Mitochondrial Fission

As with mitochondrial fusion, model organisms have provided us with vital information in the machinery provided for mitochondrial fission through the identification of the gene Dnm1p in yeast which was followed by the mammalian orthologue dynamin related protein 1 (Drp1). Drp1 is a cytosolic, microtubule associated protein containing a GTPase domain, a putative pleckstrin homology (PH)-like domain and a GTPase effector domain (Kageyama et al., 2011). Loss of Drp1 function results in long, interconnected mitochondrial networks reflective of an impairment in mitochondrial fission (Roy et al., 2015). As it is a cytosolic protein, recruitment of Drp1 to the MOM requires a number of receptors. The first of these, Fis1, was also discovered in yeast and was shown to be required for the proper assembly and distribution of Dnm1p containing fission complexes on mitochondrial tubules (Mozdy et al., 2000). Despite the finding of a mammalian homolog, further research in mammalian cells suggested that the interaction between Fis1 and Drp1 has a minor role in regulating mitochondrial fission as mitochondrial recruitment of Drp1 is not affected by Fis1 knockdown (Lee et al., 2004). Instead, it is likely that the recruitment of Drp1 to the MOM requires other receptors such a mitochondrial fission factor (Mff) and Mid49/51. Mff is tail anchored in the MOM, existing in a 200kDa complex and knockdown has been shown to inhibit fission induced by loss of mitochondrial membrane potential (Gandre-Babbe and van der Bliek, 2008) and release the Drp1 foci from the MOM while overexpression results in the mitochondrial recruitment of Drp1 and increased fission (Otera et al., 2010). Mid49/51 were discovered as a result of a mitochondrial proteomic screen and it is thought that Mid51 stimulates the GTPase activity of Drp1 in the presence of ADP implying it also acts as a metabolic sensor and regulator of fission (Losón et al., 2014).

The precise mechanism of action of Drp1 once it is recruited to the MOM is not yet known but there are a number of post translational modifications that effect its function. Phosphorylation by Cdk1/cyclin B at Ser616 increases GTPase activity but dephosphorylation by phosphatase calcineurin at Ser637 leads to mitochondrial elongation (Ni et al., 2015). Drp1 can also be ubiquinylated by

MARCH5 or sumoylated by SUMO-1 which can either regulate the stability of Drp1 or recruit Drp1 to the actual division site respectively (Karbowski et al., 2007). The fission process occurs by Drp1 assembling into higher order structures that wrap around the mitochondrial tubule, constricting and eventually severing the mitochondrial membrane by a GTP hydrolysis-dependent mechanism (Ingerman et al., 2005). It is not known if the fission of the MIM and MOM are separate events but it is plausible that the constriction of the Drp1 oligomeric ring is sufficient to sever both membranes. At present the only identified MIM protein with a role in fusion is MTP18 which is a transcriptionally regulated target of phosphatidylinositol 3-kinase (IP3 kinase) signaling and regulates mitochondrial fission coupled with the action of Drp1 so it’s precise role in MIM fission is unknown (Tondera et al., 2005). It is postulated that the ER may also be involved in the fission process as ER tubules appear to encircle and constrict mitochondrial tubules prior to the recruitment of Drp1 while Drp1 receptors are also located at the ER-mitochondria contact site (Friedman et al., 2011). Recent research has demonstrated that actin polymerization at the ER-mitochondria contact site (MAM-mitochondrion associated membrane) though the ER-localized formin 2 (INF2) protein and the recruitment of myosin II allow for efficient fission by facilitating Drp1 assembly at this site (Korobova et al., 2014, Korobova et al., 2013).

Drp1 also has multiple ways of sensing its metabolic microenvironment and adjusting mitochondrial function accordingly. The best studied of these is phosphorylation which can occur at a number of sites on the protein including two critical sites serine 616 and serine 637. Protein kinase A (PKA) phosphorylates Drp1 at S637 which inhibits its activity resulting in an elongated mitochondrial phenotype as a result of pharmacological activation, b-adrenergic stimulation (Cribbs and Strack, 2007) or mTOR inhibition which increases cAMP levels and subsequently activates PKA (Gomes et al., 2011b). On the contrary, dephosphorylation at S637 by the Ca2+ dependent phosphatase calcineurin results in a promotion of Drp1 activity and recruitment to the mitochondrial surface (Cereghetti and Stangherlin, 2008). This is relevant to metabolic activity as

dysfunction of the calcium buffering capacity of mitochondria would give rise to an increase in cytosolic Ca2+ and trigger calcineurin dependent Drp1 activation and mitochondrial fission. Another link between bioenergetics and mitochondrial fission is evident when respiration is inhibited and excess ADP binds MiD51 stimulating Drp1 spiral assembly, GTP hydrolysis and thus promoting fission (Pernas and Scorrano, 2016). Additionally AMP kinase activation results in a phosphorylation of Mff and so promotes mitochondrial fission linking energy deficiency to mitochondrial fragmentation (Toyama et al., 2016).

Thus, the dynamic nature of mitochondria, although not governed by any de novo translation, is affected by a series of intracellular sensors which can detect subtle changes in membrane potential, Ca2+ concentration and metabolism in order to alter the morphology and activity of the mitochondria through a series of post- translational modifications. The fusion/fission machinery link the metabolic state of the entire cell to mitochondrial function ensuring optimum activity depending on cell type and substrate availability.