Drosophila Marf is the evolutionary ancestor of mammalian Mfn2: a phylogenetic analysis
Year: 2011
Type: dissertation
Abstract: Mitochondria are essential organelles for cellular homeostasis. Their main function is to produce energy: mitochondrial respiration provides most of the ATP required for endoergonic reactions. Furthermore, they regulate levels and transients of cytosolic Ca2+ and are crucially involved in apoptosis, aging and oxidative stress (Dimmer and Scorrano, 2006). As much as 20% of the mitochondrial surface is in close contact with the endoplasmic reticulum (ER). This organization is important for the generation of high Ca2+ microdomains required to activate mitochondrial Ca2+ uptake under certain conditions (Rizzuto et al., 1998a). The sites where ER and mitochondria are juxtaposed form the so called mitochondria-associated membranes (MAMs), crucial for lipid and Ca2+ traffic between the two organelles and are also involved in cell death (de Brito and Scorrano, 2010). Molecular mechanisms responsible for this ER-mitochondria juxtaposition are largely unknown, but it is thought that structural changes of either organelle could regulate the interaction (Pitts et al., 1999; Simmen et al., 2005). The shape of mitochondria is determined by the equilibrium between fusion and fission processes, controlled by a family of “mitochondria-shaping proteins”. In mammalian cells, mitochondrial fusion depends on mitofusin 1 and 2 (Mfn1 and 2) in the outer mitochondrial membrane and on the inner membrane protein optic atrophy 1 (Opa1) (Liesa et al., 2009). Fission requires the additional step of translocation of dynamin-related protein 1 (Drp1) from the cytosol to mitochondria where it presumably docks on the protein fission-1 (hFis1), its adaptor in the outer membrane. Oligomerization of Drp1 is believed to provide the mechanical force to constrict mitochondrial membranes and to fragment the organelle (Hinshaw, 1999b; Smirnova et al., 2001). Translocation of Drp1 to mitochondria depends on its dephosphorylation by calcineurin and other phosphatases (Cereghetti et al., 2008). Mitofusins are dynamin-related GTPases which control mitochondrial fusion and morphology. In the past, the presence of two mammalian mitofusins raised the question of their functional divergence. Nowadays many findings support the idea that Mfn1 and 2 do not display redundant functions. Mfn2 has roles which are not shared by Mfn1, such as the control of mitochondrial oxidation (Bach et al., 2005b) and anti-proliferative function (Chen et al., 2004a). Moreover, while mitochondrial fusion mediated by the inner mitochondrial membrane GTPase OPA1 requires Mfn1 (Cipolat et al., 2004b), Mfn2 appears to be involved in the regulation of endoplasmic reticulum (ER)-mitochondria tethering (de Brito and Scorrano, 2008a). MFN2 has also been involved in several diseases including the peripheral Charcot-Marie-Tooth type IIa (CMTIIa) neuropathy (Zuchner et al., 2004a). Both Mfn1 and Mfn2 are essential for embryonic development and mice deficient in either gene die in mid-gestation, but Mfn2-/- embryos display also deficient placentation (Chen et al., 2003h).. However, post-placentation ablation of either Mfn in the mouse leads to two completely different phenotypes: Mfn1-/- mice are viable, while Mfn2-/- ones day between P1 and P17 as a consequence of massive cerebellar degeneration (Chen et al., 2007b). It is unclear whether the different phenotypes observed in vivo and in vitro can be ascribed to functional differences or simply to different expression patterns of the two Mfns . In addition, it should be noted that all vertebrates possess two Mfn, whereas only one Mfn is retrieved in almost all invertebrates reigns, with the exception of D.melanogaster. The fruitfly possesses 2 mitofusins, Fuzzy onion (Fzo) and Mitochondrial Assembly Regulatory Factor (Marf). Fzo was the first identified mediator of mitochondrial fusion (Hales and Fuller, 1997a). In spermatocytes Fzo mediates the fusion of mitochondria into two giant organelles that form a structure called Nebenkern, which is required to give energy to the flagellum of the spermatid. In fzo mutant flies, mitochondria fail to fuse and wrap each other as many fragmented organelles, giving the impression of ‘fuzzy onions’ when viewed at the electron microscopy. Altough Fzo was able to promote mitochondrial elongation, it was expressed restrictely to spermatids. This raised the question of how mitochondrial fusion was regulated in other tissues of the fruitfly. In 2002 the second mitofusin homologue Marf was identified in D.melanogaster (Hwa et al., 2002). Unlike Fzo, DMarf is expressed ubiquitously and shares 64% of homology with both mammalian mitofusins. Thus, Marf can be likely considered the single “functional” invertebrate Mfn and Drosophila melanogaster is therefore a useful model organism to investigate phylogenesis of higher vertebrate Mfns. In this thesis we set to answer to the following questions: does this model organism represent the evolutionary turning point from one to two mitofusins? Which Mfn is functionally closer to DMarf? To answer to these questions, we first tried to understand in which extension DMarf could complement mammalian mitofusins. To obtain a wider phylogenetic view, we extended our analysis on mitofusins 1 and 2 from the vertebrate Xenopus laevis (XMfn1 and XMfn2) and on mitofusin homologue from the yeast S.cerevisae (Fzo1p). The expression of Fzo1p, DMarf, XMfn1 and XMfn2, induced mitochondrial elongation in Mfn1-/- or Mfn2-/- MEFs, rescuing the fragmented phenotype caused by the absence of the Mfns and indicating that they can substitute for both Mfns. Unlike Mfn1-/- MEFs, Mfn2-/- MEFs have altered ER morphology (de Brito and Scorrano, 2008b). This defect is recovered only after expression of hMfn2 or by an ER-targeted variant of hMfn2 (hMfn2YFFT), independently from mitochondrial shape. In fact Mfn2 is partially localized on ER and is enriched in MAMs. To investigate whether DMarf could complement Mfn2 also in ER shape regulation, we cotransfected Mfn2-/- MEFs with DMarf-V5 and ER-targeted yellow fluorescent protein (ERYFP). DMarf was able to rescue ER shape in Mfn2-/-, thus displaying a functional overlap with Mfn2. We then turned our attention to an in vivo analysis of DMarf function in Drosophila. DMarf was essential for viability as shown by ubiquitous, neuronal and muscle specific downregulation of the protein, which were all lethal. In larvae depleted of DMarf mitochondria clumped in the perinuclear regions of neuronal cell bodies and muscle tissues. In addition, neuromuscular junctions (NMJs) of DMarf-RNAi individuals were severely depleted of mitochondria compared to control larvae. Given that Mitofusin-2 regulates ER morphology and tethering to mitochondria, we also investigated the effect of DMarf knockdown on ER architecture which was seriously compromised upon DMarf knockdown in muscle tissues, with loss of the sarcomeric organization of the ER. To investigate the effect of overexpression of hMfns in Drosophila, we characterized transgenic lines expressing hMfn1, hMfn2 and hMfn2R94Q, one of the most frequent mutations associated with CMT2A (Zuchner et al., 2004b). Aggregation of mitochondria in neuronal cell bodies and muscle and elongated or clumped organelles inside axons were observed upon overexpression of all hMfns. However, NMJs analysis revealed a major difference between hMfn1, hMfn2 and hMfn2R94Q expressions: while NMJs of hMfn2 and hMfn2R94Q overexpressing individuals were depleted of mitochondria, hMfn1 expression did not alter organelles distribution in the nervous system and mitochondria were retained inside junctions. Muscle ER organization was not affected in wild-type hMfns expressing larvae; interestingly, ER morphology was found altered in muscle tissues only of hMfn2R94Q individuals, showing an aspect never investigated in the study of Charcot-Marie-Tooth pathogenesis and suggesting ER morphology alteration as one of the putative factors contributing to the pathobiology of the disease. Given that DMarf was able to complement both mfns in mitochondrial morphology and Mfn2 in ER shape regulation, we tried to understand to which extent hMfn1 and -2 could surrogate Marf in Drosophila. Simultaneous expression of Mfn2 and Marf-RNAi transgenes in the nervous system or in muscle resulted in partial survival to adulthood,. while Mfn1 or Mfn2R94Q were unable to recover the DMarf-RNAi and the flies died as pupae,. However, mitochondrial clusters were still present in neuronal cell bodies and muscle and mitochondria were lacking in NMJs of individuals simultaneously expressing DMarf-RNAi and hMfn2. Thus, although Mfn2 rescued DMarf depletion lethality, this could not be ascribed to a recovery of mitochondrial morphology and distribution. We therefore assessed recovery of ER organization in individuals simultaneously expressing DMarf-RNAi and hMfns. HMfn1 had no effect on ER alteration caused by DMarf knockdown. On the contrary, hMfn2 expression recovered muscle ER architecture both when expressed ubiquitously or only in the muscle . In conclusions, in this Thesis we demonstrate that Fzo1p, DMarf, XMfn1 and XMfn2 expression could rescue mitochondrial morphology in Mfn1 -/- and Mfn2-/- MEFs. Moreover DMarf specifically complements ER shape in Mfn2-/- MEFs. Thus, mitofusins role in regulation of mitochondrial dynamics is conserved between vertebrates and invertebrates. On the other hand, in vivo experiments show that Mfn2, but not Mfn1 rescues the lethal phenotype of DMarf knock-down in Drosophila melanogaster. the rescue of DMarf-RNAi by hMfn2 seemed correlate with the correction of ER organization, whereas mitochondrial morphology and distribution were not restored. We can therefore speculate that Marf is functionally closer to Mfn2 than Mfn1, which may have diverged later during mammalian evolution. Finally, factors other than the disruption of mitochondrial distribution and morphology in the nervous system could explain the lethal phenotype of DMarf knockdown and in
Author: Valentina Debattisti
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2284363454