Molecular mechanisms of human mitochondrial dynamics : inspiration and challenge

Abstract

Mitochondria are highly dynamic organelles in cells. They frequently change the morphology via continuous fission and fusion events, referred to as mitochondrial dynamics, which includes the shape, size and number of mitochondria in the cell, as well as mitochondrial subcellular distribution and mitochondrial quality control. Mitochondrial dynamics plays critical roles in sustaining the physiological functions of mitochondria. Dysfunction of mitochondrial dynamics is associated with various human diseases. Mitochondrial dynamics is mediated by a group of mitochondria-shaping proteins in yeast and in mammals. In yeast, the mitochondrial fusion mechanism consists of three central proteins, the inner membrane-anchored Mgm1p, and the outer membrane-anchored Fzo1p and Ugo1p. Correspondingly, Mfn1/2 (the homologs of Fzo1p) and OPA1 (the homolog of Mgm1p) are the core mitochondrial fusion proteins in mammals. On the mitochondrial fission side, the dynamin-related protein Dnm1p (in yeast)/Drp1 (in mammals) is a core component of the mitochondrial fission machinery, and the recruitment of cytosolic Dnm1p/Drp1 to the mitochondrial surface is the crucial step for mitochondrial division process. In yeast, Dnm1p is recruited to mitochondria by the mitochondrial receptor Fis1p via Mdv1p or Caf4p as the adaptor. However, in mammals, Fis1 (the homolog of yeast Fis1p) is not essential for the recruitment of Drp1 (the homolog of Dnm1p), although Fis1p and Fis1 proteins are evolutionarily conserved and both can induce mitochondrial fragmentation when overexpressed. In agreement with this notion, overexpression or depletion of hFis1 does not affect Drp1 distribution in cells. Furthermore, no equivalents of Caf4p and Mdv1p have been found in mammalian cells to date. The functions of Fis1 in mammalian cells remain elusive. Instead, Mff and MIEF1/2 have been identified as new mitochondrial receptors for Drp1 in mammals. While the three receptors all can recruit Drp1 to mitochondria, whether they have distinct functions and how they work together in the mitochondrial fission process need to be further evaluated. In this thesis, we investigated the functions of Drp1 receptors Fis1, MIEF1/2 and Mff in mitochondrial dynamics of human cells and provide novel insights into the molecular mechanisms of human mitochondrial dynamics.

In study I, we explored the distinct functions of MIEF1 and its paralog MIEF2. The similarities of MIEF1 and MIEF2 are: Both MIEFs share 45% amino acid identity in human cells and are highly conserved in vertebrates. They are anchored in the mitochondrial outer membrane (MOM), associate with Drp1 and recruit Drp1 from the cytosol to mitochondria, resulting in mitochondrial elongation. However, they are dissimilar in certain aspects. For example, their expression levels are different in human tissues and various cell lines, especially during organism development. Although overexpression of either MIEF1 or MIEF2 triggers mitochondrial elongation, MIEF2 overexpression induces a higher extent of elongated mitochondrial clustering and is reverted to a lower extent by hFis1 and Mff than MIEF1. Furthermore, MIEF1 and MIEF2 proteins form distinct types of oligomers in cells and contain different oligomerization domains. All of these data imply that the mitochondrial elongation factors MIEF1 and MIEF2 partly differ in their regulation of mitochondrial dynamics.

In study II, we evaluated how Mff and MIEF1/2 coordinately work together in regulating Drp1-driven mitochondrial fission. Firstly, loss of MIEFs significantly impairs the association between Mff and Drp1, as well as the Drp1 recruitment by Mff to the mitochondrial surface, whereas knockdown of Mff does not affect the functions of MIEFs as mitochondrial Drp1 receptors. Secondly, MIEFs can bind to both Drp1 and Mff independently and serve as adaptors linking Drp1 and Mff together in a Drp1-MIEF-Mff trimeric complex, which facilitates the direct association between Drp1 and Mff. Thus, we find that MIEFs can promote the interaction of Drp1 with Mff. Furthermore, the relative amounts of MIEFs and Mff in cells govern the balance of mitochondrial dynamics. Enhanced levels of MIEFs decrease the interaction between Drp1 and Mff leading to mitochondrial elongation, while higher levels of Mff versus lower levels of MIEFs result in mitochondrial fragmentation. In sum, MIEFs and Mff work coordinately during Drp1-dependent mitochondrial fission, steering the balance between mitochondrial fission and fusion.

In study III, we addressed the role of Drp1-S637 phosphorylation in Drp1 translocation from the cytosol to mitochondria and in the regulation of mitochondrial fission. Reversible Drp1-S637 phosphorylation has been considered to regulate the Drp1-dependent mitochondrial fission and the recruitment of Drp1 to mitochondria, but the extent of this regulation is not fully understood. We confirm that Drp1 phosphorylation at S637 (Drp1-pS637) exists both in the cytosol and on mitochondria, and can be recruited and accumulated on mitochondria by MIEFs and Mff. Increased Drp1-pS637 does not affect the interaction between Drp1 and Mff whereas depletion of MIEFs decreases the binding of Mff with Drp1. Furthermore, similar to wild-type Drp1, overexpression of either phospho-deficient Drp1S637A or phosphomimic Drp1S637D mutants leads to mitochondrial fission in Drp1 deficient cells. However, Drp1S637D was less efficient than Drp1S637A and wild-type Drp1. Additionally, PKA, a kinase phosphorylating Drp1 at the S637 site, partially resides at the mitochondrial surface and is immunoprecipitated by MIEFs or Mff. However, PKA silencing does not abolish the Drp1-Mff or Drp1-MIEFs association. In brief, Drp1-S637 phosphorylation plays a finetuning but not a dominant role in governing Drp1 subcellular distribution and Drp1-mediated mitochondrial fission, whereas Drp1 receptors MIEFs and Mff coordinately regulate the process of Drp1 recruitment and mitochondrial fission.

In study IV, given a minor role of hFis1 in Drp1-dependent mitochondrial fission in human cells, we investigated the underlying molecular mechanism of hFis1-induced mitochondrial fragmentation and the roles of hFis1 in mitochondrial dynamics. Firstly, we observed that hFis1-induced mitochondrial fragmentation occurred both in presence and absence of Drp1 and Dyn2, indicating mitochondrial fragmentation promoted by hFis1 is independent of the Drp1/Dyn2-medicated mitochondrial fission process. Furthermore, immunoprecipitation revealed that hFis1 binds to the pro-fusion proteins Mfn1, Mfn2 and OPA1 at endogenous levels and inhibits the GTPase activities of these proteins specifically, suggesting that the function of hFis1 is probably to block the fusion machinery and thereby shifting the balance to mitochondrial fission. Consistent with these results, destruction of all the three pro-fusion proteins in Drp1 KO cells phenocopied the hFis1-induced mitochondrial fragmentation phenotype, and the actin cytoskeleton was partially involved in this process. In conclusion, we reveal a novel molecular mechanism of hFis1 in mitochondrial dynamics.

Collectively, this thesis develops novel insights into the molecular mechanisms of human mitochondrial dynamics, and provides more detailed knowledge for the studies of mitochondria-related diseases.