bioRxiv preprint doi: https://doi.org/10.1101/178913; this version posted January 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Microtubule dynamics regulates mitochondrial fission 2 Kritika Mehta1, Manjyot Kaur Chug1, Siddharth Jhunjhunwala1 and Vaishnavi 3 Ananthanarayanan1,* 4 1 Centre for BioSystems Science and Engineering, Indian Institute of Science, Bangalore 5 560012 6 * To whom correspondence may be addressed. Email: [email protected] 7 8 Abstract 9 Mitochondria are organised as tubular networks in the cell and undergo fission and fusion. 10 Equilibrium between fission and fusion events is necessary to maintain normal 11 mitochondrial function. While several of the molecular players involved in mediating the 12 dynamics of mitochondria have been identified, the precise cellular cues that initiate fission 13 or fusion remain largely unknown. In fission yeast, as in mammalian cells, mitochondrial 14 positioning and partitioning are microtubule-mediated. In interphase, fission yeast 15 mitochondria associate with microtubule bundles that are aligned along the long axis of the 16 cell. Here, we perturbed microtubule dynamics via kinesin-like proteins to produce cells with 17 long and short microtubules and observed that cells with long microtubules exhibited long, 18 but fewer mitochondria, whereas cells with short microtubules exhibited short, but several 19 mitochondria. We quantified that mitochondrial fission frequency correlated inversely with 20 microtubule length. In agreement with these results, upon onset of mitosis wherein 21 interphase microtubules assemble to form the spindle within the nucleus, we measured 22 increased fission of mitochondria in the absence of interphase microtubules. We confirmed 23 that partitioning of mitochondria at mitosis follows a stochastic, independent segregation 24 mechanism and that the increase in mitochondrial fission prior to cell division therefore 25 likely serves to increase the likelihood of equal partitioning of mitochondria between the two 26 future daughter cells. Further, association of mitochondria with microtubules inhibited 27 activity of the dynamin GTPase-related fission protein Dnm1, such that cells overexpressing 28 Dnm1 but containing long microtubules were protected from increased fission. Thus, we 29 demonstrate a general mechanism by which mitochondrial dynamics is dictated by the 30 dynamics of the microtubule cytoskeleton. bioRxiv preprint doi: https://doi.org/10.1101/178913; this version posted January 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 31 Introduction 32 Mitochondria are double-membraned organelles whose functions range from ATP 33 production to calcium signalling. Inside cells, mitochondrial form is dynamic and transitions 34 from tubular networks to fragmented entities depending on the activity of the mitochondrial 35 fission and fusion machinery. The major mitochondrial fission protein is dynamin-related 36 Drp1 GTPase1 (Dnm1 in yeast2,3). Multimeric Drp1 rings assemble around the mitochondrial 37 membrane and utilize the energy from GTP hydrolysis to catalyse the constriction and 38 fission of mitochondria4,5. Fusion of mitochondria requires two sets of proteins namely 39 Opa16 for the inner membrane (Mgm1 in yeast7) and Mfn1/28 for the outer membrane (Fzo1 40 in yeast9). 41 The requirement for dynamic mitochondria has been attributed to two primary 42 reasons, namely quality control and energy production10. Larger/longer mitochondria 43 resulting from fusion are hypothesized to be capable of producing more energy whereas 44 shorter/smaller mitochondria formed following a fission event are likely to undergo 45 mitophagy11. In the case of the latter, fission could serve as an efficient mechanism to 46 segregate and eliminate damaged mitochondria. Dysfunction of fission and fusion 47 processes has been implicated in neurodegeneration12,13, cancer14 and cardiomyopathies15, 48 amongst a host of metabolic disorders. 49 In mammalian cells, mitochondria are transported along microtubule tracks by the 50 activity of motor proteins kinesin-1 and dynein16. Kinesin-1 and dynein bind to the outer 51 membrane of mitochondria via the Miro/Milton complex17–19 and move mitochondria in the 52 anterograde and retrograde direction respectively. In neuronal cells, increase in calcium 53 levels results in the attachment of kinesin-1 motor to mitochondria via syntaphilin, which 54 inhibits the ATPase activity of kinesin and hence leads to stationary mitochondria on 55 neuronal microtubules20. About 70% of mitochondria in neuronal cells have been visualized 56 in this stationary state21. 57 In contrast to mammalian cells, mitochondria in fission yeast do not undergo motor- 58 driven movement along microtubules. However, the protein Mmb1 has been identified to 59 associate mitochondria with dynamic microtubules22. Upon microtubule depolymerisation 60 using methyl benzimidazol-2-yl-carbamate (MBC) or deletion of Mmb1, mitochondria have 61 been observed to undergo fragmentation3,22,23. Conversely, in cells overexpressing Mmb1, 62 mitochondria seemingly undergo reduced fission and exhibit long mitochondria22. bioRxiv preprint doi: https://doi.org/10.1101/178913; this version posted January 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 63 Cells employ several strategies to reduce partitioning error of organelles during 64 mitosis, such as ordered segregation mediated by spindle poles, or increasing copy 65 numbers of organelles prior to cell division24. In the latter, homogeneous distribution of the 66 multiple organelle copies serves to increase the probability of equal partitioning 67 stochastically. Mitochondrial inheritance has been observed to be microtubule-dependent 68 in mammalian cells25. In fission yeast, mitochondrial partitioning during cell division has 69 been proposed to be mediated by attachment of mitochondria with spindle poles26–28, 70 similar to the segregation of endosomes, lysosomes and Golgi bodies in mammalian 71 cells29,30 . However, only a portion of the observed mitochondria associated with the spindle 72 poles26. Additionally, increased mitochondrial fragmentation upon the onset of mitosis has 73 also been observed3, perhaps indicating a stochastic mechanism for mitochondrial 74 partitioning. 75 While the molecular players that effect fission and fusion have been identified in 76 several systems, the cellular signals that regulate these events are largely elusive. Here, 77 we demonstrate that mitochondria piggyback on dynamic microtubules to selectively 78 undergo fission while microtubules depolymerise. Reorganisation of interphase 79 microtubules into the nucleus when cells prepare for division also provided the cue for 80 increased mitochondrial fission. We quantified the number of mitochondria in mother cells 81 immediately after formation of mitotic spindle within the nucleus and the number of 82 mitochondria in the resulting daughter cells, and confirmed that the partitioning was indeed 83 a good fit to a binomial distribution, indicating that the mitochondria were stochastically 84 segregated into the two daughter cells. We observed that the fission protein Dnm1 was 85 excluded from regions of microtubules localisation and that the presence of long and 86 stabilised microtubules was inhibitory to uncontrolled fission even when Dnm1 was 87 overexpressed. bioRxiv preprint doi: https://doi.org/10.1101/178913; this version posted January 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 88 Results 89 1. Perturbation of microtubule dynamics leads to changes in mitochondrial 90 numbers 91 We observed that mitochondria underwent increased fission in the absence of 92 microtubules, but did not observe their subsequent aggregation as reported previously 93 (Extended Data Fig. 1a-c). Instead, the fragmented mitochondria were mobile and 94 frequently in close contact with each other (Extended Data Fig. 1b, c, Supplementary Video 95 S1). Since the depolymerisation of microtubules had a direct effect on mitochondrial fission, 96 we set out to study the consequence of modification of microtubule dynamics on 97 mitochondrial dynamics. To this end, we visualised the mitochondria and microtubules of 98 fission yeast strains carrying deletions of antagonistic kinesin-like proteins, Klp5/Klp6 and 99 Klp4 in high-resolution deconvolved images (Fig. 1a, Supplementary Videos S2, S3 and 100 S4). 101 102 Figure 1. Mitochondrial number is inversely proportional to microtubule 103 length. a, Maximum intensity projections of deconvolved Z-stack images of microtubules bioRxiv preprint doi: https://doi.org/10.1101/178913; this version posted January 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the