy and fluorescence-based probes of organelle proximity confirmed that ER-mitochondria juxtaposition was reduced by constitutive or acute Mfn2 deletion. Functionally, mitochondrial uptake of Ca 2+ released from the ER was reduced following acute Mfn2 ablation, as well as in Mfn2−/− cells overexpressing the mitochondrial calcium uniporter. Mitochondrial Ca 2+ uptake rate and extent were normal in isolated Mfn2 −/− liver mitochondria, consistent with the finding that acute or chronic Mfn2 ablation or overexpression did not alter mitochondrial calcium uniporter complex component levels. Hence, Mfn2 stands as a bona fide ER-mitochondria tether whose ablation decreases interorganellar juxtaposition and communication.he endoplasmic reticulum (ER) and mitochondria are physically coupled to control mitochondrial Ca 2+ uptake, lipid transfer, autophagosome formation, ER stress, and apoptosis (1-6). Juxtaposition is mediated by protein structures that can be visualized in electron microscopy (EM) and electron tomography (ET) studies. These physical tethers span 6-15 nm when connecting smooth, and 19-30 nm when connecting rough ER to mitochondria (7). Operationally, an ER-mitochondria tether should fulfill at least these minimal criteria: (i) it is retrieved on the outer mitochondrial membrane (OMM); (ii) it is retrieved in mitochondria-associated ER membranes, the ER subdomain involved in interaction with mitochondria; (iii) it interacts in trans with a homo-or heterotypic interactor on the opposing membrane; (iv) its deletion increases the distance between the ER and mitochondria; or (v) its deletion reduces exchange of Ca 2+ and lipids between the ER and mitochondria. The molecular nature of ER-mitochondria tethers remained elusive for many years. The scaffold protein PACS2 (phosphofurin acidic cluster sorting protein 2) modulates their extent (8), and they include the heterotypic association between the inositol triphosphate (IP3) receptor on the ER and the OMM voltagedependent anion channel (9). Another protein that fulfils the operational criteria to be defined as a tether is Mitofusin 2 (Mfn2). This OMM profusion protein is also retrieved in mitochondria-associated ER membranes and ER Mfn2 interacts in trans with Mfn1 or Mfn2 on the mitochondria to physically tether the organelles. Mfn2 ablation increases the distance between the ER and mitochondria and decreases agonist-evoked Ca 2+ transfer from the ER to mitochondria (10) that depends on the generation of high Ca 2+ microdomains at their interface (11,12). The role of Mfn2 as a tether was confirmed independently in the heart (13), in pro-opiomelanocortin neurons (14), and in the liver (15).Mfn2-dependent tethe...

Mitochondrial dynamics in neuronal injury, development and plasticity

“…However, there is currently a debate as to whether Mfn2 promotes or inhibits the apposition between ER and mitochondrial membranes. Work by the Scorrano group has suggested that Mfn2 is necessary for ER-mitochondrial association because deletion of Mfn2 decreases this interaction Naon et al, 2016). However, other groups have challenged this model, suggesting instead that deletion of Mfn2 increases ER-mitochondrial association and, in turn, are questioning whether Mfn2, indeed, localizes to the ER (Filadi et al, 2015;Cosson et al, 2012).…”

Loss of sirtuin 1 and mitofusin 2 contributes to enhanced ischemia/reperfusion injury in aged livers

“…Furthermore, SIRT1 overexpression reversed pathological events through its interaction with and subsequent deacetylation of MFN2. This mitochondrial outer membrane protein has diverse functions such as mitochondrial fusion (Chen et al., 2003), tethering between mitochondria and endoplasmic reticulum (ER) (de Brito & Scorrano, 2008; Naon et al., 2016), metabolic regulation (Bach et al., 2003; Sebastián et al., 2012), and the endocytic/secretory pathway (Daniele et al., 2014; Zhao et al., 2012). It is currently unknown how SIRT1 and MFN2 are affected by I/R in old hepatocytes.…”

From dysfunctional endoplasmic reticulum-mitochondria coupling to neurodegeneration

“…In agreement with this possibility, a recent study demonstrated an impact of the cell density at the moment of plating on the degree of ER-mitochondria juxtaposition and on expression levels of the MCU (Naon et al, 2016). …”

“…In mammals, the identity of the ER-mitochondria tethering complexes remains elusive. There is a consensus that mitofusin 2 (Mfn2) localizes both on the ER and OM membranes and regulates ER-mitochondria juxtaposition, although whether it functions as a tether (Alford et al, 2012;de Brito and Scorrano, 2008;Naon et al, 2016;Schneeberger et al, 2013) or tethering inhibitor (Cosson et al, 2012;Filadi et al, 2015Filadi et al, , 2016Leal et al, 2016) has been discussed. Controversial observations are not limited to the above mentioned studies, in which the role of Mfn2 in ER-mitochondria tethering was directly addressed by knockdown or knockout approaches in different model systems.…”

“…Measuring the colocalization of ER and mitochondrial markers is not the optimal approach for the assessment of organelle proximity, because of the limited resolution of confocal light microscopy (200 nm). Alternative fluorescent-based methods, such as Fluorescence Resonance Energy Transfer (FRET, (Csordas et al, 2010)), Proximity Ligation Assay (PLA, (Bernard-Marissal et al, 2015;De Vos et al, 2012;Hedskog et al, 2013;Stoica et al, 2016)) or dimerization-dependent fluorescence/luminescence (Alford et al, 2012;Lim et al, 2015;Naon et al, 2016), bypass this limitation by detecting specific interactions between ER and OM membranelocalized proteins. Super-resolution imaging approaches allow the assessment of changes in individual ER-mitochondria contacts, and are, so far, the most adapted method to investigate this subcellular compartment.…”