Association of mitochondria with microtubules inhibits mitochondrial fission by precluding Dnm1 assembly

Mitochondria are organized as tubular networks in the cell and undergo fission and fusion. While several of the molecular players involved in mediating mitochondrial dynamics have been identified, the precise cellular cues that initiate fission or fusion remain largely unknown. In fission yeast, mitochondria are organized along microtubule bundles. Here, we employed deletions of kinesin-like proteins to perturb microtubule dynamics, and determined that cells with long microtubules exhibited long, but fewer mitochondria, whereas cells with short microtubules exhibited short, but several mitochondria due to reduced mitochondrial fission in the former and elevated fission in the latter. Correspondingly, upon onset of closed mitosis in fission yeast, wherein interphase microtubules assemble to form the spindle within the nucleus, we measured increased mitochondrial fission. We determined that the consequent rise in the mitochondrial copy number was necessary to reduce partitioning errors while stochastically partitioning mitochondria between daughter cells. We discovered that the association of mitochondria with microtubules physically impeded the assembly of the fission protein Dnm1 around mitochondria, resulting in inhibition of mitochondrial fission. Taken together, we demonstrate a novel mechanism for regulation of mitochondrial fission that is dictated by the interaction between mitochondria and the microtubule cytoskeleton.

ls with long microtubules exhibited long, but fewer mitochondria, whereas cells with short microtubules exhibited short, but several mitochondria due to reduced itochondrial fission in the former and elevated fission in the latter.Correspondingly, upon onset of closed mitosis in fission yeast, wherein interphase microtubules assemble to form the spindle within the nucleus, we measured increased mitochondrial fission.We determined that the consequent rise in the mitochondrial copy number was necessary to reduce partitioning errors while stochastically partitioning mitochondria between daughter cells.We discovered that the association of mitochondria with microtubules phys cally impeded the assembly of the fission protein Dnm1 around mitochondria, resulting in i hibition of mitochondrial fission.Taken together, we demonstrate a novel mechanism for regulation of mitochondrial fission that is dictated by the interaction between mitochondria and the microtubule cytoskeleton..

Introduction

Mitochondria are double-membraned organelles whose functions range from ATP pro uction to calcium signalling.Inside cells, mitochondrial form is dynamic and transitions from tubular networks to fragmented entiti s depending on the activity of the mitochondrial fission and fusion machinery.The major mitochondrial fission protein is dynamin-related Drp1 GTPase (Smirnova et al., 2001) (Dnm1 in yeast (Bleazard et al., 1999;Jourdain et al., 2009)).Multimeric Drp1 rings assemble around the mitochondrial membrane and utilize the ener

from GTP hydrolysis to catalyze the constriction and fission of mitoch
ndria (Basu et al., 2017;Ingerman et al., 2005).Fusion of mitochondria requires two sets of proteins namely Opa1 (Delettre et al., 2000) for the inner membrane (Mgm1 in yeast (Sesaki et al., 2003)) and Mfn1/2 (Eura et al., 2003) for the outer membrane (Fzo1 in yeast (Hermann et al., 1998)).

The requirement for dynamic mitochondria has been attributed to two primary reasons, namely quality control and energy production (Mishra and Chan, 2016).

Larger/longer mitochondria resulting from fusion are hypothesized to be capable of producing more energy whereas shorter/smaller mitochondria formed following a fission event are likely to undergo mitophagy (Chen and Chan, 2009).In the case of the latter, fission could serve as an efficient mechanism to segregate and eliminate damaged mitochondria.Dysfunction of fission and fusion processes has been implicated in neurodegeneration (Deng et al., 2008;Hirai et al., 2001), cancer (Graves et al., 2012) and c rdiomyopathies (Ashrafian et al., 2010), amongst a host of metabolic disorders.

In mammalian cells, mitochondria are transported along microtubule tracks by the activity of motor proteins kinesin-1 and dynein (Pilling et al., 2006).Kinesin-1 and dynein bind to the outer membrane of mitochondria via the Miro/Milton complex (Glater et al., 2006;Stowers e al., 2002;van Spronsen et al., 2013) and move mitochondria in the anterograde and retrograde direction respectively.In neuronal cells, increase in calcium levels results in the attachment of kinesin-1 motor to mitochondria via syntaphilin, which inhibits the ATPase activity of kinesin and hence leads to stationary mitochondria on neuronal microtubules (Chen and Sheng, 2013).About 70% of mitochondria in neuronal cells have been visualized in this stationary state (Kang et al., 2008).In contrast to mammalian cells, mitochondria in fission yeast do not undergo motor-driven movement along microtubules (Chiron et al., 2008;Yaffe et al., 2003).However, the protein Mmb1 has been identified to associate mitochondria with dynamic microtubules (Fu et al., 2011).Upon microtubule depolymerization using methyl benzimidazol-2-yl-carbamate (MBC), mitochondria have been observed to undergo fragmentation (Fu et al., 2011;Jourdain et al., 2009;Li et al., 2015).Additionally, mitochondrial dynamics and partitioning i

fission yeast have been observed to be actin/myosin-independ
nt processes (Jourdain et al., 2009), contrary to the mechanism of mitoc ondrial partitioning in budding yeast (Fehrenbacher et al., 2004).Cells employ several strategies to reduce partitioning error of organelles during mitosis, such as ordered segregation mediated by spindle poles, or increasing copy numbers of organelles prior to cell division (Huh and Paulsson, 2011).In the latter, homogeneous distribution of the multiple organelle copies serves to increase the probability of equal partitioning stochastically.Mitochondrial inheritance has been observed to be microtubule-dependent in mammalian cells (Lawrence and Mandato, 2013).In fission yeast, mitochondrial partitioning during cell division has been proposed to be mediated by attachment of mitochondria with spindle poles (Jajoo et al., 2016;Krüger and Tolic, 2008;Yaffe et al., 2003), similar to the segregation of endosomes, lysosomes and Golgi bodies in mammali n cells (Bergeland et al., 2001;Shima et al., 1998) .However, only a portion of the observed mitochondria associated with the spindle poles (Yaffe et al., 2003).Additionally, increased mitochondrial fragmentation upon the onset of mitosis has also been observed (Jourdain et al., 2009), perhaps indicating a stochastic mechanism for mitochondrial partitioning.
hile the molecular players that effect fission and fusion have been identified in several systems, the cellular signals that regulate these events are largely elusive.

Here, we demonstrate that mitochondria piggyback on dynamic microtubules to selectively undergo fission when microtubules depolymerize.Reorganization of interphase microtubules into the nucleus when cells prepare for division also provided the cue for increased mitochondrial fission.We quantified the number of mitochondria in mother cells immediately after formation of mitotic spindle within the nucleus and the number of mitochondria in the resulting daughter cells, and confirmed that the partitioning was indeed a good fit to a binomial distribution, i dicating that the mitochondria were stochastically segregated into the two daughter cells.We determined that the presence of long a

stabilized microtubules was inhibitory to unopposed fission even when Dnm1 was
overexpressed.Finally, we discovered microtubule-bound mitochondria were unlikely to undergo fission due to the unavailability of space between microtubules and mitoch ndria for the formation of the Dnm1 ring.


Mat

ials
and Methods

Strains and media.The fission yeast strains used in this study are listed in Table S1.All the strains were grown in YE (yeast extract) media or Edinburg Minimal media (EMM) (Forsburg and Rhind, 2006) with appropriate supplements at a temperature of 30˚C.Cells that were transformed with plasmid pREP41-Dnm1 or pREP41-Dnm1-Cterm-GFP (see Table S1) were cultured in EMM with appropriate supplements and 0.05µM thiamine for partial induction of the nmt1 promoter.Strains VA076 and VA084 were constructed by crossing PT2244 with FY20823, and Dnm1Δ with G5B respectively (see Table S1), following the random spore analysis protocol (Forsburg and Rhind, 2006).

Plasmid transformation.Transformation of strains was carried out using the improved protocol for rapid transformation of fission yeast (Kanter-Smoler et al., 1994).In brief, cells were grown overnight to log phase in low glucose EMM, pelleted and washed with distilled ater.The cells were then washed in a solution of lithium acetate/EDTA (100mM LiAc, 1mM EDTA, pH 4.9) and re-suspended in the same solution.1µg of lasmid DNA was added to the suspension, followed by addition of lithium acetate/PEG (40% w/v PEG, 100mM LiAc, 1mM EDTA, pH 4.9) and then incubated at 30°C for 30 min in a shaking incubator.This was followed by a heat shock of 15min at 42°C.Thereafter, ells were pelleted down, re-suspended in TE solution (10mM Tris-HCl, 1mM EDTA, pH 7.5) and plated onto selective EMM plates.

Prepa ation of yeast for imaging.For imaging mitochondria, fission yeast cells were grown overnight in a shaking incubator at 30°C, washed once with distilled water, and stained with 200nM Mitotracker Orange CMTMRos (ThermoFisher Scientific, Cat.#M7510) diss lved in EMM for 20min.Following this, cells were washed thrice with EMM and then allowed to adhere on lectin-coated (Sigma-Aldrich, St. Louis, MO, Cat.#L2380) 35mm confocal dishes (SPL, Cat.#100350) for 20min.Unattach d cells were then removed by washing with EMM.In experiments where mitochondria were not imaged, staining with Mitotracker was omitted.

Microtubule depolymerization.For depolymerization of microtubules, cells were treated with methyl benzimidazol-2-yl-carbamate (MBC, Carbendazim 97%, Sigma Aldrich).A stock solution with a concentration of 25mg/ml was prepared in DMSO and later diluted to working concentration of 25µg/ml in EMM.


Microscopy.

Confocal microscopy was carried out using the InCell Analyzer-6000 (GE Hea thcare, Buckinghamshire, UK) with 60x/0.7 N.A. objective fitted with an sCMOS 5.5MP camera having an

-y p
xel separation of 108nm.For GFP and Mitotracker Orange imaging, 488 and 561nm laser lines and bandpass emission filters 525/20 and 605/52nm respectively were employed.Time-lapses for visualization of mitochondrial dynamics were captured by obtaining 5 Z-stacks with a 0.5µm-step size every 12s.Deconvolution was performed in images obtained using a Deltavision RT microscope (Applied Precision) with a 100×, oil-immersion 1.4 N.A. objective (Olympus, Japan).Excitation of fluorophores was achieved using InsightSSI (Applied Precision) and corresponding filter selection for excitation and emission of GFP and Mitotracker Orange.Z-stacks with 0.3µm-step sizes encompassing the entire cell were captured using a CoolSnapHQ camera (Photometrics), with 2X2 binning.The system was controlled using softWoRx 3.5.1 software (Applied Precision) and the deconvolved images obtained using the built-in setting for each channel.

3D visualization of deconvolved images.3D views of the microtubules and mitochondria in Movies S2, S3, S4, S8, S9, S10, S12 and S13 were obtained from deconvolved images captured in the Deltavision microscope using Fiji's '3D project' function, with the brightest point projection method and 60˚ total rotation with 10˚ rotation angle increment.


Estimation of volume of mitochondria. Mitochondrial volume was estimated in Fiji

by integrating the areas of mitochondria in thresholded 3D stacks of cells in fluorescent deconvolved images obtained using the Deltavision RT microscope.The total volume was then normalized to the mean total mitochondrial volume of wildtype cells.Individual mitochondrial volumes were estimated in the same fashion.

Analysis of mitochondrial dynamics.Individual mitochondria were identified in each frame of the time-lapse obtained in confocal mode of the GE InCell Analyzer after projecting the maximum intensity of the 3D stack encompassing the cell, followed by mean filtering and visualization in Fiji's 'mpl-magma' lookup table.

Following identification of mitochondria, the 'Measure' function of Fiji was employed to obtain the circularity, aspect ratio and parameters of fitted ellipse.The length of the major axis of the ellipse fitted to a mitochondrion was defined as the size of that mitochondrion.The size, circularity and aspect ratio were estimated for mitochondria at each frame and each time point.Fission and fusion frequencies of mitochondria were estimated by counting the number of mitochondria identified during each frame of the time-lapse.The difference in number of mitochondria from one frame to the next was counted, with an increase being counted as fission event and decrease as fusion event.The total number of fission events and fusion events per cell were estimated and divided by the total duration of the time-lapse to obtain the fission and fusion frequencies.

Test for fit of mitochondrial partitioning during mitosis to binomial distribution.To test the fit of mitochondrial segregation during mitosis to a binomial distribution, the data were z-transformed as previously described (Hennis and Birky, 1984).Briefly, given  mitochondria in the mother cell just prior to cell division,  and  −  mitochondria in the resulting daughter cells,  was given by 2 − /  to approximate the binomial distribution to a normal distribution of 0,1.The  values obtained for each  and  were binned into  bins of equal sizes and subjected to Chi-square test with  − 1 degrees of freedom.The  values are expected t be equally distributed among the  bins, with expected number of 1/ per bin.

Data analysis and plotting.Data analysis was performe in Matlab (Mathworks, Natick, MA).Box plots with the central line indicating the median and notches that represent the 95% confidence interval of the median were obtained by performing one-way NOVA ('anova1' in Matlab) or Kruskal-Wallis Test ('kruskalwallis' in Matlab).The former was used when data were found to be normally distributed and the latter when data were non-normally distributed (tested using 'chi2gof' in Matlab).

Following this, significant difference (p<0.05) was tested using the Tukey's Honestly Significant Difference procedure ('multcompare' in Matlab).All the plots were generated using Matlab.S1), Klp5Δ/Klp6Δ ('MT long ', strain G3B, see Table S1) and Klp4Δ ('MT short ', strain G5B, see Table S1 We observed that mitochondria underwen increased fission upon microtubule depolymerization, but did not observe their subsequent aggregation as reported previously (Fig. S1A-C).Instead, the fragmented mitochondria were mobile and frequently in close contact with each other (Fig. S1B, C, Movie S1).Since the depolymerization of microtubules had a direct effect on mitochondrial fission, we set out to study the consequence of modification of microtubule dynamics on mitochondrial dynamics.To this end, we visualised the mitochondria and microtubules of fission yeast strains ca

ying deletions of antagonistic kinesin-like proteins, Klp5/Klp6 and Klp4 in hi
h-resolution deconvolved images (Fig. 1A, Movies S2, S3 and S4).


Results


Perturbation of microtubule dynamics leads to changes in mitochondrial numbers

The heteromeric Klp5/Klp6 motor is required for maintenance of interphase microtubule length by promoting catastrophe at microtubule plus ends (Tischer et al., 2009;West et al., 2001).Cells lacking Klp5 and Klp6 exhibited long microtubule bundles ('MT long ', Fig. 1B) s reported previously due to a decreased catastrophe rate (Tischer et al., 2009).In contrast, Klp4 is required for polarized growth in fission yeast and has been sug ested to promote microtubule growth (Browning et al., 2000;Busch et al., 2004).As a res

t, in the absence of Klp4, microtubule bundles were only
bout half the length of wild-type bundles ('MT short ', Fig. 1B).

As in wild-type cells, mitochondria in Klp5Δ/Klp6Δ were in close contact with the microtubul , whereas we observed reduced association between the short microtubules and mitochondria in Klp4Δ cells (Fig. 1A, Movies S3 and S4).While wild-type cells had 4.9 ± 0.4 (mean ± s.e.m.) mitochondria per cell, we observed that Klp5Δ/Klp6Δ contained only 2.3 ± 0.4 (mean ± s.e.m.).In contrast, Klp4Δ cells had 10 ± 0.9 mitochondria per cell (mean ± s.e.m., Fig. 1C).This indicated that the number of mitochondria per cel was inversely related to the length of microtubule bundle.However, the decrease in the number of mitochondria in Klp5Δ/Klp6Δ cells and increase in Klp4Δ cells were not at the expense of mitochondrial volume, since the net mitochondrial volume in both cases was comparable to wild-type mitocho drial volume (Fig. 1D), with individual mitochondrial volumes changing to compensate for the difference in mitochondrial numbers between WT, Klp5Δ/Klp6Δ and Klp4Δ cells (Fig. S1D).


Cells with short microtubules undergo increased fission

To understand the difference in mitochondrial numbers in wild-type, Klp5Δ/Klp6Δ and Klp4Δ cells, we acquired and analyzed time-lapse videos at the single mitochondrion level in all three cases (Fig. 2A, Movies S5, S6 and S7).Similar to our observations from high-resolution images, we measured 3.6 ± 0.2, 2.7 ± 0.2, and 6.9 ± 0.8 mitochondria (mean ± s.e.m.) in wild-type, Klp5Δ/Klp6Δ and Klp4Δ cells respectively (Fig. 1C and 2B).Analysis of evolution of these mitochondrial numbers revealed no significant changes over time (Fig. 2B).Additionally, mitochondria in wild-type cells underwent ~1 fission and ~1 fusion event every minute on an average, whereas Klp5Δ/Klp6Δ cells exhibited a fission frequency that was half hat of wild-type, and Klp4Δ mitochondria had a fission frequency that was almost double that of wild-type (Fig. 2C).The fusion frequency of mitochondria in Klp4Δ cells was slightly higher than in ild-type and Klp5Δ/Klp6Δ cells (Fig. 2D), likely due to the increased number of mitochondria in Klp4Δ cells that could participate in fusion.However, the resulting ratio of the mean fission frequency to the mean fusion frequency was ~1, ~0.5 and ~1.3 in wild-type, Klp5Δ/Klp6Δ and Klp4Δ cells respectively.We therefore concluded that the difference in mitochondrial numbers between wild-type cells and Klp5Δ/Klp6Δ and Klp4Δ arose primarily due to the changes in fission frequencies of mitochondria.


Figure 2. Microtubule length determines fission frequency of mitochondria. (A)

Montage of maximum intensity projected confocal Z-stack images of wild-type ('WT', strain KI001, see Table S1), Klp5Δ/Klp6Δ ('MT long ', strain G3B, see Table S1) and Klp4Δ ('MT short ', strain G5B, see Table S1) cells represented in the intensity map indicated to the left of the images.The insets (white box) and their montages on the right of the images are representative fission and fusion events in WT ('1' and '2'), fusion event in MT long ('3') and fission event in MT short cell ('4').Time is indicated as 'mm:ss' above th montage of the insets.The normalized intensity along the mitochondrion in the inset before (magenta) and after (green) the fission or fusion Note that the rate of mitochondrial fission (per second per mitochondrion)

s comparable to the rate
of mitochondrial fusion (per second per mitochondrion) in all three cases, resulting in the constant number of mitochondria we measured over time (Fig. 2B).As observed in the high resolution deconvolved images, we also measured significant differences in t e size and morphology of the mitochondria in WT, Klp5Δ/Klp6Δ and Klp4Δ cells (Fig. 2E, Fig. S2).Mitochondrial sizes reflected microtubule bundle lengths, with the largest mitochondria in Klp5Δ/Klp6Δ cells and the smallest in Klp4Δ cells.WT cells predictably had mitochondrial sizes between that of Klp4Δ and Klp5Δ/Klp6Δ cells (Fig. 2E, Fig. S2).


Cells devoid of microtubules undergo fission with increased frequency but show unaltered fusion frequencies

While the Klp5Δ/Klp6Δ cells containing long microtubules had the same total volume of mitochondria as WT cells (Fig. 1D), their mitochondria appeared less fragmented.So, to specifically test the role of microtubules in dictating mitochondrial fission, we depolymerized microtubules in wild-type and Klp5Δ/Klp6Δ cells and measured mitochondrial dynamics in time-lapse movies (Fig. 3A, Movies S8, S9).In both WT and Klp5Δ/Klp6Δ cells, upon microtubule depolymerization, we observed a progressive increase in the number of mitochondria (Fig. 3B).We next quantified the fission and fusion events in WT and Klp5∆/Klp6∆ cells treated with MBC.We measured that the fission frequency of mitochondria in MBC-treated cells was doubled when compared to untreated c ntrol cells (Fig. 3C).At the same time, the mitochondrial fusion frequency remained unchanged (Fig. 3D), indicating that the immedi te consequence of the loss of microtubules was increased fission, without concomitant changes in mitochondrial fusion.Additionally, upon MBC treatment we measured mitochondrial sizes and morphol gies that were reminiscent of Klp4Δ cells (Fig. 3E, Fig. S3A-C).

Increase in oxidative stress via reactive oxygen species (ROS) levels has also been described to induce mitochondrial fission (Pletjushkina et al., 2006).However, we measured no difference in ROS levels between wild-type, Klp5Δ/Klp6Δ, and Klp4Δ cells (Fig. S3D).


Mitochondria undergo stochastic partitioning during mitosis

We next sought to understand the biological role for increased mitochondrial fission upon microtubule depolymerization.Fissi n yeast undergoes closed mitosis, wherein the nuclear envelope does not undergo breakdown during cell division (Ding et al., 1997).Upon onset of mitosis in fission yeast, the interphase microtubules that were previously in the cytoplasm are reorganized to form the spindle inside the closed nucleus.This natural situation mimics the depolymerization of microtubules via the chemical inhibitor MBC.Therefore, we set out to study the changes in the mitochondrial network upon cell entry into mitosis.We first obtained high-resolution deconvolved images of the microtubule and itochondria in fission yeast cells undergoing cell division (Fig. 4A, Fig. S4A, Movie S10).We observed that dividing wild-type cells had ~4x the number of mitochondria in interphase cells (Fig. 4B).

Moreover, similar to what was seen in cells lacking microtubules or Klp4 (Fig. 1A), mitochondria in dividing cells were shorter and more rounded (Fig. 4A, Fig. S4A).

There was no relationship between length of the mitotic spindle and the number of mitochondria (Fig. 4B, Fig. S4A), indicating that the increased fission likely occurred fairly early upon entry into mitosis.Analysis of time-lapse videos of wild-type cells before and 10min after entry into mitosis revealed a doubling of mitochondrial numbers in this time (Fig. 4C, D, Movie S11).The fragmented mitochondria appeared more mobile and were able to traverse distances of ~1μm in the cell (Fig. 4C).In this same period of time, non-dividing interphase cells did not show any 290 change in mitochondrial numbers (Fig. S4B, C). 291  Incre se in mitochondrial numbers prior to cell division could aid in increasing the likelihood of equal partitioning of mitochondria between daughter cells, given a stochastic mechanism.To test if mitochondria in our system underwent stochastic, independent segregation (Huh and Paulsson, 2011), with each mitochondrion in the mother cell ha ing a 50% chance of segregating to either of the future daughter cells during mitosis, we tested the fit of our data to a binomial distribution (Birky, 1983) using a Chi-square test as previously described (Hennis and Birky, 1984).Our data (Table S2) did not differ significantly from a binomial distribution with a Chi-square statistic of 7.1846 with 3 degrees of freedom and p=0.0662, indicating that mitochondria in fission yeast were indeed segregating stochastically during cell divi ion.The increase in mitochondrial numbers upon onset of mitosis also served to reduce the partitioning error of mitochondr a between the daughter cells as predicted by stochastic segregation (Fig. S4D).


Cells with long microtubules are protected from unopposed mitochondrial fission

The mitochon

ial fiss
on protein in yeast is a dynamin-related GTPase, Dnm1 (Jourdain et al., 2009).Dnm1 brings about the fission of mitochondria by selfassembling into rings or spirals around the mitochondrial outer membrane and employing its GTPase activity to effect the scission (Ingerman et al., 2005).In the absence of Dnm1, mitochondria are organised as extended, fused 'nets' (Guillou et al., 2005;Jourdain et al., 2009), which do not undergo fission even in th absence of microtubules (Fig. S5A).Further in Klp4Δ cells, which typically contain several short mitochondria (Fig. 1A), absence of Dnm1 results in a single large, fused mitochondrion (Fig. S5B).Therefore, all mitochondrial fission in S. pombe is reliant on the activity of Dnm1.Additionally, during mitosis, cells lacking Dnm1 that contained a si gle large mitochondrion relied on the cytokinesis of the mother cell to also split the mitochondrion into the daughter cells (Fig. S5C).S1) in Dnm1Δ cells (see Table S1) wild-type ('WT', strain FY7143, see Table S1) and Klp5Δ/Klp6Δ cells ('MT long ', stra n FY20823, see Table S1 Taken together, our results suggest that association of mitochondria with microtubules inhibits mitochondrial fission.Previously, cryo-electron tomographic analysis in fission yeast indicated a preferred separation distance of ~20nm for mitochondria ass ciated with microtubules (Höög et al., 2007).So too, cryo-elctron microscopy in budding yeast revealed that Dnm1 assembled into rin