Ca2+-dependent regulation of mitochondrial dynamics by the Miro-Milton complex
Calcium oscillations control mitochondrial motility along the microtubules and in turn, support on-demand distribution of mitochondria. However, the mechanism mediating the Ca2+ effect remained a mystery. Recently, several papers reported on the Ca2+-dependent regulation of mitochondrial dynamics by a Miro-Milton complex linking mitochondria to kinesin motors. Both mitochondrial motility and fusion-fission dynamics seem to be sensitive to a Ca2+-dependent switch by this complex. Evidence is emerging that calcium signaling through Miro-Milton is central to coordination of the local oxidative metabolism with the energy demands and protection against Ca2+-induced cell injury.
Miro forms a complex with Milton to recruit kinesin to the mitochondria<!>Miro-Milton complex supports mitochondrial motility along microtubules<!>Miro-Milton complex confers Ca2+ sensitivity to mitochondrial motility<!>Miro-dependent changes in mitochondrial morphology<!>Miro-Milton forms a complex with Pink1 to control mitochondrial dynamics through interaction with the fission/fusion machinery<!>Role of Ca2+-dependent regulation of mitochondrial dynamics by the Miro-Milton complex in cell function<!>Conclusions<!>
<p>Miro proteins form a mitochondrial subfamily of the Ras GTPases conserved from yeast to human. Mammals have two Miro family members, Miro1 and Miro2 (Fransson et al., 2003), whereas yeast has only one, Gem1p (Frederick et al., 2004) and the plant Arabidopsis has three, Miro1-3 (Yamaoka and Leaver, 2008). Miro1 and Miro2 consist of 618 amino acid residues with molecular masses of ~70 kDa and were found to be 60% identical to each other. Both Miro1 and Miro2 have 2 GTPase domains in the N and C terminus, and a transmembrane domain at the C terminus, which confers targeting to the outer mitochondrial membrane (OMM) (Fransson et al., 2003; Fransson et al., 2006). Ca2+ sensitivity of the Miros/Gem1p is due to the presence of a pair of Ca2+-binding EF-hand motifs (Fransson et al., 2003; Frederick et al., 2004). Miros in mammalian cells were shown to have roles in mitochondrial trafficking (Fransson et al., 2006), Gem1p defines a novel mitochondrial morphology pathway and is important for mitochondrial inheritance (Frederick et al., 2004; Frederick et al., 2008), and Miro1-3 are required for embryogenesis and influences mitochondrial morphology in pollen (Yamaoka and Leaver, 2008).</p><p>Milton family proteins are cytoplasmic proteins, which are known to bind both Miros and kinesin heavy chain (KHC, KIF5) to support mitochondrial transport (Glater et al., 2006; Rice and Gelfand, 2006; Stowers et al., 2002). Milton is a fly protein that has no identified yeast ortholog. Mammals have two Milton homologs named Grif-1 and OIP106 (Brickley et al., 2005). Milton, human Grif-1 and OIP106 consist of 1116, 914, 953 amino acid residues, respectively (Webber et al., 2008). Milton shares ~44% amino acid homology with Grif-1 and OIP106 (Brickley et al., 2005). All these 3 proteins contain a Huntingtin-associated protein1 (HAP1) N-terminal (HAPN) homologous domain, which encompasses the two coiled-coiled domains. They have no known Ca2+ binding site or transmembrane domain (Webber et al., 2008). The recruitment of KHC to mitochondria is, in part, determined by the N-terminus-splicing variant of Milton (Glater et al., 2006). Milton and Grif-1 are enriched in neuronal tissue. It was speculated that Grif-1 is a neuronal trafficking molecule because of its association with GABAA receptor (Brickley et al., 2005).</p><p>Milton and Miro localize to mitochondria in mammalian neurons and interact with each other in vivo in the brain (MacAskill et al., 2009; Wang and Schwarz, 2009) (Fig. 1.). Also, fly miro mutants phenocopy milt mutants, suggesting these two proteins function together (Guo et al., 2005). The association of Milton with mitochondria by its interaction with Miro supported a) by the ability of a truncated cytosolic form of Miro to act as a dominant negative and displace Milton from mitochondria, and b) by the ability of overexpressed Miro to recruit to mitochondria a truncated Milton (residues 1–750) that could not independently localize there. However, the C-terminal portion of Milton also localizes to mitochondria (Glater et al., 2006). Similar to Milton, the Miro1-binding domain on Grif-1 is in residues 476–700 (Macaskill et al., 2009). The interaction of Miro with OIP106 has also been confirmed (Fransson et al., 2006). Though HAP-1 shares sequence similarity with Milton, Grif-1, and OIP106, it does not localize to mitochondria or colocalize with Miro (Fransson et al., 2006).</p><p>Miro has also been shown to bind directly to KHC in a Ca2+ sensitive manner (Macaskill et al., 2009; Wang and Schwarz, 2009). Interestingly, Macaskill et al. proposed that direct association between Miro and KHC occurs at resting cytoplasmic [Ca2+] ([Ca2+]c) (<100nM) and involves the tail domain of KHC (MacAskill et al., 2009), whereas Wang and Schwarz claimed that the binding occurs when [Ca2+]c is elevated and involves the motor domain of KHC (Wang and Schwarz, 2009). These discrepant findings offer two distinct mechanisms for the molecular mechanism of the Ca2+-dependent inhibition of the mitochondria-associated KHC motors (Cai and Sheng, 2009) (Table 1.).</p><p>The Miro-Milton complex is likely to interact with several proteins (Fig. 1.). For example, Grif-1 and OIP106 interact with O-GlcNAc transferase (OGT). OIP106 can target OGT to transcriptional complexes for glycosylation of transcriptional proteins, such as RNA polymerase II, and transcription factors. Similarly, Grif-1 may serve to target OGT to GABAA receptor complexes for mediating GABA signaling cascades (Iyer et al., 2003). Also, Pink1, a mitochondrially localized serine/threonine kinase that has been implicated in Parkinson disease, can form a multi-protein complex with Miro-Milton (Weihofen et al., 2009). The binding of KHC, OGT and Pink1 does not seem to be mutually exclusive. Therefore a complex containing Miro, Milton, KHC, Pink1 and OGT might link mitochondria to microtubules. So far, the following interactions are clear: Milton connects KHC to Miro on mitochondria (Glater et al., 2006; Rice and Gelfand, 2006); Pink1 forms complex with Miro and Milton (Weihofen et al., 2009); OGT interacts with Grif-1 and OIP106 (Iyer et al., 2003) (Fig.1.). However, several points require further studies, including the mechanism of the effect of Ca2+ on the interactions among Miro, Milton and KHC, the mechanism of the binding of the Milton C-terminus to the mitochondria and the species and tissue specificity of the structure of the complexes coupling mitochondria to the kinesin motors. Furthermore, a major question remains the possible anchorage of the dynein motors to the mitochondria by the Miro-Milton complex.</p><!><p>In H9c2 cardiac cells and primary cortical neurons, silencing of Miros causes mitochondrial motility suppression, whereas overexpression of either Miros or their EF-hand mutants promotes mitochondrial movements when [Ca2+]c is maintained at the resting level (<100nM) (Saotome et al., 2008) (Table 1.). Consistently with these results, in hippocampal neurons, Miro1 overexpression increases mitochondrial motility, whereas Miro silencing or expression of a Miro-Grif-1 uncoupler exerts the opposite effect (MacAskill et al., 2009; Macaskill et al., 2009). Thus, Miros facilitate mitochondrial motility along microtubules in low [Ca]c environment irrespective of the presence of their EF-hands. Notably, altering the Miro1 GTPase activity affects mitochondrial motility by regulating its ability to recruit Grif-1 (MacAskill et al., 2009). Therefore Miro GTPase domain is involved in regulating mitochondrial movement (Table 1.).</p><p>In hippocampal neurons, overexpressed Miro shows interaction with KHC (Macaskill et al., 2009) and in Drosphila, dMiro is required for controlling anterograde transport of mitochondria to synapses (Guo et al., 2005). These data indicate that the effect of Miro on mitochondrial motility is mediated at least in part, through KHC that transports mitochondria to the anterograde direction. However, Miro is also needed for retrograde mitochondrial transport in the same model, indicating that Miro also interacts with dynein motors (Russo et al., 2009).</p><!><p>Physiological elevations of [Ca2+]c are well known to control mitochondrial motility in a variety of mammalian cells (for a review see (Graier et al., 2007; Hajnoczky et al., 2007)). Both Ca2+ entry (Quintana et al., 2006; Rintoul et al., 2003) and mobilization of ER/SR Ca2+ stores (Brough et al., 2005; Yi et al., 2004) exert inhibition on mitochondrial motility to retain mitochondria at the sites of [Ca2+]c elevations. Since KHC does not have any Ca2+ or calmodulin binding site and inhibitors of neither Ca2+/calmodulin-dependent kinases nor Ca2+-dependent protein phosphatases prevented the Ca2+-induced inhibition of mitochondrial motility, it appeared that a distinct Ca2+ sensor molecule is required to translate the Ca2+ signal for the microtubular motor proteins (Yi et al., 2004).</p><p>Now, three studies performed in different cellular models, H9c2 cells and cortical neurons (Saotome et al., 2008) and hippocampal neurons (Macaskill et al., 2009; Wang and Schwarz, 2009) provide evidence that Miro serves as a Ca2+ sensor that controls mitochondrial motility. In H9c2 cells, the [Ca2+]c-induced mitochondrial motility inhibition is Miro-dependent and requires both intact EF-hand and GTPase domains (Saotome et al., 2008). In hippocampal neurons, the Ca2+-induced motility inhibition is also dependent on the Miro's EF-hand but two different mechanisms are proposed for the molecular mechanism downstream to Ca2+ binding: a) Ca2+-binding permits Miro to interact directly with the motor domain of KHC, which prevents interactions between the motor and the microtubular track. Thus, KHC would switch from an active state in which it is bound to Miro only via Milton, to an inactive state in which direct binding to Miro prevents its interaction with the microtubules (Wang and Schwarz, 2009); b) Ca2+ binding to Miro1 would inhibit the coupling of Miro1 to KHC tail, which interaction allowed the movement of mitochondria along microtubules (Macaskill et al., 2009) (Table 1.).</p><p>In summary, the three studies reveal Ca2+-dependent mitochondrial motility regulation by the Miro/Milton complex and show the dependence on the Miro EF-hands. However, there are some differences among them, which require further studies: a) Ca2+ allows Miro to bind KHC preventing KHC from interacting with microtubules (Wang and Schwarz, 2009) or high Ca2+ concentration causes Miro to separate from the KHC motor (Table 1.) b) the [Ca2+]c that causes half-maximal effect on binding or on motility is 0.4 µM (Saotome et al., 2008; Yi et al., 2004), 1 µM (Macaskill et al., 2009), and 50µM (Wang and Schwarz, 2009),. In addition, the results also stimulate a series of related questions: How do the Miro GTPase domains that are also required for the motility inhibition, interact with the Ca2+-binding EF-hands to mediate the Ca2+ effect? Can binding of OGT, PINK1 or other factors to OIP106 and Grif-1 affect the regulation of mitochondria motility?; Are there other Ca2+ sensors involved in the Ca2+-dependent motility inhibition? Miro knockdown caused only a shift in the Ca2+ dose-response with only a small suppression of the maximal motility in H9c2 cells (Saotome et al., 2008).; What is the relationship between Miro-Milton complex and dynein? Both directions of mitochondrial movement instead of only anterograde movement were increased by Miro overexpression and were arrested at high Ca2+ concentration (Saotome et al., 2008; Wang and Schwarz, 2009).; How do direct mitochondria-microtubule tethers like syntaphilin (Kang et al., 2008) or the mitochondria-KHC linker syntabulin (Cai et al., 2007) affect the Ca2+-dependent mitochondrial motility inhibition? Neurons harboring a mutant syntaphilin exhibit enhanced short-term facilitation during prolonged stimulation, probably by affecting calcium signaling at presynaptic boutons (Kang et al., 2008).</p><!><p>Movements control mitochondrial distribution and position mitochondria to allow fusion and perhaps, participate in fission, which effects are relevant for mitochondrial morphology. As Miro controls mitochondrial motility, the question comes up if Miro can regulate mitochondrial morphology in motility-dependent or independent way. Miro1 overexpression causes accumulation of mitochondria at the microtubule-organizing center, and a Miro1 GTPase domain mutation (Miro-1/Val-13) induces mitochondrial aggregation (Fransson et al., 2003). This response to the Miro1 mutation can be separated to two distinct components that are often visible in the same cell: the appearance of thread-like mitochondria and the formation of mitochondrial aggregates. Clustering of mitochondria requires the first GTPase domain of Miro1, but is not dependent on the EF-hand (Fransson et al., 2006). Miro2 overexpression only induced aggregation in Cos-7 cells (Fransson et al., 2006). In H9c2 cells, overexpression of both Miro1 and Miro2 induce mitochondrial thread formation and condensation, and dominant-negative Miro constructs and Miro knock-down cause mitochondrial fragmentation and condensation. Thus, the length of the mitochondria seems to be directly proportional to the availability of Miro. The fragmentation seems to involve Drp1-mediated mitochondrial fission. In non-stimulated cortical neurons, overexpression of Miro or Miro EF-hand mutant also induces mitochondria elongation (Saotome et al., 2008). Collectively, these results indicate that the Miro proteins promote the formation of elongated mitochondria and in this process, utilizes their GTPase domain but their EF-hand is not required. A target of this Miro effect seems to be the suppression of Drp1-mediated mitochondrial fission, though recruitment of fusion promoting cytoplasmic proteins may also be affected. Notably, long mitochondria show less movement activity than the short ones in H9c2 cells (Liu and Hajnóczky, unpublished), indicating that the Miro effect on mitochondrial fusion-fission can not account for the Miro effect on motility. Restricted transport of the elongated mitochondria promotes mitochondrial aggregation (S. Das and G. Hajnóczky, unpublished) but this has to be complemented with some other aggregation mechanisms (e.g. in Miro2 overexpressing Cos-7 cells).</p><p>Ca2+ was suggested to control Drp1-dependent mitochondria fission through calcineurin and CaM kinase.(Cereghetti et al., 2008; Cribbs and Strack, 2007; Han et al., 2008). The Miro-dependent Ca2+-induced mitochondrial fission also seems to involve Drp1 (Saotome et al., 2008). Thus, it seems possible that the effect of two discrete Ca2+ sensors (calcineurin and Miro) mediate activation of Drp1.</p><p>Yeast cells lacking Gem1p contain collapsed, globular, or grasp-like mitochondria. Both GTPase domains and EF hands are required for Gemp1 function. However, Gem1p is not an essential component of the characterized pathways that regulate mitochondrial dynamics (Frederick et al., 2004).</p><p>Milton's effect on mitochondrial distribution is well documented but only scarce information is available on the Milton-dependence of the morphology of individual mitochondria. In milton flies axonal and synaptic mitochondria are absent but mitochondria normally distributed in the cell bodies (Glater et al., 2006; Gorska-Andrzejak et al., 2003). Milton overexpression causes mitochondrial redistribution or aggregation (Stowers et al., 2002; Weihofen et al., 2009). In milton spermatocytes, mitochondria are distributed normally; however, after meiosis, the mitochondrial derivatives do not elongate properly (Aldridge et al., 2007). Along this line, overexpression of Grif-1 or OIP106 in H9c2 cells seems to support mitochondrial elongation and aggregation (S. Das and G. Hajnóczky, unpublished). The similarity between the effects of Miro and Milton on mitochondrial elongation and aggregation indicates that a common mechanism might mediate some effects of these factors on mitochondrial morphology. The effects of Miro-Milton-Pink1 on mitochondrial morphology and dynamics are summarized in Table 2.</p><!><p>Loss-of-function mutations in the Pink1 or Parkin, which encode a mitochondrially localized serine/threonine kinase and a ubiquitin-protein ligase, result in recessive familial forms of Parkinson disease. The Pink1/Parkin pathway was shown to promote mitochondrial fission and/or to inhibit fusion by negatively regulating Mfn and Opa1 function, and/or positively regulating Drp1 (Deng et al., 2008) (Fig. 1.). The loss of mitochondrial and tissue integrity in Pink1/parkin mutants seems to derive from reduced mitochondrial fission (Poole et al., 2008). In fly and mammalian cells, Pink1 overexpression promotes mitochondrial fission, whereas inhibition of Pink1 leads to excessive fusion (Table 2.). Genetic interaction results suggest that Fis may act in-between Pink1 and Drp1 controlling mitochondrial fission (Yang et al., 2008).</p><p>Based on a recent report, Pink1 forms a complex with Miro-Milton. Miro and Milton overexpression increases the mitochondrial Pink1 pool. Pink1 without a mitochondrial targeting sequence can still be targeted to the OMM (Fig. 1.). Miro and Milton overexpression rescues the altered mitochondrial morphology induced by loss of Pink1 function. However, differently from previous studies, Pink1 silencing induced mitochondria fragmentation instead of elongation in this study (Weihofen et al., 2009). Pink1 was also shown to promote mitochondrial fission and/or to inhibit fusion. Miro1 has also been involved in Drp1-mediated mitochondrial fission. It is interesting that Miro2 and Milton-1 expression could rescue the altered mitochondrial morphology induced by Pink1 loss of function. This suggests that Miro/Milton/Pink1 would function together to regulate the fusion-fission dynamics (Weihofen et al., 2009).</p><p>Pink1 also interacts with Omi in the mitochondria and both proteins are components of the same stress-sensing pathway. Pink1-dependent phosphorylation of Omi might modulate its proteolytic activity, thereby contributing to an increased resistance of cells to mitochondrial stress (Plun-Favreau et al., 2007).</p><!><p>In neurons, Ca2+ influx occurs at presynaptic terminals and postsynaptic dendrictic spines, where mitochondria are commonly retained to maintain the Ca2+ homeostasis. In flies, Milton is required for synaptic accumulation of mitochondria (Stowers et al., 2002). In hippocampal neurons, the Miro-and Ca2+-dependent mitochondrial dynamics regulation allows activation of NMDA receptors with exogenous or synaptically released glutamate to regulate mitochondrial movement and thus to recruit mitochondria to activated synapses. Thus, Miro is a key determinant of how energy supply is matched to energy usage in neurons (Macaskill et al., 2009). Regulation by Miro-Milton can enable neurons to efficiently retain mitochondria at the sites with high Ca2+, providing a neuronal protection mechanism. The EF-hands of Miro mediate glutamatergic regulation of mitochondrial motility and are protective during excitotoxic stresses (Wang and Schwarz, 2009).</p><p>In primary cortical neurons, Miro also caused an increase in dendritic mitochondrial mass and enhanced mitochondrial calcium signaling. Miro mediated redistribution and fusion of mitochondria increased their ability to accumulate Ca2+ independently of the Ca2+ binding activity of the protein (Saotome et al., 2008). Increased Ca2+ transfer by the mitochondria in cortical neurons provides a means to enhance the mitochondrial Ca2+ buffering and to stimulate the Ca2+-dependent reactions in mitochondrial energy production. Thus, Miro proteins both mediate the effects and regulate calcium signaling to coordinate the mitochondrial dynamics and contribution to cell function.</p><p>Increased Ca2+-dependent cell death might accompany the upregulation of Miro due to higher sensitivity of Ca2+-mediated mitochondrial fragmentation. Overexpression of a Miro mutant cased increased apoptotic activity in cultured cells (Fransson et al., 2003). However, Gem1p is not required for pheromone-induced yeast cell death (Frederick et al., 2004). Thus, further experimental work is necessary to determine the role of Miro proteins under pathological conditions.</p><!><p>Miro forms a complex with Milton, KHC, Pink1 and OGT, which couples mitochondria to the microtubules and allows Ca2+-regulated transport of mitochondria along the microtubules. In the complex, Miro recruits KHC to mitochondria via Milton or directly. The Miro-Milton complex supports mitochondrial motility irrespective of Miro EF-hands at resting [Ca2+]c, and controls the [Ca2+]c elevation-induced mitochondrial motility inhibition by a both Miro EF-hand-and GTPase domain-dependent mechanism. Furthermore, the Miro-Milton complex affects the mitochondrial morphology both at low and high [Ca2+]c, which seems to occur through a Drp1-mediated fission mechanism, while Pink1 also promotes mitochondrial fission and/or inhibits fusion. The regulation of the mitochondrial motility and fusion-fission dynamics by the Miro-Milton complex would provide a mechanism that helps to match the energy production with the demand throughout the cells, including the dendrites and axons and protects neurons during excitotoxic challenges.</p><!><p>This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. 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