Miro (Mitochondrial Rho)
The Ras superfamily family of small GTPases consists of GTP binding-dependent molecular switches with diverse cellular functions. Two isoforms of an “atypical” Rho GTPases, named mitochondrial Rho (Miro), are the first identified mitochondrial-associated Ras superfamily members (Fransson et al. 2003). Evolutionary conserved Miro GTPase has been identified in humans (Fransson et al. 2003), yeast (Frederick et al. 2004), Drosophila (Guo et al. 2005), as well as plant (Yamaoka and Leaver 2008).
Functional studies of Miro in various organisms have suggested critical roles of Miro in regulating mitochondrial morphology (Fransson et al. 2003), inheritance (Frederick et al. 2004), and mitochondrial calcium homeostasis (Lee et al. 2016). It is also a key regulator of cytoskeleton-mediated, long-range mitochondrial transport in neuronal axon through as part of a multi-protein complex (Glater et al. 2006).
As a key regulator of mitochondrial transport, dynamics, and calcium homeostasis, Miro would be expected to be involved in neurological diseases, where defects in these mitochondrial processes have been implicated. Studies have so far implicated Miro in the pathogenesis of Alzheimer’s disease (Iijima-Ando et al. 2012) and Parkinson’s disease (Liu et al. 2012; Wang et al. 2011).
Structural Overview of Miro
Two genes encoding Miro GTPase exist in human: Miro1 and Miro2 (also known as RhoT1 and RhoT2). Human Miro1 and Miro2 both consist of 618 amino acid residues and share 60% sequence identity. Miro protein contains two GTPase domains separated by a linker region containing a pair of helix-loop-helix calcium-binding domains called EF-hands. Miro was originally identified as an atypical Rho GTPase based on sequence similarity of the N-terminal GTPase domain, but lacks the conserved G12 and Q61 residues, indicating an altered GTP hydrolysis activity (Fransson et al. 2003). In addition, the two GTPase domains of Miro lack the Rho-specific insert region (a unique feature of the Rho superfamily), the conserved Switch 2 (G-3 motif), and the CAAX box for lipid modification (Fransson et al. 2003; Frederick et al. 2004). A recent study of Miro in Drosophila showed that its C-terminal GTPase domain is most structurally similar to Rheb, a Ras subfamily member (Klosowiak et al. 2013).
A significant difference between the Miro proteins and the other Rho GTPase family is the absence of the CAAX-motif, which is typically used for membrane targeting of Rho GTPases (Fransson et al. 2003). Instead of a CAAX-domain, the Miro proteins harbor a C-terminal transmembrane domain that tail anchors them to the outer mitochondrial membrane, exposing their other domains to the cytosol (Fransson et al. 2003; Frederick et al. 2004).
Biological Functions of Miro: Mitochondrial Dynamics
Proteins of the Miro family act as potent regulators of mitochondrial dynamics in several ways. Yeast homologue of Miro, Gem1p, is essential for the maintenance of tubular mitochondrial morphology and contributes to mitochondrial inheritance, a process depending on actin cables. Yeast lacking Gem1p showed altered mitochondrial distribution described as collapsed, globular, or grape-like (Frederick et al. 2004). Miro was shown not to be required for the canonical mitochondrial fission and fusion events, although it influences mitochondrial morphology and inheritance in yeast (Frederick et al. 2004).
Drosophila homozygous for dMiro mutations die during larval and early pupal stages due to various developmental defects. Loss of dMiro causes a derangement of the axonal mitochondrial transport, resulting in impaired larval locomotion and disrupted subcellular distribution of mitochondria in neurons and muscles. In fly larvae lacking dMiro, mitochondria are restricted to the cell body and are depleted from axons and presynaptic terminals (Guo et al. 2005). Recent studies in larval axons based on overexpression or loss of function of dMiro suggests that dMiro is required for both anterograde and retrograde mitochondrial trafficking by regulating kinesin and dynein motor protein directed movements along the microtubule system (Russo et al. 2009; Wang et al. 2011). dMiro shows binding to an adaptor protein called Milton, and together they form a Miro/Milton/KHC (kinesin heavy chain) transport machinery to direct mitochondrial trafficking (Russo et al. 2009).
Overexpression of two mammalian Miro isoforms, Miro-1 and Miro-2, caused mitochondrial aggregation and increased apoptotic index (Fransson et al. 2003). A Recent study showed that mammalian Milton homologs, TRAK1 and TRAK2, are required for axonal and dendritic mitochondrial motility. These coiled coil domain-containing proteins have been reported to interact with kinesin and dynein family members (Brickley and Stephenson 2011).
In Arabidopsis, three genes encoding putative Miro-related GTPases have been described. The EMB2473/MIRO1 mutant exhibits abnormally enlarged or tube-like mitochondrial morphology, resulting in the disruption of continuous streaming of mitochondria in the growing pollen tube. Furthermore, it was shown that MIRO1 plays a decisive role in the process of embryogenesis, whereas mutations in the MIRO2 gene do not have any obvious influence on plant development (Yamaoka and Leaver 2008).
Biological Functions of Miro: Mitochondrial Calcium Homeostasis
Mitochondria are known to take up calcium from the cytosol, on one hand, to maintain proper levels of mitochondrial calcium for the metabolic functions, as many metabolic enzymes in the mitochondrial matrix require calcium for optimal activity; on the other hand, calcium uptake by mitochondria helps to maintain cellular calcium homeostasis and guard against the toxicity of high cytosolic calcium (Lee and Lu 2014). Mitochondria can associate directly with endoplasmic reticulum (ER) membranes via the mitochondria-ER contact sites (Rowland and Voeltz 2012). A physical link, in the form of the ER-mitochondria encounter structure (ERMES) tethering complex, is known for its role in phospholipid exchange between the two organelles. In addition, calcium channels on the ER and mitochondrial membrane constitute a path for mitochondrial calcium uptake from ER (Rizzuto et al. 2012).
Recent studies in yeast provided an intriguing subcellular localization of yeast Gem1p to the ERMES (Kornmann et al. 2011). Studies in Drosophila and mammalian cells both revealed that Miro overexpression leads to increased calcium uptake by mitochondria upon ER store depletion (Lee et al. 2016). Also, Polo-like kinase 1 (PLK1)-mediated phosphorylation of Miro contributes to the stabilization of the ERMCS, enhancement of ER-mitochondrial Ca2+ transfer, and detrimental consequence of mitochondrial calcium overload and loss of neural stem cells in the developing Drosophila nervous system (Lee et al. 2016).
Molecular Interactions Suggesting Miro’s Potential Role in the Pathogenesis of Brain Disorders
Miro performs its biological roles in mitochondrial dynamics, transport, and calcium homeostasis through interacting with a number of cellular proteins. Miro interacts with the kinesin adaptor Milton (TRAK1 and TRAK2), as well as the conventional kinesin-1 system, to perform its mitochondrial transport function (Glater et al. 2006). The Miro/TRAK complex also associates with a myriad of other factors, such as the mitochondrial fusion factors mitofusins 1 and 2, the PTEN-induced putative kinase 1 (PINK1), the neuron-enriched member of the Armcx gene family, Armcx3, and disrupted in schizophrenia 1 (DISC1) (Lee and Lu 2014). A recent report also showed that MIRO interacts with and recruits centromere protein F (Cenp-F) to the mitochondria to facilitate mitochondrial transport to the periphery of daughter cells after mitosis (Kanfer et al. 2015). Miro is also known to be a substrate of the E3 ubiquitin ligase Parkin (Liu et al. 2012; Wang et al. 2011). Mutations in PINK1 and Parkin have been linked to familial Parkinson’s disease (Kitada et al. 1998; Valente et al. 2004). Genetic studies in Drosophila PINK1 model support the functional relevance of the interaction between Miro and PINK1-Parkin in disease pathogenesis. The localization of Miro to mitochondria-ER contact sites and its interaction to calcium transporters at those sites, which have been shown to interact with proteins implicated in Alzheimer’s disease (Schon and Area-Gomez 2013), indicated that Miro is possibly involved in the pathogenesis of a broad spectrum of neurological diseases and represents a potential therapeutic target.
Miro family proteins are atypical Rho GTPases associated with mitochondria. Compared to the typical Rho GTPases, Miro proteins are understudied. However, the high evolutionary conservation of these proteins from yeast to humans, their critical roles in regulating fundamental mitochondrial functions, and their association with human diseases suggest that we are so far only seeing the tip of the iceberg in terms of Miro function and regulation in health and disease. Looking ahead, we expect to see exciting new developments in studies of Miro’s physiological function in different tissues and cell types and the contribution of Miro dysregulation to various human diseases, from cancer and metabolic diseases to neurodegenerative diseases.