principles of the mitochondrial fusion and fission cycle...
TRANSCRIPT
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Principles of the mitochondrial fusion and fission cycle in neurons
Michal Cagalinec1, Dzhamilja Safiulina1, Mailis Liiv1, Joanna Liiv1, Vinay Choubey1,
Przemyslaw Wareski1, Vladimir Veksler1,2,3, Allen Kaasik1
1Department of Pharmacology, Centre of Excellence for Translational Medicine, University of
Tartu, Ravila 19, Tartu, Estonia,
2INSERM, U-769, Châtenay-Malabry F-92296, France,
3Univ Paris-Sud, Châtenay-Malabry F-92296, France.
Running title: Principles of the mitochondrial fusion and fission cycle
To whom correspondence should be addressed: Allen Kaasik, Department of Pharmacology,
Centre of Excellence for Translational Medicine, University of Tartu, Ravila 19, Tartu, Estonia.
Tel: 372 7374361; Fax: 372 7374352; Email: [email protected]
© 2012. Published by The Company of Biologists Ltd.Jo
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JCS Advance Online Article. Posted on 22 March 2013
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Abstract
Mitochondrial fusion-fission dynamics play a crucial role in many important cell processes.
These dynamics control mitochondrial morphology, which in turn influences several important
mitochondrial properties including mitochondrial bioenergetics and quality control, and it
appears to be affected in several neurodegenerative diseases. However, an integrated and
quantitative understanding of how fusion-fission dynamics controls mitochondrial morphology
has not yet been described. Here, we took advantage of modern visualisation techniques to
provide a clear explanation of how fusion and fission correlate with mitochondrial length and
motility in neurons.
Our main findings demonstrate that: 1) the probability of a single mitochondrion fissing
is determined by its length; 2) the probability of a single mitochondrion fusing is determined
primarily by its motility; 3) the fusion and fission cycle is driven by changes in mitochondrial
length and deviations from this cycle serves as a corrective mechanism to avoid extreme
mitochondrial length; 4) impaired mitochondrial motility in neurons overexpressing 120Q Htt or
Tau suppresses mitochondrial fusion and leads to mitochondrial shortening whereas stimulation
of mitochondrial motility by overexpressing Miro-1 restores mitochondrial fusion rates and
sizes.
Taken together, our results provide a novel insight into the complex crosstalk between
different processes involved in mitochondrial dynamics. This knowledge will increase
understanding of the dynamic mitochondrial functions in cells and in particular, the pathogenesis
of mitochondrial-related neurodegenerative diseases.
Keywords: mitochondrial dynamics, mitochondrial fusion, neurons, neurodegeneration.
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INTRODUCTION
Mitochondria are dynamic organelles that constantly fuse with each other and then split apart
(i.e., undergo fission). Fusion serves to mix and unify the mitochondrial compartment whereas
fission generates morphologically and functionally distinct organelles.
The balance of these two processes determines organelle shape, size and number and is
critical for organelle distribution and bioenergetics. The latter is particularly important in
neurons, which have a unique bioenergetic profile due to their dependence upon energy from
mitochondria and their specialised, compartmentalised energy needs. Beyond the control of
morphology, the mitochondrial fusion-fission cycle appears to be also critical in regulating cell
death and mitophagy. Mitochondrial fission contributes to quality control by favouring removal
of damaged mitochondria via mitophagy (Gomes, et al., 2011) and may facilitate apoptosis in
conditions of cellular stress (Suen, et al., 2008). Failure of mitochondrial fusion-fission dynamics
has been linked to several diseases. Perturbations of the mitochondrial fusion-fission cycle cause
autosomal dominant optic atrophy and Charcot-Marie-Tooth type 2A and appear to be involved
in the pathogenesis of several neurodegenerative diseases (Westermann, 2010).
The molecular mechanisms underlying mitochondrial fusion and fission events are
relatively well-known. The key molecules responsible for fusion are Mitofusin-1 and -2 (Mfn1
and 2, respectively), which are located in the outer mitochondrial membrane, and OPA1, which
is located in the inner mitochondrial membrane. The outer membrane protein Fis1 and the
cytoplasmic protein Drp1 are responsible for fission events (for a review, see Knott et al., 2008).
Recent papers have also revealed interplay between fusion and fission; in most cases, they
appear to be sequential, cycle-forming events (Twig et al., 2008; Wang 2012). Nevertheless,
despite these advances, there remains no clear understanding of how mitochondrial fusion and
fission events interact at the cellular systems level. If the fusion and fission events are cyclic then
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how do they sense each other? How do the fusion or fission events sense mitochondrial size? Do
these events have feedback mechanisms to maintain the size of the mitochondrial population? To
what extent do they depend upon mitochondrial motility or can they regulate mitochondrial
motility themselves? Perhaps most relevantly, to what extent is this network of events affected in
neurons known to be most vulnerable to mitochondrial impairment?
To answer to these questions, using living neurons, we examined the dynamics in
morphology of mitochondrial populations together with fusion and fission characteristics. We
reveal key rules governing mitochondrial dynamics and show the role that mitochondrial length
and motility play in the feedback that regulates mitochondrial homogeneity. Finally, we show
that in models of neurodegenerative diseases, we can rescue an impaired mitochondrial
phenotype by correcting mitochondrial motility.
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MATERIALS AND METHODS
Neuronal culture. Primary cultures of rat cortical cells were prepared from neonatal Wistar rats.
Briefly, cortices were dissected in ice-cold Krebs-Ringer solution (135 mM NaCl, 5 mM KCl, 1
mM MgSO4, 0.4 mM K2HPO2, 15 mM glucose and 20 mM HEPES, pH = 7.4) containing 0.3%
BSA and then trypsinised in 0.8% trypsin for 10 min at 37°C. The cells were then triturated in a
0.008% DNase solution containing 0.05% soybean trypsin inhibitor. Cells were resuspended in
Basal Medium Eagle with Earle’s Salts (BME) containing 10% heat-inactivated FBS, 25 mM
KCl, 2 mM glutamine, and 100 µg/ml gentamicin and then plated onto 35 mm glass-bottom
dishes (MatTek, MA, USA), which were pre-coated with poly-L-lysine, at a density of
approximately 106 cells/ml (2 ml of cell suspension per dish). After incubating for 3 h, the
medium was changed to NeurobasalTMA medium containing B-27 supplement, 2 mM
GlutaMAXTM-I, and 100 µg/ml gentamicin. Over 60% of the cells showed a neuronal
phenotype when the described procedure was followed.
To prepare primary cultures of cerebellar granule cells, the cerebella from 8 day-old
Wistar rats were dissociated by trypsinising in 0.25% trypsin at 35°C for 15 min followed by
trituration in a 0.004% DNase solution containing 0.05% soybean trypsin inhibitor. Cells were
resuspended in BME containing 10% FBS, 25 mM KCl, 2 mM glutamine, and 100 µg/ml
gentamicin. Neurons were plated onto 35 mm glass-bottom dishes that were pre-coated with
poly-L-lysine at a density of 1.3 x 106 cells/ml. Ten µM cytosine arabinoside was added 24 h
after plating to prevent the proliferation of glial cells.
The PC12 cell line was maintained in RPMI medium supplemented with 10% horse
serum and 5% FBS on collagen IV (Sigma) -coated plastic dishes. All of the culture media and
supplements were obtained from Invitrogen (Carlsbad, CA, USA).
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Plasmids. Mitochondrial-KikGR1 (mito-KikGR1) was constructed by PCR amplification of the
mitochondrial targeting signal from pdsRed2-Mito (Human COXVIIIa, Nucleotides 597-683)
and cloning in-frame between the EcoRI and HindIII sites of the Kikume vector, which was
obtained from Amalgaam Co. (Tokyo, Japan). The plasmid expressing mito-CFP was obtained
from Evrogen (Moscow, Russia), and mitochondrial-pDsRed2 was obtained from Clontech (CA,
USA). Plasmids expressing shRNA targeted against Drp1, Miro1, and Miro2 were from
SABiosciences (Frederick, MD, USA). Wild-type (wt) and dominant negative Mfn2ΔTM were
generous gifts from Dr. S. Hirose (Honda et al., 2005), Drp1 was from Dr. G. Szabadkai
(Szabadkai et al., 2004), Fis1 was from Dr. J-C. Martinou (Mattenberger et al., 2003), Map2c-
EGFP was from Dr. A. Matus (Kaech et al., 1996), Miro1 was from Dr. P. Aspenström
(Fransson et al., 2003), Tau was from Dr. G. Johnson (Krishnamurthy and Johnson, 2004), and
mutant Htt was from Dr. L. Hasholt (Hasholt et al., 2003).
Transfections. Cultures were transiently transfected on the 2nd day after plating using
LipofectamineTM 2000 (Invitrogen). Briefly, conditioned medium was collected and 120 µl of
Opti-MEM® I medium containing 2% LipofectamineTM 2000 and 1 µg of total DNA (1 µg of
mito-KikumeGR1 in the control; 0.33 µg of mito-KikumeGR1 and 0.67 µg of the desired DNA
in the other groups) were added directly to cells grown on 35 mm glass-bottom dishes, and the
cells were then incubated for 3 h at 37°C in a humidified atmosphere containing 5% CO2/95%
air. At the end of this incubation, 2 ml of fresh (or conditioned medium for granular neurons)
NeurobasalTM medium was added per dish. The cells were then cultured for 3-4 d to enable
expression of the transfected DNA with the exception of 120Q Htt (4-5 d), Tau wt (5-6 d), and
Miro shRNA (8 d). During acquisition, the cells were maintained in Krebs-Ringer solution
supplemented with calcium (1 mM) and glucose (15 mM).
Immunohistochemistry. Neurons were fixed using 4% paraformaldehyde solution in
NeurobasalTM-A containing 5% sucrose for 10 min at 37°C. Fixed cells were permeabilised
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using 0.3% Triton in PBS for 5 min and then blocked using 10% normal goat serum and 3%
BSA for 60 min at room temperature. The neurons were then incubated with the primary
antibodies mouse anti-FLAG (1:500, F1804, Sigma-Aldrich), rabbit anti-Mfn2 (1:100, ab50838,
Abcam, USA), rabbit anti-TTC11 (1:100, ab71498, Abcam), rabbit anti-Myc (1:500, ab9106,
Abcam), mouse anti-DNML (1:250, ab56788, Abcam), rabbit anti-RHOT1 (1:50, HPA010687,
Sigma-Aldrich, Germany), mouse anti-DRPLA-35Q (1:10, MW2, Developmental Studies
Hybridoma Bank at the University of Iowa, USA), or mouse anti-Tau (1:50, 5A6,
Developmental Studies Hybridoma Bank) in the presence of 3% normal goat serum at 4°C for 48
h. After washing, the cells were further incubated with respective Alexa 488- or 594-conjugated
secondary antibodies at room temperature for 1 h and subsequently examined using confocal
microscopy.
Western blotting. For western blotting, cells were lysed in a buffer containing 50 mM Tris-Cl, 1
mM EDTA, 150 mM NaCl, 1% NP-40, 1 mM Na3VO4, 1 mM NaF, 0.25% sodium
deoxycholate, and 5% protease inhibitor cocktail (Roche) for 30 min on ice. Equivalent amounts
of total protein were separated by SDS-PAGE on 10% polyacrylamide gels and then transferred
to Hybond-P PVDF transfer membranes (Amersham Biosciences, UK) in 0.1 M Tris-base, 0.192
M glycine, and 10% (v⁄v) methanol. The membranes were blocked with 5% (w⁄v) non-fat dried
milk in TBS containing 0.1% (v⁄v) Tween-20 at room temperature for 1 h and then probed
overnight with mouse monoclonal anti-β-actin (1:4000, Sigma), mouse monoclonal anti-DNM1L
(1:2000, Abcam), mouse monoclonal anti-RHOT1 (1:2000, Abcam), or rabbit polyclonal anti-
RHOT2 (1:1000, ProteinTech Group Inc.). The membranes were then incubated with appropriate
HRP-conjugated secondary antibodies (1:4000, Pierce, USA) for 1 h at room temperature.
Immunoreactive bands were detected by enhanced chemiluminescence (ECL, Amersham
Biosciences, UK) using medical x-ray film blue (Agfa, Belgium). The probed blots were
analysed by densitometry using MicroImage software (Media Cybernetics, Bethesda, MD).
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Image acquisition. A laser scanning confocal microscope (LSM 510 Duo, Zeiss) equipped with a
LCI Plan-Neofluar 63x/1.3 immersion-corrected DIC M27 objective was used in this study. The
temperature was maintained at 37°C using a climate chamber. Mitochondrial fusion and fission
events were followed using photoconvertable mitochondrial-targeted mito-KikGR1.
Mitochondria labelled with mito-KikGR1 were first visualised using a 488-nm Argon laser line
and BP 505-550 emission filters. Selected green-emitting mitochondria were then
photoconverted to red using a 405-nm Diode laser (50 mW, 1.5% power, 20 iterations). For the
images, which typically comprised 10 - 20 mitochondria per neurite, four to five mitochondria
were photoactivated using two separate bleaching regions to facilitate detection of fusion events.
All mitochondria were then illuminated using the 488 nm Argon laser (for green mitochondria;
30 mW, 1.5% power) and a 561-nm DPSS laser (for red mitochondria; 15 mW, 7% power).
Images were acquired simultaneously to avoid movement distortions during scanning using a BP
505-550 and LP575 filters to separate green- and red-emitting mitochondria, respectively.
For cycle analysis, images were taken every 10 s for 2 h. To study the fusion events, fast
time-lapse experiments were conducted where images taken in ~250 ms intervals were acquired
over 10 mins. To compare the different transfection conditions, images were taken at 10-s
intervals for 10 min. For colocalisation studies of mito- KikGR1 and mito-CFP, mito-CFP
fluorescence after excitation with a 458-nm Argon laser line (1% power) was split using HFT458
and then separated using a BP465-510 filter.
Image processing and analysis. The fate of all photoactivated mitochondria was followed
throughout the time-lapse and the fusion and fission events recorded. Changes in mitochondrial
lengths were measured using LSM5 Duo version 4.2 software (Carl Zeiss MicroImaging Gmbh,
and EMBL Heidelberg, Germany). The resulting database was then used to analyse fusion and
fission cycle parameters, length dependency, and for twin analysis. Mitochondria were further
tracked for motility analysis using Retrack version 2.10.05 (freeware provided by Nick Carter).
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In experiments that compared different conditions, 4 fields per dish were imaged and 4
dishes per condition were used, and all experiments were performed in at least duplicate. Thus,
the mitochondrial population presented always originates from at least 32 different neurons. The
number of fusions, fissions, and contacts with other mitochondria of an photoactivated
mitochondrion was summarised per dish and then averaged over 8-24 dishes. To compare the
mitochondrial velocities, 10-20 mitochondria per neurite (including non-activated mitochondria)
were tracked and the dish medians averaged.
Length analysis was performed using MicroImage software (Media Cybernetics,
Bethesda, MD, USA) and also included other non-activated mitochondria from studied axons.
The presented images and videos were 2D deblurred and deconvoluted using the AutoDeblur and
Autovisualise X software package (Media Cybernetics Inc, Bethesda, MD, USA).
Statistics. The D'Agostino-Pearson omnibus test was used to test for normality. The Mann-
Whitney U test, Student’s t-test, one-way ANOVAs followed by Newman-Keuls posthoc tests or
Kruskal-Wallis tests followed by Dunn’s test were used to compare differences between
experimental and control groups. Correlations were calculated using either the Spearman or
Pearson tests. The Χ2 test was used to determine whether the observed distribution was
significantly different from the expected distribution. P values <0.05 were considered
statistically significant.
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RESULTS
Visualisation of fusion and fission in neurons. For visualisation of fusion and fission events, we
used mitochondrially targeted KikGR1 protein. Mitochondrial localisation of this protein was
confirmed by its colocalisation with the well-known mitochondrial markers mito-CFP (Figure
1A) or mito-pDsRED2 (data not shown). Mito-KikGR1 demonstrated a relatively high
mitochondrial/cytoplasmic intensity ratio (> 30). The photoconversion properties of KikGR1
remained unaltered after mitochondrial targeting.
The fusion events between red (photoactivated) and green (non-activated) mitochondria
were easily recognizable because the contacting mitochondria turn yellow after exchanging their
matrices (Figure 1B, Video 1). The exchange of matrix occurred quickly and to completion and
typically, was complete in less than 10 s. Fission events occurred where mitochondria split and
the daughter mitochondria moved away from each other. During the 2 h observation period, we
detected several (up to 9) sequential fusion or fission events of photoactivated mitochondria and
their progenies.
Fusion and fission events are cyclic. Mitochondrial fusion and fission rates were estimated in
axons of two morphologically distinct subtypes of neurons: cortical neurons and cerebellar
granule neurons. In both types of cell, the fusion rate matched the fission rate perfectly although
in cortical neurons, these rates (0.023 ± 0.003 fusions/mitochondria/min and 0.023 ± 0.003
fissions/mitochondria/min; n = 17 dishes) were lower than in cerebellar granule neurons (0.045 ±
0.006 fusions/mitochondria/min, p = 0.001, and 0.039 ± 0.005 fissions/mitochondria/min, p =
0.0072; n = 12 dishes).
We also attempted to measure the mitochondrial fusion rate in the soma of cortical
neurons. However, mitochondria are packed very densely in the soma, and in the majority of
cases, the laser beam photoactivated mitochondria located in the vicinity of the mitochondrion of
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interest. Nevertheless, we estimated the fusion rate in soma of cortical neurons, which was
significantly higher (0.149 ± 0.021 fusion/mitochondria/min, n = 10; p < 0.0001) than in axons.
Our next aim was to determine whether the fusion and fission events occurred randomly
or in a regulated manner (for example, cyclic, as suggested by Twig et al., 2008). Theoretically,
each fusion could be followed by fission or by a secondary fusion, or similarly, each fission
could be followed by fusion or a secondary fission (Figure 1C). Analysis of the combinations of
these events demonstrated that in cortical neurons, a fusion was followed by a fission in 86.4%
of cases and by a second fusion in only 13.6% of the cases. In addition, fission was followed by
fusion in 83.5% of cases and by a second fission in 16.5% of cases. We performed the same
analysis using cerebellar granule neurons and obtained similar results (Figure 1C). These data
suggest that fusion and fission events are sequential events that form a cycle rather than
independent, randomly occurring events. However, an alternating fusion-fission cycle was not
consistently observed, and there were deviations from this rule (fission-fission or fusion-fusion
events) in 15% of the cortical neurons and 24% of the cerebellar granule neurons.
We next measured the duration of the two components of the fusion-fission cycle in
cortical neurons. The mean time interval between fusion and fission was 4.7 ± 0.6 min, which
was significantly shorter than the interval between fission and the next fusion (15.3 ± 2.0 min; p
< 0.0001, Mann-Whitney U test). The duration of the entire cycle was approximately 20 min. It
should be noted that this analysis was only applied to the active subpopulation of mitochondria,
which may explain why the cycle duration found here was shorter than that calculated from the
fusion or fission rates of the total mitochondrial population (approximately 43 min).
Nevertheless, these values suggest that mitochondria spend approximately ¼ of their time in
between fusion and fission events and ¾ of their time in between fission and the next fusion
event.
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Rules governing mitochondrial fusion and fission. Fusions almost always occurred between
stationary (or passive) and moving (or active) mitochondria. Indeed, of 34 fusion events
analysed, 28 events (82%) showed clearly recognisable active and passive partners, 3 events
(9%) occurred between two active mitochondria, and 3 events (9%) occurred between two
passive mitochondria.
To further study how the mitochondria are juxtaposed at the beginning of fusion, we
performed fast time-course experiments with 250-ms intervals between frames. This approach
enabled accurate examination of the exact positions of two mitochondria relative to each other.
Although we expected that the head of the active partner would collide with the passive partner
to merge the matrices of each mitochondrion, this movement was not common. Such head-on
fusions were only observed in 2% of all cases (2 of 89 analysed events) whereas in most cases,
the active partner passed the passive partner, which lead to side-to-side fusions (43%) or rear-end
fusions (55%). It should be mentioned that the moving head passed the body of the passive
mitochondrion in 88% of all cases (68 out of 77 analysed events) and remained behind the head
of the passive mitochondrion in only 12% of cases (p < 0.0001, Χ2 test).
This complicated behaviour of fusing mitochondria may be related to the localisation
and/or activity of fusion-controlling proteins. We investigated the distribution of Mfn2 in
mitochondria and found that its localization was indeed focal and non-homogenous and localized
to specific spots (Figure 3A; Karbowski et al., 2006).
The passing process itself was often sophisticated and comprised numerous stops
including nudging, turn-aways, and unexpected returns that ended with fusion. The videos show
two examples (Video 2 and 3) where after the initial contact, the active partner moved away
from the passive one and then turned back to make the final contact. The duration of these
dances was very variable with some interactions being very short and others lasting up to a few
minutes (mean value 55 ± 4 s, n = 171). The dance ended with a firm grip between the partners
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that lasted between 10-80 s (mean value 58 ± 4 s, n = 172) and ended with an exchange of
matrix. The duration of the grip appeared to be controlled by proteins responsible for fusion
because in neurons transfected with a dominant negative Mfn2, this parameter was prolonged
(100 ± 14 s, p = 0.001 compared with control).
When fused mitochondria subsequently underwent fission, in most cases, one of the
resulting daughters remained passive whereas the other moved away. Interestingly, the active
daughter almost always followed the same direction as the active parent had. We analysed 43
such events, and in 41 cases, the active daughter continued in the same direction, and in only 2
cases, the direction was reversed (p < 0.0001, Χ2test). These results suggest that the “moving
head” of active mitochondria is conserved before fusion and moves in the same direction after
fission.
Fission rate is length dependent. Both fusion and fission change mitochondrial length. In
consideration of the alternating nature of these events, it is reasonable to suggest that the
negative feedback(s) controlling the fusion and/or fission rates involves mitochondrial length. To
assess this hypothesis, we performed a “twin study”, where daughter mitochondria originating
from the same parent mitochondrion were compared. We tracked 30 “families” up to the moment
when one of the daughters underwent a second fission. The results demonstrated that the average
length of the daughter undergoing a second fission was greater than twice the length of its
nonfissing twin (Figure 2A). In addition, we followed 42 pairs of twins until one of the daughters
fused. The average length of the fusing daughter matched perfectly the length of the non-fusing
daughter (Figure 2B).
These results suggest that longer mitochondria enter fission more readily. To confirm this
conclusion, we next divided the total population of mitochondria in cortical and cerebellar
granule neurons based on their length and determined the fission and fusion rates for each
subgroup. Figure 2C demonstrates that in both neuron types, the fission rate was very low in
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short mitochondria; however, the fission rate increased dramatically in long mitochondria.
Therefore, in contrast to the fission rate, the fusion rate appears to be relatively independent of
length.
Globally, the fusion rate exceeded the fission rate in shorter mitochondria whereas the
fission rate exceeded the fusion rate in longer mitochondria. Thus, the fusion-fission balance
shifted towards fusion in shorter organelles and towards fission in longer organelles. Therefore, a
shorter mitochondrial length decreases the probability of fission being the next event and
consequently increases the probability of fusion (Supplementary Table 1). This finding suggests
that the mitochondria of greatest length after fission are more likely to undergo secondary,
“corrective” fission. In contrast, post-fusion mitochondria that are too short are more likely to
undergo secondary fusion. We analysed particular cases to determine whether these “errors”
indeed correlated with mitochondrial size. The results depicted in Figure 3D and E show that
post-fission mitochondria that entered an “erroneous” cycle-breaking second fission were
significantly longer than “normal” mitochondria that entered fusion. In agreement with this
observation, post-fusion mitochondria that entered an “erroneous” second fusion were
significantly shorter that “normal” mitochondria that entered fission. Thus, this type of feedback
mechanism may serve as a quality control mechanism to correct mitochondria that are
excessively short or long.
The next important question concerned the mechanism underlying the fission rate-length
relationship. This relationship was quite steep (Figure 2C), and its non-linearity suggested a
cooperative interaction between factors/molecules responsible for fission provided that the
number of these molecules increased with mitochondrial length. To determine the putative role
of Drp1 in the fission rate-length relationship, we modified Drp1 expression in cortical neurons.
As expected, overexpression of Drp1 together with Fis1 led to mitochondrial shortening whereas
Drp1 silencing was associated with dramatic mitochondrial elongation (Figure 3). Interestingly,
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neither Drp1 silencing nor Drp1 and Fis1 overexpression led to consistent changes in fission or
fusion rates. Analysis of the fission rate-length relationship demonstrated that Drp1 and Fis1
overexpression increased the sensitivity of the fission rate to mitochondrial length whereas Drp1
silencing (using specific shRNAs) led to a dramatic drop in this sensitivity (Figure 4A). The role
of Drp1 was specific because no change was observed following the overexpression of wild-type
(wt) Mfn2 or dominant negative Mfn2.
Impairment of the feedback mechanism in Drp1-suppressed neurons was associated with
high variability in mitochondrial length. The coefficient of variation for length was considerably
higher in Drp1 shRNA-expressing neurons compared with control (Figure 4B). Importantly,
Mfn2 overexpression-mediated elongation of mitochondria did not affect length variability.
Together, these results suggest that the length-fission feedback loop is Drp1-dependent.
Fission rate adapts itself to the fusion rate. The efficiency of this feedback control mechanism
was assessed in experiments that studied the adaptation of the fission rate to a switched fusion
rate and vice versa. We first overexpressed wt Mfn2 and found that it induced a 76% increase in
mitochondrial fusion rate (Figure 3E). This finding was accompanied by a 68% increase in
fission rate but a relatively modest 36% increase in mitochondrial length. Because Mfn2 has also
been implicated in mitochondrial transport (Misko et al., 2010), we also performed a separate
experiment that showed that overexpression of Mfn1 induced similar effects to those of Mfn2
(Figure 3E). Inversely, inhibition of the mitochondrial fusion rate by overexpressing dominant
negative Mfn2 decreased the fusion rate to half that of the control level. This effect was
accompanied by almost perfectly matched changes in the fission rate and a 29% decrease in
mitochondrial length. These results demonstrate that the fission rate adapts to the fusion rate; i.e.,
augmented or decreased fusion rates favour the formation of longer or shorter mitochondria, that
are either more or less likely to enter fission, respectively.
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Fusion depends on the contact rate. Fusion can only take place when two mitochondria meet.
However, not every contact results in fusion. In cortical neurons, of 1187 analysed mitochondria
from 66 separate fields, the axonal mitochondria made an average of 0.45 ± 0.02 contacts per
mitochondrion per min, and only 7.0 ± 0.4% resulted in fusion. However, in cerebellar neurons,
axonal mitochondrial made an average of 0.24 ± 0.03 contacts per mitochondrion, of which 20%
resulted in fusion. Higher fusion efficiency in cerebellar neurons may be due to the 1.65-fold
increase in Mfn2 expression (p = 0.014) that was observed in these cells compared with cortical
neurons.
We measured the average number of contacts made by mitochondria and the number of
fusions in a further 232 neurites. Not surprisingly, the number of fusions correlated well with the
number of contacts (Spearman r = 0.33, p < 0.0001), and a higher number of contacts increased
the likelihood of fusion.
Apparently, the number of contacts depends upon the number of partners available, and
the probability of a mitochondrion meeting another is higher in crowded neurites. We compared
the contact rate in different axonal regions with different mitochondrial densities. As expected,
an increase in the density of mitochondria was associated with an increase in contact rate
(Spearman r = 0.144, p = 0.020).
Motility determines fusion rate. It is clear that mitochondrial partners must approach each other
for a contact to occur, and therefore, a contact should depend upon movement characteristics.
Therefore, we measured the motility of individual mitochondria and the number of contacts they
made. As expected, increased motility was associated with an increase in contact rate (Spearman
r = 0.482, p < 0.0001). To confirm the relationship between the motility and fusion rate, we
analysed the velocity of twin mitochondria where only one of the daughters fused. The results
showed that the fusing daughter had a significantly higher velocity than its non-fusing twin
(Figure 5A). In additional experiments, we inhibited mitochondrial motility by suppressing Miro
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proteins, which are responsible for mitochondrial attachment to molecular motors. Suppression
of these proteins led to a proportional inhibition of mitochondrial velocity and contact rate,
which in turn was associated with a reduction in fusion rate (Figure 5B-F). These results showed
that motility, at least in part, determines the rate of fusion (Figure 5B-F). Inhibition of
mitochondrial motility by Miro shRNA also inhibited fusion and decreased the average
mitochondrial length (Figure 5G), an effect that was similar to the effect of dominant negative
Mfn2. Similarly, overexpression of the axonal docking protein syntaphilin inhibited
mitochondrial motility and fusion rate (data not shown).
Mitochondrial motility during the fusion-fission cycle. Considering that mitochondrial velocity is
an important determinant of mitochondrial fusion, we next studied the velocity of single
mitochondria during the course of a fusion-fission cycle. We first traced the velocity of those
mitochondria that later entered fusion. Figure 6A and B show that at approximately 10 min
before a fusion event, both the active and passive partners had the same mean low velocity.
However, later, the velocity of the active partner increased progressively up to the moment of
fusion whereas no change was observed in the velocity of the passive partner. Remarkably, the
fusion event induced a dramatic drop in mitochondrial motility, and the fusion product was
relatively immobile compared with the active parent. In contrast to the pre-fusion state, no
change in mitochondrial velocity was observed in the pre-fission state. Interestingly, after
fission, the velocity of the active daughter that moved away was similar to that of the active
partner prior to fusion (Spearman r = 0.425 and p = 0.0062). However, the high velocity slowed
rapidly but was nevertheless higher than the velocity of the second daughter for a period of time
(Figure 6C and D). Thus, the fusion-fission cycle is associated with clear changes in the velocity
of individual mitochondria.
Fusion-fission dynamics in models of neurodegenerative diseases. Recent reports demonstrate
that overexpression of mutant Huntingtin (Trushina et al., 2004; Chang et al., 2006; Song et al.,
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2011) and Tau (Ebneth et al. 1998) impair mitochondrial dynamics. To test whether the fusion
and fission dynamics are altered in these disease models, we first overexpressed the 1st exon of
mutant Huntingtin, containing 120 polyglutamines (120Q-Htt), in cortical neurons. As expected,
expression of this construct led to a decrease (by 40 ± 7%) in average mitochondrial velocity and
a concomitant reduction (by 35 ± 9%) in the number of contacts between mitochondria.
Importantly, these changes were associated with decrease in fusion and fission rates (by 57 ± 6%
and 46 ± 10%, respectively). Similar to the reduced motility induced by suppression of Miro
protein, the reduced fusion rate was related to mitochondrial shortening (Figure 7).
To test the causal relationship between reduced motility and fragmentation, we next tried
to increase the motility in 120Q-Htt-expressing neurons using Miro-1. The results depicted in
Figure 7 demonstrate that Miro-1 overexpression restored mitochondrial velocity completely in
addition to the rate of mitochondrial contacts. These changes were followed by a recovery in
fusion rate and an amelioration of the mitochondrial fragmented phenotype.
Finally, we overexpressed Tau protein, which is also known to inhibit mitochondrial
motility in neurons. Similar to the results obtained following 120Q-Htt overexpression, Tau
overexpression reduced mitochondrial motility and led to a decreased contact rate. It was also
associated with a decrease in fusion rate that led to mitochondrial shortening. The co-expression
of Miro-1 and Tau improved mitochondrial motility, contact, and fusion rates and rescued the
mitochondrial phenotypes (Figure 7).
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DISCUSSION
Mitochondrial fission and fusion are often viewed as being in a finely-tuned balance within cells;
however, an integrated and quantitative understanding of how these processes interact with each
other and with mitochondrial motility and morphology has yet to be formulated. Our main
findings demonstrate that:
• the probability of a single mitochondrion fissing is determined by its length;
• the probability of a single mitochondrion fusing is determined primarily by its motility;
• the fusion and fission cycle is driven by changes in mitochondrial length – an increase in
mitochondrial length after fusion increases the probability of fission whereas a decrease in
mitochondrial length after fission reduces this probability;
• deviations from the fusion and fission cycle serve as a corrective mechanism to avoid extreme
mitochondrial length;
• impaired mitochondrial motility in neurons overexpressing 120Q Htt or Tau suppresses
mitochondrial fusions and leads to mitochondrial shortening; stimulation of mitochondrial
motility by overexpressing Miro-1 restores mitochondrial fusion rates and sizes under these
conditions.
Self-regulation of mitochondrial length. Our major novel finding was that mitochondrial length
controls the rate of mitochondrial fission. Longer mitochondria enter fission more readily than
shorter mitochondria. For example, a 5-µm mitochondrion showed a 10-fold higher fission rate
than a 2-µm mitochondrion. Similar results were also observed in our “twin study“ that
minimised the involvement of other factors, such as different DNA or protein composition, or
cytoplasmic environment, which may affect the fission rate independent of length. Regardless of
the molecular mechanism underlying the observed length-fission relationship, this phenomenon
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appears to control mitochondrial length in neurons and also appears to be the main feedback tool
enabling neurons to sense and correct mitochondrial length.
This type of control also explains why the fission rate is related to the fusion rate such
that these two rates are balanced perfectly. None of the interventions used in our experiments
changed the fusion rate/fission rate ratio significantly. On average, mitochondria in axons of
cortical neurons underwent fusion or fission 33 times per day. The slightest imbalance (one
fusion less or more per day) should therefore eventually lead to complete mitochondrial
fragmentation or the formation of a few megamitochondria in the cell; however, this was not the
case. Overexpression of wt Mfn2 doubled the fusion and fission rates and led to only a slight
increase in mitochondrial length. It is most likely that increased fusion activity induced a slight
elongation in mitochondria, which in turn activated the fission machinery that attempted to block
a further increase in mitochondrial length.
In addition, these findings provide a clear explanation as to why manipulations of fission
machinery failed to change the fission rate. For example, Drp1 upregulation did not increase the
rate of fusion although it did lead to mitochondrial shortening. An attempt to increase the fission
rate and thereby shorten mitochondria is likely to be counterbalanced by an inhibition of the
fission machinery by that same mitochondrial shortening. Suppression of the fission rate by
inhibition of Drp1 induced the opposite effect, which leads to mitochondrial elongation that in
turn activates fission machinery. Mitochondrial length is thus self-regulated via Drp1-dependent
fission.
Fusion rate is determined by the availability of partners – “it takes two to tango”. Fusion can
only take place when two mitochondria meet. The number of contacts a mitochondrion makes
depends on its motility and the number of mitochondria in the vicinity. Higher motility increases
the likelihood of a mitochondrion finding a partner and consequently the fusion rate. In turn,
lower motility decreases the number of contacts and the fusion rate. The latter conclusion is
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consistent with previous findings that inhibition of mitochondrial movement by nocodazol and
vasopressin in H9c2 cells also inhibited mitochondrial fusion (Liu et al., 2009). Similarly, a
recent study by Twig et al. (2010) suggested that mitochondrial motility facilitates mitochondrial
fusion in H9c2 and INS1 cells. Our results support this hypothesis with quantitative data
demonstrating that mitochondria entering fusion show two-fold higher motility compared with
their nonfusing sisters. Moreover, our results demonstrate that direct slowing of mitochondrial
motility by suppressing mitochondrial Miro proteins (required for mitochondrial antero- and
retrograde transport (Russo et al., 2009)) or by overexpressing the axonal docking protein
syntaphilin inhibit the rate of mitochondrial fusion.
Obviously, the interaction between mitochondrial motility and fusion/fission dynamics is
rather sophisticated. The velocity of mitochondria that are about to fuse begins to increase
several minutes prior to the fusion and increases progressively to the point of fusion. It is unclear
whether this increase in motility reflects an intrinsic “intention to fuse” or whether the fusion is a
simple consequence of the higher motility. Recent research has demonstrated that Mfn2 interacts
with Miro-2 protein and is required for mitochondrial transport (Misko et al., 2010), which
suggests that the Mfn-Miro interaction is used by mitochondria to inform the transport
machinery of the readiness to fuse.
It is also relevant to note that after the fusion event, the new mitochondrion remains
relatively immobile. This cannot be explained by its increased length because we did not observe
any correlation between mitochondrial length and motility. It is more likely that passive
mitochondria are anchored so strongly that they will also hold the moving mitochondria.
However, after fission, the mitochondria that formed from a previously active segment move
away and slow down. Thus, the velocity of individual mitochondria changes with the phase of
the fusion/fission cycle (Figure 6).
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Another factor that may increase the mitochondrial fusion rate is the number of
surrounding mitochondria. For example, the fusion rate in a neuronal soma, which shows a
higher mitochondrial density, is greater than 5-fold that of axons (where mitochondrial density is
relatively low). This finding could be one of the reasons why the fusion rate changes between
different neuronal compartments and may explain why mitochondria in the soma are 50% (1 µm)
longer than in the periphery. Nevertheless, we cannot rule out the possibility that the increased
fusion rate is also caused by the higher accessibility or activity of the fusion-fission proteins.
The fusion-fission cycle is driven by changes in mitochondrial length. In their paper, Twig et al.
(2008) elegantly demonstrated that fusion and fission, typically, are sequential events that form a
cycle. However, in a recent report, Wang et al. (2012) showed that the fusion and fission events
were not always sequential (35% of events in Hela cells and 40% of the events in MEF cells
were not cyclic). We have shown that in neurons, the fusion and fission events were also not
always sequential: 15% of events in cortical neurons and 25% in cerebellar neurons were not
cyclic. Importantly, the dependency of the fission rate on length discovered in our study provides
a very simple explanation as to why mitochondrial fusion and fission alternate and why these
events are not always cyclic. After each fusion, the mitochondrial length doubles. This doubling
causes an 8-fold increase in the probability of fission and predicts a short lifetime for the post-
fusion mitochondrion. In contrast, fission halves the mitochondrial length and therefore
diminishes the likelihood of a secondary fission and increases the probability of a fusion being
the next event. This mechanism enables alternating fusion and fission events and also explains
why mitochondria spend approximately ¼ of their time in a post-fusion state and ¾ of their time
in a post-fission state. If fusion occasionally leads to the formation of a mitochondrion that is too
short or a fission product that is too long, the cycle has been compromised. Deviations from the
cycle (two consecutive fusions or two consecutive fissions) serve as quality control mechanisms
to correct small or oversized mitochondria. This mechanism may have an important
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physiological relevance in the maintenance of optimal mitochondrial size. Mitochondria that are
too long have been shown to exhibit a compromised bioenergetic capacity (Benard et al., 2007)
and may have difficulties in delivering energy to the cell.
Mitochondrial fusion-fission dynamics in neurodegenerative diseases. Recent reports suggest
that mutant Huntingtin impairs the balance in mitochondrial fission and fusion and thereby
causes neuronal injury. Mutant Huntingtin triggers mitochondrial fragmentation in rat neurons
and fibroblasts from individuals with Huntington's disease (Song et al., 2011). Increases in Drp1
and Fis1 and decreases in Mfn1 and Mfn2 expression have been observed in cortical samples
from HD patients (Shirendeb et al., 2011).
Our experiments demonstrated that mutant Huntingtin impaired mitochondrial fission-
fusion balance by inhibiting fusion activity. These data suggest that mutant Huntingtin may also
induce mitochondrial fragmentation indirectly by inhibiting mitochondrial motility (Trushina et
al., 2004; Chang et al., 2006). This effect could inhibit the number of contacts between
mitochondria and consequently reduce the rate of fusion, which would lead to mitochondrial
shortening. Mutant Huntingtin-mediated mitochondrial fragmentation, defects in the fusion and
fission rates, and a decrease in the number of contacts were all rescued by increasing
mitochondrial motility via the overexpression of Miro1 protein. Interestingly, this effect was not
specific to mutant Huntingtin. Ebneth et al. (1998) reported that overexpression of Tau resulted
in a failure of the cell to transport mitochondria to peripheral compartments, which may be of
relevance to Alzheimer's disease. In our settings, overexpression of wt Tau suppressed
mitochondrial motility and mitochondrial fusion and induced mitochondrial fragmentation.
Similar to mutant Huntingtin, all of these parameters were restored following Miro-1
overexpression. These results suggest that motility likely plays a key role in mitochondrial
fusion-fission dynamics and morphology, and its restoration may constitute a new treatment
option for Huntington's and Alzheimer’s disease.
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Together, our results provide a novel insight into the complex crosstalk between
mitochondrial fusion and fission, length, and motility. This knowledge will provide better
understanding of the dynamic mitochondrial function in cellular physiology and the pathogenesis
of mitochondrial-related neuronal diseases.
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Acknowledgements. We thank Dr. S. Hirose, Dr. N. Nakamura, Dr. E. Bampton, Dr. J-C.
Martinou, Dr. A. van der Bliek, Dr. G. Szabadkai, Dr. A. Matus, Dr. P. Aspenström, Dr. A.
Fransson, Dr. G. Johnson, and Dr. L. Hasholt for providing the plasmids. We also thank Dr.
Miriam A. Hickey for her assistance with proofreading.
This research was supported by the institutional research founding grant (IUT2-5) from the
Estonian Research Council, grants from the Estonian Science Foundation (grant numbers 7991,
7175, 8810, and 8125), the European Community, the European Regional Development Fund,
and the Estonian-French research program Parrot. M.C. was supported by MOBILITAS
(MJD35).
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LEGENDS TO FIGURES
Figure 1. Fusion and fission are sequential events. A. Photoactivation of mito-Kikume-Green.
Mitochondria expressing Kikume-Green were co-transfected with mito-CFP and demonstrated
clear colocalisation of both proteins (left panels). Selected mitochondria were irradiated using a
405-nm laser line (marked with a yellow rectangle) and mito-Kikume-Green was converted into
mito-Kikume-Red. Note that the neighbouring mitochondria retained their green fluorescence. B.
A fusion event between mito-Kikume-Green and photoactivated, mito-Kikume-Red
mitochondria. The fusion product became yellow after mixing of the contents of the red and
green mitochondrial matrices (see also Figure S1). C. Each fusion (left) was followed by a
fission or by a secondary fusion, and each fission (right) was followed by a fusion or a secondary
fission. To determine the extent of fusion and fission events that were sequential, we analysed
the number of event pairs (111 event pairs starting with fusion and 103 starting with fission in
cortical neurons, and 63 pairs starting with fusion and 55 starting with fission in cerebellar
granule neurons). The figure shows the number of each type of event. A Χ2 test was used to
determine whether the observed distribution was significantly different from the expected
distribution.
Figure 2. Mitochondrial fission is length dependent. A,B. Mitochondrial twin analysis.
Comparison of mitochondrial length within families, where one of the daughters underwent
fission (A) or fusion (B) after the primary fission (n = 30 and 45, respectively; the Wilcoxon test
was used to calculate the p value). C. Length dependency of mitochondrial fusion (black circles)
and fission (red circles) rates. Mitochondria from cortical neurons (1277) and cerebellar granule
neurons (413) were sub-grouped based on their lengths, and the fission and fusion rates were
determined for each sub-group. D. Length analysis of the post-fission mitochondria entering
either fusion (normal cycle, n = 86) or fission (erroneous, cycle-breaking, n = 17, Mann-Whitney
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test). E. Length analysis of the post-fusion mitochondria entering either a fission (normal cycle,
n = 96) or fusion event (erroneous, cycle-breaking, n = 15, Mann-Whitney test).
Figure 3. Manipulation of fusion proteins but not fission proteins alters the fusion-fission
dynamics. A. Overexpressed Mfn-FLAG (detected using an anti-FLAG antibody, green) is
located in the ends of mito-DsRed-expressing mitochondria. B. Expression of a specific shRNA-
encoding plasmid suppressed Drp1 levels in PC12 cells by 93%. C. Overexpressed Drp1 (anti-
Dnml) demonstrates classical cytoskeletal localisation. D. Overexpressed Fis1 (anti-TTC11,
green) was homogenously distributed throughout the mitochondrial membrane (mito-DsRed). E.
Fusion and fission rates and lengths of mitochondria in control, wild-type (wt) Mfn2-, dominant
negative (dn) Mfn2-, wt Mfn1-, wt Drp1 + wt Fis 1-overexpressing and Drp1-suppressed
neurons (see also Figure S2). Data are shown as the mean ± SEM. *p < 0.05, **p < 0.01 and ***
p < 0.001 vs. control. The number of dishes analysed (for fusion and fission rate) or the number
of mitochondria analysed (for length) is shown in brackets.
Figure 4. Knockdown of Drp1 abolishes the length dependency of mitochondrial fission. A.
The length dependency of mitochondrial fission in Drp1 shRNA- (red line), Drp1- and Fis 1-
expressing (black line), and control (dotted line) neurons. Mitochondria (513) from the Drp1
shRNA group and Drp1 and Fis 1 (531) groups were analysed. B. The average coefficient of
variability of mitochondrial length in control, Drp shRNA-, and Mfn2-overexpressing neurons.
Coefficients were calculated for individual neurites (27 in control and 12 in other groups) and
then averaged. *** p < 0.001 vs. control.
Figure 5. Fusion is correlated with mitochondrial motility. A. Motility analysis of twin
mitochondria from families where one of the daughters fused during the observation time.
Columns show the mean mitochondrial motility for fusing and non-fusing daughters. B. Both
Miro-1 shRNA (left) and Miro-2 shRNA (right) effectively suppressed the expression of
endogenous Miro-s. C. Kymograms derived from videos obtained from control and Miro-
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silenced neurons. The upper panels show the starting point and the lower panels show the
movement of mitochondria (red or green) over the following 10 min. A fusion event led to the
formation of a yellow signal, and the movement of fission products is designated by the dotted
line. D,E. Parameters of mitochondrial dynamics and morphology in control and Miro-silenced
neurons. Data are shown as the mean ± SEM. *p < 0.05, **p < 0.01, and *** p < 0.001 vs.
control. See also Figure S3.
Figure 6. Mitochondrial motility changes at different stages of the mitochondrial fusion
and fission cycle. A. Mitochondrial velocity starting at 10 min prior to the fusion event up to 10
min after the fusion event. B. The mean mitochondrial velocity measured 10 min prior to the
fusion event, immediately before the fusion event and immediately after the fusion event. C. The
mitochondrial velocity from 10 min before the fission event up to 30 min after the fission event.
D. The mean mitochondrial velocity measured immediately before the fission event, immediately
after the fission event and 20 min after the fission event.
Figure 7. Miro-1 restores mitochondrial dynamics in 120Q-Htt and Tau-expressing
neurons. A. Overexpressed 120Q-Htt-EGFP co-localised with staining for an anti-Htt antibody
(red) in the cytoplasm and showed typical inclusion bodies in the nucleus (stained using DAPI,
blue). B. Overexpression of wt Tau (anti-tau 5A6, red) in neurons expressing the microtubule
marker MAP2c-EGFP. C. Overexpression of Miro-1 was confirmed using an anti-Rhot1
antibody (green). Miro-1 was distributed homogenously in mitochondria (mito-DsRed ). D. The
suppression of mitochondrial motility, kiss rate, fusion rate, and mitochondrial length in neurons
overexpressing 120Q-Htt were all reversed following the co-expression of wt Miro-1 (Htt +
Miro). E. The suppression of mitochondrial motility, kiss rate, fusion rate, and mitochondrial
length in neurons overexpressing wtTau wt was reversed following the c0-expression of wt
Miro-1 (Tau + Miro). *p < 0.05, **p < 0.01 and ***p < 0.001 compared with control and # p <
0.05, # # p<0.01, and # # # p < 0.001 compared with Htt or Tau group.
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Figure 8. Summarised principles of the feedback that controls mitochondrial length in
neurons. Mitochondrial number and motility positively correlates with the kiss rate, which in
turn, acts as a pacemaker for fusions. The fusion rate may also be affected by the expression or
activity of fusion proteins. Increases or decreases in the fusion rate in turn leads to a
corresponding increase or decrease in mitochondrial length, respectively, which controls the
fission rate. Alterations in the expression or activity of fission proteins also influences
mitochondrial length, which balances the fission rate via feedback. These controls enable the
equilibration of fusion and fission rates.
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mito-CFP
mito-Kik-Red
mito-Kik-Red
mito-Kik-Green
mito-Kik-Green
merge
merge
before activation
before fusion
after activation
after fusion
2µm
A
B
C FISSIONFUSION
Event pairs Cerebellar neuronsCortical neurons
Fusion - Fission 96
33 86
49
p < 0.0001
p < 0.0001p < 0.0001
p = 0.0017
15
12 17
14Fusion - Fusion
Fission - Fission
Fission - Fusion
2µm
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FUSION-FISSION FUSION-FUSION
FISSION-FUSION FISSION-FISSION
normal
normal
erroneous
erroneous
Len
gth
(m
m)
normal
normal
erroneous
erroneous
p = 0.000
p = 0.018
Len
gth
(m
m)
0
1
2
3
4
0
1
2
3
4
A
C
B
D
E
Len
gth
(m
m)
Len
gth
(m
m)
non- FUSING
non- FUSING
non-FISSING
non-FISSING
FUSING
FUSING
FISSING
FISSING
p = 0.92
p = 0.000
0
1
2
3
4
0
1
2
3
4
0 2 4 6 80.0
0.2
0.4
0.6
CerebellarCortical
Fu
sio
n o
r F
issio
n e
ven
t p
er
min
Fu
sio
n o
r F
issio
n e
ven
t p
er
min
0 2 4 6 8 10 120.0
0.2
0.4
0.6
0.8
1.0
1.2
FISSIONFUSION
length (mm)length (mm)
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
0.029 ± 0.002 (71) 0.028 ± 0.003 (71) 2.03 ± 0.06 (1857)
0.051 ± 0.003 (27) ***
0.057 ± 0.004 (8) ***
0.047 ± 0.002 (27) ***
0.051 ± 0.006 (8) *
2.77 ± 0.10 (282) ***
2.45 ± 0.10 (178) *
0.014 ± 0.001 (17) *** 0.015 ± 0.001 (17) ** 1.45 ± 0.04 (556) ***
0.029 ± 0.004 (13) 0.032 ± 0.004 (13) 1.31 ± 0.02 (850) ***
0.032 ± 0.003 (16) 0.025 ± 0.003 (16) 7.03 ± 0.52 (201) ***
control
fusion rate (fusion/mito/min)
fission rate (fission/mito/min)
length (µm)
wt Mfn2
wt Mfn1
dn Mfn2
wt Drp1 + wt Fis1
Drp1 shRNA
ctrl
ctrl
Drp1 shRNA
Drp1 shRNA
b-actin
Drp1
Rela
tive D
rp1/b
-act
in r
atioA
C D
B
E
0
50
100
**
2ìm
2ìm
3ìm
10ìm 10ìm
1ìm
Mnf2-FLAG Mitochondria
Fis1 MitochondriaDrp1
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
0 2 4 6 8 100.0
0.2
0.4
0.6 control
Drp1 shRNADrp1, Fis1
contr
ol
DRP s
hRNA
MFN
20.0
0.2
0.4
0.6
length (µm)
Fis
sio
n r
ate
(even
t/m
in/m
ito
)
Co
eff
icie
nt
of
vari
ab
ilit
y0.8
***
A B
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
0
5
10
contr
ol
nonFUSING
FUSING
contr
ol
contr
ol
contr
ol
Miro
shR
NAs
Miro
shR
NAs
Miro
shR
NAs
Miro
shR
NAs
0.0 0.0
0.2
0.4
0.6
0.8
Mit
och
on
dri
al
velo
cit
y (m
m/s
)
0.1
0.2
0.3
0.4
Co
nta
ct
rate
(conta
ct/m
itoch
ondria/m
in)
0.0
0.5
1.0
1.5
2.0
2.5
Mit
oc
ho
nd
ria
l le
ng
th (m
m)
0.000
0.005
0.010
0.015
0.020
5 mm
Fu
sio
n r
ate
(fusi
on/m
itoch
ondria/m
in)
D E F G
*
*
** * ***
0
0.3
0.6
0.9
1.2
Mit
och
on
dri
al velo
cit
y
(m
m/s
)
non- FUSING
FUSING
A B
b-actinb-actin
Miro-2Miro-1
Miro-2 shRNAMiro-1 shRNA
Miro-1 shRNA + Miro-2 shRNA
ctrlctrl
controlC
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
-10 -8 -6 -4 active
active
active
active
passive
passive
passive
passive
fused
fused
immediately before fusion
immediately after fission
10 min beforefusion
time after fusion (min)
time after fission (min)
Avera
ge
mit
oc
ho
nd
ria
l v
elo
cit
y (m
m/s
)
Avera
ge
mit
oc
ho
nd
ria
l v
elo
cit
y (m
m/s
)
Avera
ge m
ito
ch
on
dri
al
velo
cit
y (m
m/s
)
Avera
ge m
ito
ch
on
dri
al
velo
cit
y (m
m/s
)
20 min afterfission
-2 0
0 0 0
1 1 1
2 2 2
3 3 3
2 4 6 8 10
-10 -8 -6 -4 -2 0
0
1
2
3
fusion
fission
5 10 15 20 25 30
0
1
2
3
0
1
2
3
***
*** *** ***
* **
A B
C D
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
D
* *
*
***
###
###
cont
rol
Htt
Htt+
Miro
cont
rol
Htt
Htt+
Miro
cont
rol
cont
rol
Htt
Htt
Htt+
Miro
Htt+
Miro
0.0
0.5
1.0
Mit
och
on
dri
al velo
cit
y (m
m/s
)M
ito
ch
on
dri
al velo
cit
y (m
m/s
)
0.0
0.1
0.2
0.3
0.4
0.5
Co
nta
ct
rate
Co
nta
ct
rate
0.00
0.01
0.02
0.03
0.04
Fu
sio
n r
ate
Fu
sio
n r
ate
(fusi
on/m
itoch
ondria/m
in)
(fusi
on/m
itochondria/m
in)
0
1
2
3
Mit
och
on
dri
al le
gh
th (m
m)
Mit
och
on
dri
al le
gh
th (m
m)
E
cont
rol
Tau
Tau+
Miro
0.0
0.5
1.0
1.5
cont
rol
Tau
Tau+
Miro
0.0
0.1
0.2
0.3
0.4
0.5
cont
rol
Tau
Tau+
Miro
0.00
0.01
0.02
0.03
0.04
cont
rol
Tau
Tau+
Miro
0
1
2
3
** **
*
# # ## ###
10 µm 2 µm
A
5µm 1µm
CB
25ìm
#
(conta
ct /m
itoch
ondria/m
in)
(conta
ct /m
itoch
ondria/m
in)
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
FUSION RATE
KISS RATE
MOTILITYNUMBER
FISSION RATE
LENGTH
+ wt hMiro1- shRNA Miro1+2
+ Mfn2- nd Mfn2
+Drp1+Fis1- shRNA Drp1 and nd Drp1
= ++
++
(in steady state)
_
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t