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Mitochondrial fusion

Subtítulo

intro

Membrane fusion is a fundamental biological process in mitocondrial dynamics. During fusion, the two membranous compartments, initially separated and delimited by lipid bilayers, have to connect and merge in order to mix their membrane and aqueous components. Biological membranes are designed to be stable, therefore fusion events are energetically costly and require the intervention of specialized proteins which help membranes to advance through the successive stages leading to fusion. These four stages  include: (i) membrane recognition and docking, (ii) membrane approach and deformation, (iii) membrane disruption and merging, with the potential formation of a hemi-fusion structure where the outer leaflets of the lipid bilayers have merged, while the inner leaflets and the aqueous compartments remain separated and (iv) the formation and growth of a fusion pore, leading to mixing of the two aqueous compartments [1].

In particular, mitochondrial fusion is described as a two-step process that starts with the GTP-dependent fusion of the outer mitochondrial membrane (OMM) that is subsequently followed by the fusion of the inner mitochondrial membrane (IMM) [2]. The first step is directed by the mitochondrial fusion proteins Mitofusin 1 and 2 (Mfn1 and Mfn2) [3], whereas the fusion of IMM is controlled by the optic atrophy type 1 protein Opa1 [4]. Although both fusion events are independent, they need to strictly be coordinated to prevent mitocondrial dysfunction [5]. These proteins might be also assisted by various regulatory factors (lipids or proteins), which facilitate fusion (for example, by inducing local membrane curvature), and/or ensure that fusion occurs at the right time and place (for example, by trapping and releasing the fusion machinery in response to particular signals).

 

Our first goal is to unveil the minimal components necessary for Mfn-dependent fusion and the pathway it occurs. To address this question we reconstitute the full-length Mfn2 protein into lipid vesicles. In vitro experiments with Mfn2 in small unilamellar vesicles (Mfn2-SUVs) show that GTP, but not GDP, induces the fusion of SUVs in the second timescale. Moreover, the presence of physiological concentration (up to 30 mol %) of dioleylphosphatidylethanolamine (DOPE) was shown to be a requisite to observe GTP induced Mfn2-dependent fusion. Also, the protein-to-lipid ratio for efficient fusion was determined to be ≈ 103. The direct visualization of the fusion process was tracked by reconstituting the fusion protein into giant unilamellar vesicles (GUVs). High-speed video-microscopy demonstrates that Mfn2-dependent fusion follows an uncommon pathway where the adhesion patch of apposing membranes does not progress to a hemifusion diaphragm but rather grows through a zipper mechanism at the rim of the contact interface or septum. Finally, the adhesion patch opens nearby the rim of the septum and expands fast to eventually complete the fusion of two apposing membranes [6].

Our synthetic platform opens the way to combined reconstitutions of Mfn1 and Mfn2 or the addition of other regulating proteins for a better insight on  the mitochondrial fusion process. Structural aspects of Mfn2 are also being explored using both single molecule techniques and scattering-based methods.

references

  1. L. V. Chernomordik, M. M. Kozlov. Cell 123, 375-382 (2005).
  2. S. Meeusen, J. M. McCaffery, J. Nunnari. Science 305, 1747-1752 (2004).
  3. Y. Eura, N. Ishihara, S. Yokota, K. Mihara. Journal of biochemistry 134, 333-344 (2003).
  4. A. Zorzano, M. Liesa, D. Sebastian, J. Segales, M. Palacin. Semin. Cell Dev. Biol. 21, 566-574 (2010)
  5. N. Ul Fatima, V. Ananthanarayanan. Current Opinion in Cell Biology 80, 102150 (2023).