Dual role of Miro protein clusters in mitochondrial cristae organisation and ER-Mitochondria Contact Sites

Background

Mitochondrial Rho GTPase (Miro) proteins link mitochondria to kinesin and dynein motors, enabling the transport of mitochondria along microtubules. Miro proteins are located on the outer mitochondrial membrane (OMM) and recruit trafficking kinesin protein (TRAK) adaptors, shown to bind to both kinesin-1 and dynein/dynactin (Birsa et al., 2013; van Spronsen et al., 2013). Interestingly, the yeast and the Drosophila homologues of Miro proteins are thought to be associated with the mitochondrial contact site and cristae organizing system (MICOS). The MICOS complex is involved in the maintenance of mitochondrial cristae and inner membrane (IMM) architecture. Moreover, MICOS complex interacts with Sam50 protein on the OMM, building a bridge between the OMM and the IMM. This structure is called mitochondrial intermembrane space bridging complex (MIB).   

Representation of the MICOS and the MIB complex. The MICOS complex in humans is formed by Mic60/mitofilin, Mic10, Mic19/CHCHD3, Mic25/CHCHD6, Mic23, Mic27, Mic13, Mic14/CHCHD10 and DnaJC11 (Kozjak-Pavlovic, 2017).

Some recent findings have challenged this classical role of Miro proteins, since in their complete absence kinesin and dynein motors and the TRAK adaptors can still localise to the mitochondrial membrane (Lopez-Domenech et al., 2018). In this preprint, the authors continue to investigate alternative roles of Miro proteins in mitochondrial biology combining biochemical, super-resolution and electron microscopy techniques.

Key findings

The authors had already observed that in Miro double-knockout (DKO) MEFs mitochondria were preferentially accumulated perinuclearly, suggesting a defect in mitochondrial transport. When looking more closely at mitochondrial structure using electron microscopy in DKO MEF cells, the authors observed alterations in IMM structure and cristae architecture. However, no significant changes in MICOS components, Sam50 protein, motor proteins or other mitochondrial proteins were observed. This suggests that Miro proteins may play a role in IMM organization. Indeed, Miro1 and Miro2 co-immunoprecipitate with core components of the MICOS complex, responsible for mitochondrial cristae organization, and with Sam50, an outer mitochondrial membrane that interacts with MICOS complex to bridge both membranes. Using super resolution microscopy techniques, Miro proteins were shown to localize to discrete domains along the mitochondrial network forming nanoclusters. Miro1 and Miro2 nanoclusters were found to have a similar average diameter of 100 nm, which was conserved across different cell types. Using dual color dSTORM imaging, authors found that Mic60/Mitofilin nanoscale pattern was partially overlapping with the pattern found for Miro proteins. 

Therefore, the authors were able to show that Miro proteins a) form clusters that associate with MICOS clusters, and b) interact with MICOS complex and Sam50. But still some questions remained, such as the role of Miro1 and Miro2 in MICOS complex stabilization/association and their interaction with Sam50. Interestingly, Miro proteins were not essential to maintain the core of the MICOS complex, but their loss could destabilize certain interactions between components of the MICOS complex. So what happens with MICOS complex distribution upon Miro proteins loss? The authors found that Mic19/CHCHD3 could still form clusters, but those were severely affected, with some mitochondrial areas devoid of them, indicating that Miro proteins are important for the distribution of MICOS clusters.

It was previously established that Miro1 and Miro2 regulate mitochondrial transport along microtubules by interacting with TRAK adaptors. Interestingly, Mic19/CHCHD3 and TRAK1/2 co-immunoprecipitate in the presence of Miro proteins. However, loss of Miro proteins hampers the interaction between MICOS complex and TRAK proteins. This unveils a new function of Miro proteins linking motor machineries with the inner membrane structure of mitochondria. Interestingly, loss of Miro proteins resulted in distally-transported mitochondria almost devoid of Mic19/CHCHD3 clusters. To further support the hypothesis that Miro proteins are able to ensure the concerted transport of OMM and IMM components by interacting with the transport machinery and the MICOS complex, authors analysed the distribution of an IMM component of the OXPHOS system, ATP5α versus an OMM, Tom40. By forcing the redistribution of mitochondria to the periphery, authors observed that in cells lacking Miro proteins, the distribution pattern of Tom40 (OMM marker) and ATP5α (IMM marker) was shifted. Tom40 was preferentially distributed towards the periphery of the cell while ATP5α signal was accumulated in more proximal structures. 

Why I like this preprint

One thing that is particularly novel in this preprint is the use of super resolution imaging techniques to visualize submitochondrial compartments. This is especially important for mitochondria since their diameter is generally close to the resolution limit of conventional light microscopy, making it necessary to use super resolution techniques to image submitochondrial protein distributions. Using those techniques, this preprint advances our knowledge in the submitochondrial organization of different complexes. Moreover, the authors continue to challenge the accepted model of mitochondrial trafficking mediated by Miro proteins and evidence for an alternative function of Miro proteins in regulating not only what happens “on the outside” of mitochondria but also “on the inside”. Therefore, Miro proteins are emerging as major regulators in the concerted transport of both outer and inner mitochondrial membranes. 

Questions for the authors

• Do you think that loss of Miro proteins destabilizes MICOS complex, altering the mitochondrial inner membrane organization and cristae structure? Is it possible that losing the tether between the mitochondrial transport machinery and MICOS complex affects the transport rate between inner and outer mitochondrial membranes resulting in cristae disassembling?
• Does the loss of inner membrane organization and cristae structure impact OXPHOS function in DKO cells? Is there any defect in mitochondrial respiration, increased ROS production or loss in membrane potential?
• Do you think that this bridge between motor proteins and inner mitochondrial membrane complexes is particularly important in those cells where mitochondria have to travel long distances, such as neurons?

References

• Birsa N. et al. Mitochondrial trafficking in neurons and the role of the Miro family of GTPase proteins. Biochem Soc Trans. 41(6):1525-31 (2013).
• Kozjak-Pavlovic V. The MICOS complex of human mitochondria. Cell Tissue Res. 367(1):83-93 (2017).
• López-Doménech G. et al. Miro proteins coordinate microtubule- and actin-dependent mitochondrial transport and distribution. EMBO J. 37(3):321-336 (2018).
• van Spronsen M. et al. TRAK/Milton motor-adaptor proteins steer mitochondrial trafficking to axons and dendrites. Neuron. 77(3):485-502 (2013).

 

Tags: super resolution techniques, transport

doi: https://doi.org/10.1242/prelights.11613

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