Mitochondria-cytoskeleton associations in mammalian cytokinesis
© Lawrence et al. 2016
Received: 25 August 2015
Accepted: 2 March 2016
Published: 18 March 2016
The role of the cytoskeleton in regulating mitochondrial distribution in dividing mammalian cells is poorly understood. We previously demonstrated that mitochondria are transported to the cleavage furrow during cytokinesis in a microtubule-dependent manner. However, the exact subset of spindle microtubules and molecular machinery involved remains unknown.
We employed quantitative imaging techniques and structured illumination microscopy to analyse the spatial and temporal relationship of mitochondria with microtubules and actin of the contractile ring during cytokinesis in HeLa cells.
Superresolution microscopy revealed that mitochondria were associated with astral microtubules of the mitotic spindle in cytokinetic cells. Dominant-negative mutants of KIF5B, the heavy chain of kinesin-1 motor, and of Miro-1 disrupted mitochondrial transport to the furrow. Live imaging revealed that mitochondrial enrichment at the cell equator occurred simultaneously with the appearance of the contractile ring in cytokinesis. Inhibiting RhoA activity and contractile ring assembly with C3 transferase, caused mitochondrial mislocalisation during division.
Taken together, the data suggest a model in which mitochondria are transported by a microtubule-mediated mechanism involving equatorial astral microtubules, Miro-1, and KIF5B to the nascent actomyosin contractile ring in cytokinesis.
KeywordsMitochondria Cytoskeleton Cytokinesis Microtubules Actin Miro KIF5B
Mitochondria are essential organelles that function in ATP energy production, calcium buffering, and the generation of reactive oxygen species (ROS) [1–4]. The distribution of mitochondria within the cytoplasm is determined by interactions with the cytoskeleton. In mammalian cells, mitochondria are transported over long distances on microtubules, while short-range movement and docking require actin [5–7]. However, the role of the cytoskeleton and associated proteins in determining the distribution of mitochondria during cytokinesis, the final stage of cell division, is poorly understood.
Cytokinesis begins in anaphase with the assembly of a contractile ring composed of filamentous actin (F-actin) and Myosin II at the equatorial cortex. Constriction of the actomyosin ring results in a cleavage furrow that physically divides the parent cell into two daughter cells . Microtubules of the mitotic spindle specify the assembly of the actomyosin ring by delivering the small GTPase RhoA to the equatorial cortex [9–11]. Subsequently, RhoA triggers local actin polymerisation and Myosin II contractility via downstream effectors such as the diaphanous-related formin mDia1, Rho kinase, and citron kinase [12–14].
We recently reported that mitochondria localize to the cleavage furrow during cytokinesis in mammalian cells using a mechanism that is dependent on microtubules of the mitotic spindle . However, while we found that mitochondrial transport to the cleavage furrow during cytokinesis was microtubule-dependent, the subset of spindle microtubules and molecular machinery remained unknown. In contrast, a recent RNAi screen for new actin-based motors that function in division also identified the mitochondrial motor Myosin XIX (Myo19) as being involved in the regulation of mitochondrial distribution in dividing HeLa cells . Indeed, when Myo19 expression was knocked down in dividing cells, mitochondria were mislocalized and asymmetrically inherited. The authors reported a similar phenotype for cells treated with Latrunculin B  and concluded that both Myo19 and actin were necessary for appropriate mitochondria distribution during cell division. In contrast, we previously showed, through systematic spatial and temporal analysis, that mitochondria accumulated at the cell equator following anaphase onset with no overall quantifiable differences in mitochondrial distribution between DMSO- or Latrunculin A-treated cells . Thus, the mechanism of mitochondrial redistribution in dividing cells remains controversial.
The kinesin-1 family of motor proteins play a key role in transporting mitochondria in the anterograde direction along microtubules [17–20]. Mammals possess three isoforms of kinesin-1 (KIF5A, B and C). KIF5A and KIF5C are neuronal, whereas KIF5B is ubiquitously expressed . KIF5B binds to mitochondria via an adaptor complex composed of the mitochondrial Rho GTPase (Miro) and the cytoplasmic adaptor protein Milton . Miro is an highly conserved atypical GTPase that is anchored in the mitochondrial membrane [21–23]. Miro binds to Milton, which in turns binds to KIF5B, thereby linking mitochondria to microtubules [24, 25]. It is not known if the KIF5B-Miro machinery mediates microtubule-based transport of mitochondria to the furrow during cytokinesis.
Herein, we used a combination of live spinning disk and superresolution structured illumination microscopy (SIM) to gain mechanistic insights into the previously reported relationships of mitochondria with microtubules and actin in mammalian cytokinesis. The mechanism of microtubule-based transport of mitochondria to the furrow was investigated using dominant-negative mutants of KIF5B and Miro-1. Finally, systematic spatial and temporal quantification of control- and cell-permeable C3 transferase treated-cells, allowed us to investigate the relationship of mitochondria with the contractile ring during cytokinesis.
Mitochondria associate with astral microtubules during cytokinesis
Miro-1 localizes with mitochondria at the cleavage furrow during cytokinesis
Miro-1 and KIF5B are required for mitochondrial distribution during cytokinesis
Mitochondrial enrichment at the cell equator occurs simultaneously with the formation of the contractile ring
Next, we sought to quantify the time of onset of F-actin and mitochondria enrichment at the cell equator. To measure equatorial enrichment, equator: pole F.I. ratios for both F-actin and mitochondria were calculated at 30 s intervals following metaphase exit. The mean equator: pole F.I. ratio of eight cells (four quarters for each cells) ± SEM was plotted against time (Fig. 4c). Analysis revealed that the equatorial enrichment of both actin and mitochondria initiated at 1-min post-metaphase exit (Fig. 4c, arrow). Thus, the onset of mitochondrial enrichment at the cell equator occurs simultaneously with the onset of the formation of the actomyosin contractile ring.
Inhibiting contractile ring formation prevents mitochondrial enrichment at the cell equator
The involvement of microtubules and Miro-1 in the re-distribution of mitochondria during the transition from late cytokinesis to interphase has also been recently confirmed by Kanfer et al., which provided evidence that, upon entry into mitosis, CENP-F redistributes to mitochondria via Miro-1 binding and links mitochondria to microtubule plus-tips . Herein, we investigated the role of KIF5B in linking mitochondria to microtubules during mitosis and found that inhibition of KIF5B activity caused mislocalized mitochondria in late cytokinesis but not during earlier stages of cytokinesis. One interpretation is that KIF5B mediates mitochondrial transport only during late cytokinesis. Alternatively, since our transfections were stringently optimized to minimize cytotoxic effects, dominant negative expression levels may not have been adequate to completely inhibit endogenous KIF5B activity. Another possibility is that other members of the kinesin-3 family members, such as KIF1B and KLP6, or CENP-F may compensate for KIF5B inhibition [36, 37]. In this regard, it would be interesting to assess the role of CENP-F and other kinesins in mediating the microtubule-mitochondria interaction at earlier stages of cytokinesis.
Following inhibition of Miro-1 activity and mitochondrial mislocalization, we did not observe obvious defects in cytokinesis or mitochondrial inheritance. This may be because mitochondria were only partially mislocalized and hence were still present, although reduced, at the furrow. It is also worth noting that our protocol did not allow for the observation of the effect of disrupted Miro-1/KIF5B over multiple generations, which may have yielded more robust mitochondrial mislocalization.
The fact that mitochondria polarize towards the cell equator simultaneously with contractile ring formation further supports, albeit indirectly, a microtubule-based mechanism of mitochondrial distribution during cytokinesis since microtubules are also required to stimulate contractile ring assembly at the equatorial cortex . In addition, our data show that mitochondrial distribution is coordinated with a key event of cytokinesis, contractile ring assembly. Whether the enrichment of mitochondria in the vicinity of the contractile ring or retention of mitochondria at the furrow during ingression has functional consequences for cytokinesis remains to be established. While we previously reported that mitochondria are transported to the cleavage furrow via an actin-independent mechanism [15, 26], a recent study showed that depletion of the actin-based motor Myo19 by RNAi in dividing HeLa cells caused mitochondria to aberrantly localize to the spindle poles during cytokinesis and resulted in defects in mitochondrial inheritance and cytokinesis . However, whether Myo19 anchors mitochondria to the contractile ring remains unknown since, in contradiction with our present and previous observations , they did not report mitochondria enrichment at the cleavage furrow in their control cells. The same group also failed to show enrichment of mitochondria to the equatorial portion of dividing cell in presence of Latrunculin B. The reason for this is unknown, but may relate to methodological differences in quantification methods, seeding density, or cell health. Indeed, we have reason to believe that the aberrant mitochondrial and F-actin distribution we sometimes observed during cell division in transfected cells (Additional file 6: Figure S3), may have been may be linked to altered cell shape or adhesion. Indeed, Fink et al.  suggested that adhesion geometry can bias dynamic subcortical actin structures in mitotic cells. Spindle positioning have been shown to be actin-dependent in a number of other models , and subcortical actin clouds and retraction fibres have been suggested to be involved in centrosome positioning . Under our proposed model, aberrant spindle positioning due to aberrant retraction fibres (as seen in the aggregated phenotype of Additional file 6: Figure S3) would produce equally aberrant mitochondria distributions.
Interestingly, we also observed the colocalization of mitochondria with a subcortical pool of F-actin at the cleavage furrow. The source of this subcortical F-actin pool is unknown, but may represent pre-formed F-actin filaments that have been delivered to the cleavage furrow for incorporation into the contractile ring . Alternatively, actin may be directly polymerized on the surface of furrow-enriched mitochondria. A recent paper by Li et al., showed the formation of actin filament on the outer mitochondrial membrane in interphase and mitotic cells . Therefore, it is possible that actin at the cleavage furrow may promote mitochondrial docking and further enrich mitochondria at the furrow.
Mitochondria were retained at the cell poles and the enrichment of mitochondria at the cell equator was reduced when RhoA was inhibited. Interestingly, RhoA has previously been shown to reduce mitochondrial motility by promoting mitochondrial tethering to actin via mDia1-activation and local actin polymerization [43, 44]. In the context of dividing cells, local RhoA-triggered actin polymerization at the equatorial cortex in dividing cells may act similarly to inhibit mitochondrial motility in the vicinity of the cleavage furrow. However, it is difficult to discern the effects of RhoA inactivation since such cells exhibit a dramatically altered cell morphology. Additionally, since mDia1 has been shown to regulate microtubules at the furrow , we cannot exclude that mitochondrial mislocalization following RhoA inhibition may be due to the perturbation of microtubules at the furrow. This would however, still be compatible with microtubule-mediated transport of mitochondria to the cleavage furrow.
Finally, while there is now considerable evidence for microtubule-mediated transport to the cleavage furrow, it is also possible for mitochondria to be passively distributed. An example of such a mechanism would involve the mitochondria being squeezed toward the equator by the growth of the spindle in anaphase, telophase and early cytokinesis. Considering the importance of mitochondrial inheritance in ensuring daughter cell viability, it may be envisaged that mitochondria distribution during cell division relies on both passive and active, microtubule-mediated processes.
Herein, we demonstrate, for the first time, an association of mitochondria with astral microtubules of the mitotic spindle and their co-localization with the nascent contractile ring in early cytokinesis. Furthermore, we provided evidence that mitochondrial localization to the furrow requires KIF5B and Miro-1 and the formation of a contractile array. In this model (Fig. 6), mitochondria are delivered to the cleavage furrow along equatorial astral microtubules in a mechanism involving the KIF5B/Miro-1 trafficking. A similar a model of microtubule-dependent delivery and actin-dependent docking at a contractile ring has been observed in the context of the immune synapse in T-cells .
Enriching mitochondria at the cell equator may contribute to the robustness of cytokinesis by promoting actomyosin assembly and contractility at the equatorial cortex. Cytokinesis failure leads to centrosome amplification and tetraploidy and is a hallmark of certain cancers [46, 47]; therefore, a complete understanding of the mechanisms underlying cytokinesis may have important therapeutic implications.
Cell culture and drug treatments
HeLa cells purchased from ATCC were maintained at 37 °C in a 5 % CO2 incubator and grown in Minimum Essential Media (MEM; Invitrogen) supplemented with 2 mM GlutaMAX (Gibco) and 10 % foetal bovine serum (FBS; Invitrogen). To stain for mitochondria, live cells were incubated for 15 min with 20 nM MitoTracker Red CMX Ros or MitoTracker Deep Red FM (Molecular Probes) as indicated in the text. To inhibit RhoA, cultured HeLa cells were incubated with 2 μg/ml of a commercially available, cell-permeable version of C3 transferase (Cytoskeleton Inc.) for at least 4 h prior to live cell imaging. C3 transferase is an ADP ribosyl transferase that selectively ribosylates RhoA, RhoB and RhoC proteins on asparagine residue 41, rendering them inactive . It has extremely low affinity for other members of the Rho family such as Cdc42 and Rac1 and does therefore not affect these GTPases. Analysis was limited to metaphase cells exhibiting the hallmarks of RhoA inhibition: a spindly, non-round morphology [49, 50].
The cDNA encoding the wild-type, full-length human Miro-1 as well as Miro-1 lacking the transmembrane binding domain (Miro-1Δ593-618, hereafter referred to as Miro-1ΔTM) were purchased from Addgene (Addgene plasmids 47888 and 47895; originally published in . The cDNA encoding the motor domain-less KIF5B (hereafter referred to as KIF5BTail) was a kind gift from C. Hoogenraad, Utrecht University, The Netherlands, and was successfully used to study microtubule trafficking [51, 52]. Miro-1ΔTM and KIF5BTail cDNA sequences were amplified by polymerase chain reaction using the Expand High Fidelity polymerase (Roche). Miro1ΔTM was amplified with the primer pair: 5′-CCGCTCGAGCGGATGAAGAAAGACGTGCGGAT-3′ and 5′-CCGGAATT.
CCGGTCAAAACGTGGAGCTCTTGAGGT-3′. KIF5BTail was amplified with the primer pair: 5′-CCGCTCGAGCGGATGTGGCGTAATGGGGAGACG-3′ and 5′-CCGGAATTCCGGTCAAGTTGGAGAAGCTGCTGGAT-3′. The underlined sequences correspond to Xho1 (forward primer) and EcoR1 (reverse primer) restriction sites. Full length Miro1, Miro1ΔTM, and KIF5BT were subcloned into a pCIG2-ires-eGFP bicistronic vector (a kind gift from M. Cayouette, IRCM, Montreal). GFP-Tubulin and GFP-UtrCH in the pCS2eGFP vector were kind gifts from W. Bement, University of Wisconsin, Madison.
HeLa cells were plated 1 day prior to transfection on 35 mm glass-bottom dishes (1.5 mm thickness; MatTek) in antibiotic-free MEM media. Transfections were performed using Effectene Transfection Reagent according to the manufacturer’s protocol (Qiagen). Complexes were washed out after 6 h to minimize cytotoxicity. To achieve maximal numbers of mitotic cells for imaging, transfections were performed at least 30 h before imaging. To detect microtubules and filamentous actin, cells were transfected with 200 ng GFP-Tubulin and GFP-UtrCH, respectively. For dominant negative experiments, cells were transfected with 300 ng of the relevant construct and transfected cells were identified by GFP expression from the bicistronic pCIG2.ires.eGFP vector. Transfected cells with an average GFP fluorescence intensity of <1400 arbitrary units were used for analysis.
For fixed confocal imaging, cells were grown on #1.5 coverslips. For super resolution microscopy, cells were grown on Zeiss high precision #1.5 coverslips (a kind gift from the CIAN microscopy facility) prepared for superresolution imaging by washing with spectroscopic grade ethanol and methanol. First, live cells were stained for mitochondria, then fixed with 3.2 % paraformaldehyde in phosphate buffered saline (PBS) for 10 min, permeabilized with PBS containing 0.1 % Triton X-100 and blocked with 2 % bovine serum albumin (BSA) in PBS for 30 min. Primary antibodies were incubated overnight at 4 °C and secondary antibodies were incubated for 45 min at room temperature in the dark. The following primary antibodies were used: rabbit anti-alpha-tubulin (Abcam, ab18251) and rabbit anti-RhoT1(A16) (Santa Cruz, sc-102083). Alexa Fluor 488- and 647-conjugated secondary antibodies were purchased from Invitrogen. DNA was stained with DAPI (Invitrogen). For fixed confocal microscopy, coverslips were mounted onto slides using PermaFluor (ThermoScientific). For superresolution SIM, coverslips were mounted onto slides with Prolong Gold (Invitrogen) and left to cure for 60 h.
Microscopy and imaging
Fixed confocal imaging was performed using a Confocor LSM 510 META confocal system on a Zeiss Axiovert 200 M inverted microscope using a 1.4 NA 63× oil-immersion objective and Zen imaging software (Zeiss). MitoTracker Red fluorescence was excited with 514 nm laser, Alexa 488 fluorescence was excited with 488 nm laser and DAPI fluorescence was excited with 405 nm laser. Confocal stacks were acquired with a 0.3 μm step size.
Superresolution SIM was performed using an ELYRA PS.1 superresolution system combined with an LSM 710 laser scanning confocal microscope (Zeiss). Images were acquired using a 1.4 NA 63× oil-immersion objective and captured with a pco.edge sCMOS camera controlled by Zen imaging software (Zeiss). Alexa-488 fluorescence was excited with a 488 nm laser, MitoTracker Red fluorescence was excited with a 561 nm laser and Alexa 647 fluorescence was excited with a 642 nm laser. Emission was collected with the appropriate filter sets optimized for each laser line. Stacks were acquired with a 0.2 μm step size. SIM images were reconstructed from the raw data using Zen imaging software.
Live imaging was performed using a Quorum WaveFX-X1 spinning disk confocal system on a Leica DMI6000B inverted microscope (Quorum Technologies Inc.). HeLa cells were grown on 35 mm glass-bottom dishes (1.5 mm thickness; MatTek) and maintained on the microscope stage in complete phenol-free MEM media (Invitrogen) at 37 °C and 5 % CO2 using a Chalamide TC environmental control system (Live Cell Instruments). All Images were acquired using a 1.4 NA 63× oil-immersion objective and captured with a Hamamatsu ImagEM EM-CCD camera controlled with Metamorph software (Molecular Devices). MitoTracker Deep Red FM fluorescence was excited with a 643 nm laser and 25 ms exposure time and collected with an ET 700/75 emission filter set. GFP fluorescence was excited with a 491 nm laser line and 100 ms exposure time and collected with an ET 525/50 emission filter set. For time-lapse experiments, images were collected every 30 s. At each time point, 30 z-series optical slices were obtained with a step size of 1.0 μm using an ASI MS-2000 piezo stage.
For each cell fluorescence intensities were systematically quantified both spatially (linescan from cell pole to cell equator; all four quadrants) and at five representative phases of division (metaphase to late cytokinesis) as described previously  with the following modifications: analysis was performed on average z-stack projections and a 40 pixel-wide linescan was used to measure average fluorescence intensity. Fluorescence intensity measurements from multiple cells were normalized and averaged using custom algorithms in MATLAB (mathworks) and transferred to GraphPad (Prism) for graph plotting. This analysis scheme allows for the direct and statistical comparison of average fluorescence intensity between division stages or culture conditions.
Linescan data were also used to calculate mitochondria and F-actin equator: pole fluorescence intensity ratios. Briefly, ratios were calculated using the first five (pole) and last five (equator) data points of each linescan at 30-s intervals from metaphase exit (t = 0 min) to late anaphase (t = 4.5 min) and then averaged across multiple cells.
The brightness, contrast and background were adjusted using Metamorph software (Molecular Devices). In order to aid the visualization of weakly fluorescent astral microtubules from live imaging data (Fig. 1a), the gamma was altered for presentation purposes, but was left unchanged for analysis.
Paired F-tests were used to compare mitochondrial distributions at five representative division stages in control cells versus KIF5BTail- or Miro-1ΔTM-expressing cells and DMSO- versus C3-treated cells. Mitochondria and F-actin distributions were also compared at five representative division stages using paired F-tests. All analyses were performed using GraphPad Software version 6.0 for Mac. Significance was set at p < 0.05.
reactive oxygen species
mitochondrial Rho GTPase
structured illumination microscopy
EL carried out the confocal and SIM experiments, participated in the design of the study, performed the statistical analyses and helped draft the manuscript. EB participated in the analyses and helped draft the manuscript. CM participated in the design of the study and helped draft the manuscript. All authors read and approved the final manuscript.
We are grateful to Casper Hoogenraad for generously providing the KIF5BTail cDNA and Michel Cayouette for the pCIG2 vector. We also thank Gregory Twork for the custom Matlab scripts used in the quantitative analysis.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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