Screening for genetic interactors of myo2(LQ) revealed that mutations blocking mitochondrial fusion are synthetic lethal in a myo2(LQ) background (Figure 26). myo2(LQ) cells with the conditional fzo1-1 allele show an almost complete block of mitochondrial inheritance under restrictive conditions (Figure 28), which explains the growth defect. The growth defect is rescued when mitochondrial fragmentation by mitochondrial division is prevented (Figure 26C), demonstrating that the fusion activity is not essential for myo2(LQ) mutants. Rather, these mutant cells depend on a wild type-like mitochondrial morphology. Blockage of mitochondrial division in myo2(LQ) mutant cells with fusion-competent mitochondria results in enhanced mitochondrial inheritance and augmented growth compared to myo2(LQ) single mutants (Figure 26 and Figure 27). Thus, components of two
72 antagonistic pathways, mitochondrial fusion and division, genetically interact with myo2(LQ) in divergent ways regarding growth and mitochondrial inheritance. Moreover, the temperature-sensitive fzo1-1 allele and deletion of mitochondrial fusion components lead to many buds devoid of mitochondria in a wild type MYO2 background (Figure 28B, C and Figure 29), indicating that fragmentation of mitochondria per se results in compromised inheritance. Notably, this is the first study observing an inheritance defect in yeast fusion mutants, although these have been characterized for more than 15 years (Hermann et al., 1998; Rapaport et al., 1998).
The question remains why mitochondrial morphology is important for inheritance. Possibly, a hyper-connected mitochondrial network alleviates the anterograde movement since much mitochondrial mass can be transported into the bud by pulling on a single mitochondrial tip. This would explain the effects of the DNM1 knock-out. However, deletion of NUM1, which also results in net-like mitochondria (Cerveny et al., 2007), had no effect on mitochondrial inheritance in a myo2(LQ) background (Figure 25A and B), suggesting that the hyper-connection of the network alone is not sufficient to ease the transport. On the other hand, basal fission activity in num1 cells might be strong enough to slightly disconnect the mitochondrial network and thus to obscure the effect.
In the case of fragmented mitochondria many transport events of single mitochondria are required to ensure the inheritance of a large organellar volume. This might already be difficult for cells with a functional transport machinery and be further aggravated by compromising the machinery with the myo2(LQ) allele. However, it is counterintuitive that smaller mitochondria are harder to transport and that no mitochondrion at all is inherited by the bud in the myo2(LQ) fzo1-1 mutant. Smaller mitochondria are expected to be the simpler cargo. Accordingly, secretory vesicles are transported by Myo2 with 3 µm/s (Schott et al., 2002), whereas the bigger mitochondria move with a velocity of clearly below 1 µm/s (Boldogh et al., 2004; Förtsch et al., 2011). One possible explanation for the defect is that the density of mitochondrial Myo2 receptors is too low on fragmented mitochondria in order to pull them into the bud. If this was the case, overexpression of the two proposed Myo2 receptors Mmr1 and Ypt11 might rescue the inheritance defect of fusion mutants and the growth defect of myo2(LQ) fzo1-1 double mutants. Moreover, the growth defect of myo2(LQ) fzo1-1 double mutants may prove valuable as a tool to search for additional mitochondrial Myo2 receptors. A screen comprising all yeast ORFs should be able to uncover genes the overexpression of which rescues the growth defect. Potentially, this screen can enable the identification of proteins that recruit Ypt11, Mmr1 or Myo2 itself to the mitochondrial surface and thereby promote anterograde transport.
Although the effects of mitochondrial dynamics on inheritance have not attracted much attention in yeast, it is known that mitochondrial distribution in neurons depends on fusion and fission (Hollenbeck and Saxton, 2005). Neurons are an ideal model to study mitochondrial transport since mitochondrial biogenesis occurs mainly in the cell body and mitochondria then have to be transported into the tips of dendrites and axons (reviewed in Sheng, 2014). These transport processes cover distances of up to one meter. Interestingly, there are conflicting results on the issue which morphologies are easier to transport. Defects in mitochondrial fusion and division have both
73 been shown to impact on mitochondrial motility and both pathways were demonstrated to be necessary for the transport of mitochondria to their destination (Li et al., 2004; Verstreken et al., 2005). Thus, a balance between fusion and division appears to ensure a mitochondrial morphology suitable for neuronal transport, which is highly important for the functionality of neurons. Miro, the human homolog of Gem1, connects mitochondria with kinesin and microtubules for mitochondrial transport. Remarkably, overexpression of Miro results in interconnection of mitochondria and boosts their transport, whereas compromising the functionality of Miro leads to mitochondrial fragmentation and less transport activity (summarized in Sheng, 2014), resembling the situation in myo2(LQ) cells. There, mitochondrial hyper-connection augments mobility, whilst fragmentation hampers transport. Furthermore, Miro directly interacts with the human homolog of Fzo1, Mfn2, and knock-down of Mfn2 results in less mitochondrial anterograde and retrograde movement (Misko et al., 2010).
Obviously, mitochondrial dynamics is an important factor for the movement of mitochondria. It is yet unclear, which role the dynamics exactly plays. One can speculate that fragmented mitochondria are less likely to be inherited to the daughter because this morphology is a hallmark of mitochondria with reduced functionality and these mitochondria would be detrimental to the daughter’s health.
Mitochondria undergo division and fragment in yeast and mammalian cells when they are damaged and stressed, for instance with H2O2, and this results in heavily reduced mitochondrial motility in yeast (Baker et al., 2014; Zhou et al., 2014). Moreover, yeast cells accumulate fragmented, dysfunctional mitochondria concomitantly with replicative age (Scheckhuber et al., 2007; Hughes and Gottschling, 2012; Wang et al., 2014). Possibly, mitochondria with declined metabolic capacity adopt a shape by fragmentation that is less likely to be transported into the daughter cell, thus providing a potential quality control filter in addition to mitophagy. The biogenesis of Mgm1, the MIM component required for mitochondrial fusion, offers a putative mechanism, since the processing of Mgm1 by the protease Pcp1 depends on ATP (Herlan et al., 2004). Mitochondria with reduced oxidative phosphorylation and ATP thus might lack an Mgm1 isoform which is essential for fusion.
Hence, these mitochondria cannot refuse with the mitochondrial network and are not inherited. In future experiments, this hypothesis can be tested by observation of mitochondrial motility via time-lapse fluorescence microscopy in stressed and aged yeast cells.
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