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Restricted diffusion of OXPHOS complexes in dynamicmitochondria delays their exchange between cristaeand engenders a transitory mosaic distribution

Verena Wilkens, Wladislaw Kohl and Karin Busch*Department of Biology/Chemistry, Division of Mitochondrial Dynamics, University of Osnabrueck, 49069 Osnabrueck, Germany

*Author for correspondence (busch_k@biologie.uni-osnabrueck.de)

Accepted 5 September 2012Journal of Cell Science 126, 103–116� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.108852

SummaryMitochondria are involved in cellular energy supply, signaling and apoptosis. Their ability to fuse and divide provides functional andmorphological flexibility and is a key feature in mitochondrial quality maintenance. To study the impact of mitochondrial fusion/fission

on the reorganization of inner membrane proteins, oxidative phosphorylation (OXPHOS) complexes in mitochondria of different HeLacells were tagged with fluorescent proteins (GFP and DsRed-HA), and cells were fused by polyethylene glycol treatment. Redistributionof the tagged OXPHOS complexes was then followed by means of immunoelectron microscopy, two color super-resolution fluorescencemicroscopy and single molecule tracking. In contrast to outer membrane and matrix proteins, which mix quickly and homogeneously

upon mitochondrial fusion, the mixing of inner membrane proteins was decelerated. Our data suggest that the composition of cristae ispreserved during fusion of mitochondria and that cristae with mixed OXPHOS complexes are only slowly and successively formed byrestricted diffusion of inner membrane proteins into existing cristae. The resulting transitory mosaic composition of the inner

mitochondrial membrane illuminates mitochondrial heterogeneity and potentially is linked to local differences in function andmembrane potential.

Key words: Mitochondrial fusion and fission dynamics, Super-resolution tracking and localization microscopy, Oxidative phosphorylation, Membrane

protein diffusion, Mitochondrial microcompartments, Immunoelectron microscopy (IEM)

IntroductionMitochondria provide the vast majority of the cellular energy by

oxidative phosphorylation (OXPHOS). Their role in calcium

homeostasis, in the process of aging, their significance for cell

life and death and their participation in the progress of diverse

diseases emerged only recently, though (Beal, 2005; Lu, 2009).

Mitochondrial fusion and fission dynamics (Bereiter-Hahn and

Jendrach, 2010) play a crucial role in these processes. In general,

the balance between fusion and fission determines the

mitochondrial morphology and facilitates the exchange of

metabolites, mtDNA and proteins (Nakada et al., 2001; Ono

et al., 2001; Malka et al., 2005; Twig et al., 2006). Also the

maintenance of a healthy mitochondrial population is directly

linked to ongoing fusion/fission (Twig et al., 2008; Campello and

Scorrano, 2010). The immediate outcome of mitochondrial fusion

and fission such as the redistribution of membrane compounds as

well as the problem of concerted membrane fusion and division

have to still be worked out in the field, though.

Mitochondria possess two membranes: the outer (OMM) and the

inner (IMM) mitochondrial membrane. The inner membrane harbors

one fifth of total mitochondrial proteins, of which the protein

complexes of the respiratory chain and the mitochondrial F1FO ATP

synthase provide 55%. NADH dehydrogenase (NADH:ubiquinone

oxidoreductase; CI) (Harmon et al., 1974), succinate dehydrogenase

(CII), cytochrome bc1 complex (cytochrome c reductase; CIII),

cytochrome c oxidase (cytochrome c:oxygen oxidoreductase, CIV)

and the F1FO ATP synthase (complex V) constitute the oxidative

phosphorylation (OXPHOS) system. Multiple intrusions of the inner

mitochondrial membrane leaflet enlarge the surface of the IMM

many fold (Mannella et al., 1994; Perkins et al., 2010). As a

consequence, the inner mitochondrial membrane is basically

composed of two parts: the inner boundary membrane (IBM)

facing the OMM and the intruding cristae membrane (CM),

separated by the so called cristae junctions (CJ). Probably, the

CM is the principal site of oxidative phosphorylation (Gilkerson

et al., 2003). Beside ATP synthesis, maintenance of a membrane

potential by the OXPHOS is essential for mitochondrial and cell

survival. To guarantee this, different levels of mitochondrial quality

control are established: (1) mitochondrial proteases degrade

dysfunctional proteins (Arnold and Langer, 2002); (2) damaged

organelles lose their ability to fuse and are decomposed via a process

called mitophagy (Scherz-Shouval and Elazar, 2007; Kurz et al.,

2008; Twig et al., 2008; Mouli et al., 2009); and (3) fusion and

fission dynamics provide a mechanism to avoid the accumulation of

damaged proteins by facilitating the regular remixing of

mitochondrial compounds (Ishihara et al., 2003; Arimura et al.,

2004). Despite this, the distribution and function of mitochondria

within a cell is not necessarily homogeneous (Bereiter-Hahn and

Voeth, 1998; Collins et al., 2002; Busch et al., 2006; Muster et al.,

2010) and protein and membranous structures might be unevenly

distributed (Wikstrom et al., 2009; Wurm et al., 2011). To further

dissect this, we here analyzed the distribution of OXPHOS

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complexes in recently fused mitochondria by means of super-

resolution fluorescence and immunoelectron microscopy (IEM).

Our data suggest that during mitochondrial fusion and fission cristae

are predominantly preserved resulting in patchy mitochondria at

first. Restricted mobility of OXPHOS complexes in cristae

membranes engenders this transitory mosaic composition of

recently fused mitochondria.

ResultsRecently, we showed that mitochondria displayed a patchy

distribution of OXPHOS complexes for at least some hours in

fused cells (Muster et al., 2010) (Fig. 1). We here asked whether

the patches from fluorescence images correspond to cristae with

primarily unmixed composition of OXPHOS complexes. This

question is firsthand related to the dynamics of the CM in the

context of mitochondrial fusion and fission. Little is known about

how CMs conduct in these processes. Certainly, cristae rearrange

in the immediate fusion zone as observed by correlative

microscopy (Bereiter-Hahn and Voth, 1994; Busch et al.,

2006), and cristae also remodel after apoptotic stimuli (Frezza

et al., 2006). However, the (re-)organization of cristae more distal

from the fusion site is still concealed. In general, two scenarios

are possible: (i) single cristae are preserved when mitochondria

fuse; or (ii) cristae are completely rebuilt and recomposed to form

new hybrid cristae (Fig. 1). To dissect this, cristae have to be

characterized by their OXPHOS complex composition, which

can be done by means of IEM. Therefore, OXPHOS complexes

from different mitochondria have to be distinguished, which is

not possible for endogenous complexes. For the same reason

photoswitchable proteins – which are useful for analyzing

spreading in single cells – are not suitable for IEM analysis of

mitochondrial dynamics. Thus, cells expressing OXPHOS

complexes with different tags were fused and mixing was

analyzed on the mitochondrial level according to an established

method (Ishihara et al., 2003).

To visualize individual OXPHOS complexes, complex I was

tagged with monomeric eGFP (eGFPm) C-terminally at the 30 kDa

subunit, CII was tagged with eGFPm at the subunit B (SDHB) and

CIII at the subunit 10 (6.4 kDa subunit). CIV was tagged with

DsRed–HA at the Cox8a subunit that faces the IMS, and complex

V was labeled at the c-subunit. The successful assembly of the

tagged subunits into complexes was shown before (Muster et al.,

2010; Sukhorukov et al., 2010). For immunodetection, antibodies

against the eGFPm- or HA-tagged OXPHOS complexes were used,

and the secondary antibody was probed with a gold nanoparticle

(Fig. 1; supplementary material Fig. S1). Combinations of 6 nm/

12 nm and 12 nm/20 nm gold were used. Control cells without

labeled OXPHOS complexes were negative in IEM.

OXPHOS complexes are primarily localized in thecristae membrane

First, a distribution profile of OXPHOS complexes in the different

IMM microcompartments CM, CJ and IBM was compiled.

OXPHOS complexes were visualized by immunogold labeling on

cryosections. Generally, the arrangement of cristae in HeLa

mitochondria was rather regular, with .90% of cristae

perpendicular to the longitudinal axis of the respective

mitochondrion (Fig. 2A). The mean width of single cristae was

rather constant with 1762 nm (6s.d.), and the distance between

two adjacent cristae was about 51615 nm in average (Fig. 2A9).

For allocation of the probed OXPHOS complexes in the IMM

microcompartments, a typical crista with a length according to the

average length (413 nm) was delineated. The preferential

distribution of all complexes in the CM microcompartment is

obvious (Fig. 2B–F). CI showed a rather homogeneous distribution

in the middle part of the CM (Fig. 2B). Similar longitudinal

distributions were obtained for all complexes, whereas the absolute

distance of the gold labels to the crista membrane varied as predicted

from the likely position of the tagged subunit.

OXPHOS complexes display different localization profilesin the CM

To dissect the distribution of the OXPHOS complexes in the

different microcompartments IBM, CJ and CM quantitatively, a

Fig. 1. Experimental outline to determine the

consequence of mitochondrial fusion on the

reorganization of the inner mitochondrial

membrane. In cryosections of fused cells with

differently tagged OXPHOS complexes, the

distribution of labeled proteins was studied by

immunoelectron microscopy to determine the

mode of inner membrane fusion. Two states are

feasible: (i) entire cristae are preserved during

fusion with no exchange of compounds between

adjacent cristae, and (ii) cristae of mixed

composition emerge, which result either from

reformation of cristae or diffusion of compounds

into existing cristae. Scale bar: 3 mm.

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window was moved alongside the IMM. Within this sliding

window, particles were counted and respective protein

localization profiles were generated as recently described (Rabl

et al., 2009) (supplementary material Fig. S2). All five

investigated OXPHOS complexes were predominantly located

in the CM-microcompartment (Fig. 2G). The comparison of the

Fig. 2. See next page for legend.

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distribution profiles clearly shows an overlap of CI, CIII, CIV

and CV (Fig. 2G): 80% of CI coincided with 80% of CIII, CIV

and CV. Analysis of the CJ region revealed that in contrast to the

other complexes CI hardly was found in the immediate CJ region,

while CII showed the highest abundance in close proximity to the

CJ (65% of the molecules were found within the proximal region

of the CJ region). Also the number of CII molecules decreased

distal from the CJ (Fig. 2E,G). CV was homogeneously distributed

within the CM including the CJ. We also plotted the relative

distribution of CV under consideration of the true length of the

respective crista and found a homogeneous distribution of CV

along the CM (supplementary material Fig. S2C).

Taken together these data show that (1) OXPHOS complexes

were predominantly found in the CM microcompartment; (2) the

distribution profiles of CI, CIII, CIV and CV along the CM were

similar; and (3) CII had a significant quota of molecules localized

in the IBM.

OXPHOS complex mixing in single mitochondria depends

on fusion and fission dynamics

Next, cells with differently labeled OXPHOS complexes were

fused and analyzed at different time points after cell fusion. The

mixing of OXPHOS complexes in mitochondria was quantified

by calculating the Pearson cross correlation coefficient rP for

both fluorescence distributions alongside the respective

mitochondrion as described earlier (Muster et al., 2010). The

overview fluorescence images already show that the number of

fused mitochondria increased with time: 4 h after cell fusion the

area covered by yellow mitochondria was generally larger than in

a specimen analyzed 2 h after fusion (Fig. 3A,D). Also the

corresponding rP - values in exemplary mitochondria were higher

(rP 4h, fusion50.42 versus rP 2h, fusion50.26).

We next asked how an imbalance in fusion/fission would

influence the distribution pattern. Overexpression of hFis1

resulted in fragmented mitochondria due to an increase in

fission (James et al., 2003, Yoon et al., 2003). Hardly any mixing

of mitochondrial compounds was observed in these cells

(supplementary material Fig. S3). Opposite, it is known that

certain stresses such as starvation can induce elongated

mitochondria (Tondera et al., 2009). This stress-inducedmitochondrial hyperfusion (SIMH) likely results from a shift ofmitochondrial dynamics towards relative more fusion than fission

events. Probably, the number of active Drp1 molecules acting atmitochondria is lowered, resulting in a reduction of fission(Gomes et al., 2011; Rambold et al., 2011). We suspected thatunopposed fusion would increase the patchiness of mitochondria,

since more and more fusion events enhance the amount of addedmitochondrial material.

To induce SIMH, the cells were incubated under serum and

amino acid deprivation in PBS for 2 hours after PEG-inducedfusion. This form of starvation induced a relative highermitochondrial fusion rate in surviving cells with a tendency toan overemphasized mitochondrial network (Fig. 3G,H). Barely at

the fusion zones of the cells expressing CI-DsRed–HA and CIII-eGFPm, respectively, the mitochondria fused and became hybrid,while in the peripheral zones mitochondria formed an

interconnected network without mixing of mitochondrialcompounds (Fig. 3G). These observations resemble the situationin aging cells, where increased fusion (relative to fission) was

combined with a reduced exchange of mitochondrial material (Maiet al., 2010). A detailed view of the fusion area shows that themitochondria sparsely mixed their components resulting in larger

patches within mitochondria and red and green mitochondriaexisting next to each other (Fig. 3H). A line scan for fluorescencedistribution in a mitochondrion where green as well as redfluorescence is detectible (dotted line in Fig. 3H) revealed a

Pearson cross correlation coefficient of rP 2h,SIMH50.18, indicatingvery low mixing (Fig. 3I). The mean Pearson cross correlationcoefficient for fusion areas in SIMH cells was rP

2h,SIMH50.2260.14 (6s.d.), whereas the mean values for thenormal fused cells were rP 4h,fusion50.4760.1 for 4 hours afterfusion and rP 2h,fusion50.3860.17 for 2 hours after fusion (Fig. 3J),

indicating less mixing in hyperfused mitochondria.

Since the amount of fused mitochondria with mixedcomposition depends on the number of fused cells in asyncytium, the time and the position within the syncytium, a

quantitative analysis to determine the quota of fused-mixedversus original mitochondria is not apposite. Rather theindividual analysis of fused-mixed mitochondria in more detail

is appropriate to learn about the redistribution of IMM proteinsand IMM dynamics during fusion and fission.

Preserved cristae exist next to hybrid cristae

Next we analyzed the distribution pattern of OXPHOS complexesin cristae of fused mitochondria by means of IEM. Gathering ofcristae composed of only one species of labeled OXPHOScomplexes in fused mitochondria indicates preservation of cristae

rather than remodeling of cristae. So, the analysis of cristaecompositions will in addition elucidate inner membranedynamics. We determined the distribution of labeled OXPHOS

complexes in cristae in mitochondria of fused HeLa cells by IEM(Fig. 1). Fused cells were identified by the existence of at leasttwo nuclei. Areas of at least three to five cristae that exclusively

displayed only one type of gold particles were characterized as aregion of preserved cristae. Single cristae with mixedcomposition were identified by the presence of a smaller and a

larger gold particle on one crista. By screening hundreds of singlemitochondria, the following pattern of distribution evolved:preserved regions with one species of labeled OXPHOS complex

Fig. 2. Localization profiles of OXPHOS complexes I, II, III, IV and V in

the inner membrane compartments of mitochondria from human HeLa

cells. Labeled OXPHOS complexes were localized by immunogold detection in

numerous cryosections of cells, and their absolute position was plotted on a

schematic crista. The secondary antibody was probed with 12 nm gold.

(A) Design of a representative crista membrane model derived from .100

analyzed cristae in cryosectioned HeLa cells. (A9) Average proportions for

cristae. (B) Distribution of complex I (CI) in the IMM determined by

immunogold labeling of eGFPm fused to its 30 kDa subunit. (C) Distribution of

CIII probed by immunogold detection of the 6.4 kDa eGFPm subunit of the

complex. (D) Distribution of CIV obtained after immunogold labeling of

DsRed–HA fused to subunit Cox8a of the complex. The secondary antibody

was probed with 20 nm gold in this case. (E) Distribution of immunogold

labeling CII, where subunit SDHB was tagged for probing. (F) Distribution of

CV revealed by immunogold labeling of eGFPm fused to the c-subunit of the

complex. (G) Box-and-whisker plot showing the distribution of CI (n5137),

CII (n5121), CIII (n5136), CIV (n5146) and CV (n5148) along the IMM.

The boxes represent the 10th to 90th percentiles and 25th to 75th percentiles

within the gradient shaded boxes. The vertical lines in the gradient shaded boxes

represent the median values. The square symbols in the boxes denote the

respective mean values. The error bars denote the 1st and 99th percentile values.

The minimum and maximum values are denoted by x. IBM, inner boundary

membrane; CJ, cristae junction; CM, cristae membrane. Scale bar: 200 nm (A).

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were repeatedly found. These parts were habitually alternating

with preserved regions with the complementary OXPHOS

complex of different origin indicating generally non-mixed

parts. These non-mixed parts retained their original state within

fused mitochondria (Fig. 4A–D, green and red arrowheads). In

some fused mitochondria we found larger regions composed of

Fig. 3. Different states of mitochondrial hybridization examined by OXPHOS fluorescence distribution in fused cells. (A) Cells expressing CI-eGFPm and CIII-

DsRed were fused by PEG treatment and incubated for another 4–5 h before analysis. The fluorescence image shows a fused cell with at least three nuclei. Yellow areas

indicate the presence of hybrid mitochondria with a mixed composition of OXPHOS complexes. (B) A more detailed view reveals that in the hybrid mitochondria the

OXPHOS complexes CI-eGFPm and CV-DsRed are not homogeneously distributed. (C) Line plot of fluorescence distribution of CI-eGFPm and CV-DsRed along a single

mitochondrion shown in B (dotted line). (D) Overview of fused cells with CI-DsRed and CIII-eGFPm 2 h after cell fusion. (E) Fused CI-DsRed and CIII-eGFPm cells

display a patchy appearance of the mitochondria indicating inhomogeneous distribution of the protein complexes within the mitochondria. (F) Line plot of fluorescence

distribution of CI-DsRed and CIII-eGFPm along a single mitochondrion shown in E (dotted line). (G) Overview of cells expressing CI-DsRed–HA and CIII-eGFPm 2 h

after cell fusion under stress conditions to induce mitochondrial hyperfusion (SIMH). (H) SIMH cells show a patchy outline at the zone where the mitochondria of the

differently labeled cells fused. (I) Line plot of fluorescence distribution of CI-DsRed–HA and CIII-eGFPm along a single mitochondrion shown in H (dotted line).

(J) Determination of the complexes mixing degree in single mitochondria 4 h and 2 h after cell fusion and in stress-induced hyperfused mitochondria. Mean Pearson

cross correlation coefficients were calculated from 37 mitochondria (*P50.023; ***P53.5361025). Scale bars: 10 mm (A,D,G); 2 mm (B,E,H).

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several cristae with mixed composition (colored yellow), flanked

by regions with cristae that harbor only one type of OXPHOS

complex (Fig. 4A, arrowheads; supplementary material Fig.

S4Aa). These conserved regions constituted up to 25% of the

mitochondrial area. So, mixed cristae were embedded in regions

where only one species of tagged OXPHOS complex was present.

Within one cell, also original non-fused mitochondria were found

next to fused mitochondria (Fig. 4C, green asterisk). Rarely, also

mitochondria with good mixing and numerous hybrid cristae side

by side were identified (Fig. 4E). Projection of the distribution of

different OXPHOS complexes related to single cristae on the

longitudinal axis of the respective mitochondria generated an

outline that conclusively corresponded to the patterned dispersion

found in fluorescence images before (supplementary material Fig.

S4). In SIMH cells fused mitochondria with mixed OXPHOS

complex composition were definitively less frequent as already

expected from the fluorescence analysis. Also the number of

hybrid cristae with mixed composition in single mitochondria was

reduced (Fig. 4F,G). The overall labeling degree was comparable

with normally fused cells, though.

For comparison, the distribution of OXPHOS complexes in

cells coexpressing two differently tagged fusion proteins was

determined by IEM. From previous fluorescence microscopic

analyses we expected a more homogeneous distribution of

OXPHOS complexes in coexpressing cells (Muster et al., 2010).

Indeed, we found significantly more hybrid cristae with mixed

composition in mitochondria of coexpressing cells compared to

fused mitochondria (Fig. 4H,I; supplementary material Fig.

S4B). This observation was independent of the particular

OXPHOS complex combination: CI-DsRed–HA transiently

cotransfected with CII-eGFPm showed numerous hybrid cristae

(Fig. 4H), which was also the case for cells stable coexpressing

CI-DsRed–HA and CIII-eGFPm (Fig. 4I). In addition, projections

of stable cotransfected cells emphasized the well-mixed hybrid

composition of the cristae (supplementary material Fig. S4B).

Cristae with mixed composition of OXPHOS complexes

occur only occasionally in fused cells

We next determined the number of hybrid cristae per

mitochondrion that univocally were probed with both sizes of

Fig. 4. Ultrastructural analysis of OXPHOS

complex allocation in fused mitochondria

reveals an overall low number of hybrid cristae

in close proximity to preserved cristae. In fused

and coexpressing HeLa cells, the distribution of

OXPHOS complexes was analyzed by IEM.

(A) A fused mitochondrion harboring several

hybrid cristae with a mixed composition of

OXPHOS complexes (yellow) indicated by the

presence of 6 nm (CI-DsRed–HA) and 12 nm

(CIII-eGFPm) gold nanoparticles. The cristae with

mixed composition were post-colored in yellow.

(B) CI-DsRed–HA (20 nm gold nanoparticle)

fused with CIII-eGFPm (12 nm gold

nanoparticle). (C) A long, fused mitochondrion

with two small regions of mixed composition

(yellow) identified by the presence of 20 nm

(CI-DsRed–HA) and 12 nm (CIII-eGFPm) gold

nanoparticles. The asterisk marks a non-fused

original mitochondrion expressing CIII-eGFPm

with exclusively 12 nm gold nanoparticles.

(D) Fused CI-DsRed–HA/CIII-eGFPm (6 nm anti-

HA and 12 nm anti-GFP). (E) Fused

mitochondrion with well mixed CI/CIII

throughout single cristae with 6 nm and 12 nm

gold antibodies. (E9) Subsequently colored image

showing the large amount of hybrid cristae with

mixed OXPHOS composition (yellow).

(F,G) Hyperfused (SIMH) cells. Immunogold

labeling against CI-DsRed–HA (20 nm) and CIII-

eGFPm (12 nm) indicates the low mixing degree

resulting in patchy mitochondria (red and green

arrowheads). (H) Mitochondrion of a cell

expressing CI-DsRed–HA co-transfected with

CII-eGFPm and immunogold labeled with 6 nm

(CI-DsRed–HA) and 12 nm (CII-eGFPm) gold

nanoparticles. (I) Mitochondrion of a cell stable

coexpressing CI-DsRed–HA and CIII-eGFPm

immunolabeled with 6 nm (CI-DsRed–HA) and

12 nm (CIII-eGFPm) gold antibodies. Scale bars:

100 nm.

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gold particles. This quantification confirmed that the relative

amount of hybrid cristae with mixed composition was

significantly lower (33623%) in fused cells than in cells

coexpressing OXPHOS complexes (46627%, P53.961022).

SIMH cells possessed even less hybrid cristae (5611%,

P51.361028; Fig. 5A). The quota of preserved cristae in single

mitochondria of fused cells was higher (49%) than in coexpressing

cells (46%). Eighteen percent of the cristae in mitochondria of

fused cells were not labeled compared to only 8% within those of

coexpressing cells. Since labeled OXPHOS complexes are diluted

out by non-tagged OXPHOS complexes from the fusing partner

cells, this is reasonable. To quantify the internal mixing degree, the

distances between nearest neighbor gold particles of different sizes

were determined in fused and hyperfused mitochondria (Fig. 5B).

Indeed, the OXPHOS mixing was less in SIMH mitochondria

reflected by significantly larger distances between gold particles of

different size (6 nm/12 nm; 12 nm/20 nm; P-value of 9.661024).

The mean value for gold particle distances in SIMH cells was

2006140 nm (6s.d.) compared to 1306100 nm (6s.d.) in

normally fused cells. 80% of the measured distances for the

SIMH cells ranged from 70 nm to 390 nm compared to a range

from 40 nm to 280 nm in normal fused cells. Obviously, the

mingling of OXPHOS complexes from different origin is

handicapped in SIMH cells.

The mixing degree of OXPHOS complexes in single hybrid

cristae in fused mitochondria is low

We next analyzed the mixing of OXPHOS complexes in single

hybrid cristae that derived from mitochondrial fusion/fission

dynamics. The distance between different gold particles

(respective different OXPHOS complexes) is an indicator for

the mixing degree: If hybrid cristae in fused cells develop by the

spreading and mixing of previous separated OXPHOS complexes

from different cristae, this will be reflected in the distance

between OXPHOS complexes from different origins. If the model

is right, then the distance between small and large gold particles

in cristae in fused cells should be larger than in cristae from

coexpressing cells. In coexpressing cells, a uniform dispersion of

Fig. 5. Analysis of OXPHOS allocation in coexpressing, fused and

hyperfused mitochondria. (A) Fraction of identified hybrid cristae in cells

coexpressing CI-DsRed–HA/CII-eGFPm and CI-DsRed–HA/CIII-eGFPm for

48 h and in syncytia of fused cells expressing CI-DsRed–HA and CIII-

eGFPm. The number of analyzed mitochondria was 34, which included 280

cristae in fused and 190 cristae in co-transfected cells and 36 mitochondria

including 267 cristae in SIMH cells. The plotted values are the percentage

(6s.d.) of hybrid cristae per single fused mitochondrion with mixed

composition. More than 700 gold particles were analyzed. (B) Quantification

of the distances between two different sized gold nanoparticles within single

mitochondria of fused cells expressing CI-DsRed–HA and CIII-eGFPm (6 nm

to 12 nm, 12 nm to 20 nm, nearest neighbor). The distances between gold

particles in fused mitochondria is significantly larger in SIMH cells than in

fused cells without starvation. Box-and-whisker plot showing mean distances

(n576) between the different sized gold nanoparticles. (C) Box-and-whisker

plot showing the mixing of OXPHOS complexes in hybrid cristae

characterized by the mean distance (n566) between 6 nm and 12 nm sized

gold nanoparticles within single hybrid cristae of fused cells expressing CI-

DsRed–HA and CIII-eGFPm and cells coexpressing CI-DsRed–HA and CIII-

eGFPm or CI-DsRed–HA and CII-eGFPm (see Fig. 2G). Asterisks indicate

statistical significance determined by a one-way-ANOVA (*P,0.05;

**P,0.01; ***P,0.001).

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OXPHOS complexes and thus a short distance between large andsmall gold particles is expected.

Data presentation as a box-and-whisker plot shows that 80% ofthe measured distances for fused cells (CI+CIII) cover thedistance range from 35 nm to 290 nm, while in coexpressing

cells 80% of the measured distances range from 39 nm to 160 nm(CI+CIII) and from 20 nm to 90 nm (CI+CII), respectively(Fig. 5C). In mitochondria of fused cells with immunogold

labeled CI and CIII the mean distances between 6 nm and 12 nmsized gold particles was 123699 nm (6s.d.; n565). In contrast,the mean distance between 6 nm and 12 nm gold particles on

single cristae of coexpressing cells was significantly smaller:81649 nm (n565) for cells coexpressing CI and CIII(P52.861023). Interestingly, in cells coexpressing CI and CIIthe mean distance was even smaller with 50628 nm (n565;

Fig. 5C). The observed difference in the distances between CI+CIIcompared to CI+CIII in cotransfected cells was also significant(P51.861025), indicating a more homogeneous distribution of

CII. In sum, (1) the percentage of identified cristae with mixedcomposition in cotransfected cells was higher than in fused cells;(2) SIMH mitochondria possessed less hybrid cristae than

normally fused mitochondria; and (3) in single hybrid cristae infused mitochondria the mixing of different complexes was sparse.

The diffusion of OXPHOS complexes in the IMMcompartments is restricted

The data so far strongly support the model of a preponderant

preservation of cristae and their composition duringmitochondrial fusion and fission cycles. The patterneddistribution of OXPHOS complexes and the low amount of

hybrid cristae with mixed composition also indicate that proteindiffusion apparently is no doorway to overcome thisheterogeneity. To prove this experimentally we applied single

molecule tracking and localization microscopy (TALM) anddetermined the spatiotemporal behavior of OXPHOS complexesin mitochondria in live cells (Appelhans et al., 2012).

We analyzed the diffusion behavior of CII and CV. CII waschosen because our results indicated a specific distribution profileof CII combined with better mixing behavior compared to the other

OXPHOS complexes. CV is specific in its role to shape cristaecurvature (Strauss et al., 2008; Davies et al., 2012), probably bydimerization and oligomerization. Thus a mobility analysis withinthe cristae membrane microcompartment is tantalizing.

For tracking, respective subunits were tagged C-terminallywith the HaloTag7 and covalently labeled with HaloTag-

ligand conjugated with tetramethylrhodamine (HTL-TMR).Substoichiometrical labeling enabled the visualization of singlemolecules. Trajectory maps of CII in mitochondria weregenerated from time series (1000 frames) by reconnecting

single molecules from frame to frame by use of the multiple-target tracing algorithm (Serge et al., 2008). Single trajectorieswere plotted on an averaged image of the raw data image series

(Fig. 6A). To determine diffusion coefficients, step lengths wereplotted, and diffusion constants were determined from gradingthe obtained jump size histograms by a mixture model of

increasing complexity. The final complexity was validated byapplying a x2-test (Fig. 6B). By analyzing the jump sizedistributions, three populations characterized by different

diffusibilities could be gauged: (1) mobile CII freely diffusingalong mitochondria (dark blue curve); (2) a less mobile fraction(light blue curve); and (3) rather immobile CII (red curve). To

prove whether the position of the tag had an effect on the stabilityand mobility of the complex, subunit SDHD was labeled in a

control, but no difference of the diffusion coefficients was found(supplementary material Fig. S5).

For tracking, CV-Halo was specifically labeled with HTL-TMR at subunit-c. Here, 3000 subsequent image frames with

single molecules were recorded for further analysis. Thegenerated trajectory map was overlaid with an averaged imageof the raw data series (Fig. 6C). Immediately, the difference in

the course of trajectories with respect to the longitudinal axis ofthe mitochondria is visible (Fig. 6A,C). In this example, the jumpsize distribution diagram revealed three sub-populations of

complex V with different mobility, respectively: (1) a mobilefraction with a diffusion coefficient of Dapp50.070 mm2/s(indicated by the dark blue fitting curve); (2) a less mobilefraction with an apparent diffusion constant Dapp50.019 mm2/s

(light blue curve); and (3) an immobile fraction with an apparentdiffusion constant Dapp50.005 mm2/s. For more detailedanalysis, selected trajectories of CV were plotted and their

course was evaluated with respect to the position within amitochondrion (Fig. 6E). About 60% of the CV trajectoriesshowed a restricted course perpendicular to the longitudinal axis

of the respective mitochondrion (depicted in red). Bluetrajectories represent free diffusion or a course along thelongitudinal axis of the mitochondrion.

As we recently demonstrated, the orthogonal trajectories most

likely reflect the movement of OXPHOS complexes in CM. Incontrast, we assigned the molecules with the free respectivelongitudinal movements to diffusion in the IBM. The trajectories

of most of the CV fell in either of these categories. In rare cases(estimated ,1%), a change in course from orthogonal to random(light blue trajectories) or vice versa was found. When the

perpendicular part of the course overlaid with the perpendicularcourse of a different molecule, we assigned this part to berestricted diffusion within one crista and the overall course to

reproduce a transition between CM and IBM microcompartment.Analysis algorithms to identify these specific events forautomatic quantification are not yet developed. We next askedwhether differences in diffusion constants could be assigned to

different localizations of molecules (IBM versus CM). So,histograms of single trajectories of CV molecules wereconstructed using their diffusion constants calculated from their

mean square displacements (MSD, supplementary material Fig.S6). The frequency plot shows clearly two subpopulations:Subpopulation A with a mean Dapp50.056 mm2/s and

subpopulation B with a mean Dapp50.008 mm2/s. Thesesubpopulations were independently plotted in two trajectorymaps. In the trajectory map of the molecules with highermobility, trajectory courses were rather fortuitous, but some

perpendicular courses were also present not unlike a possibleconfinement in cristae (supplementary material Fig. S6A). Incontrast, for the majority of reduced mobile molecules the

displacement was quasi-punctual or was restricted inperpendicular courses (supplementary material Fig. S6B).Although the tendency that the diffusion coefficient is related

to the localization within a definite microcompartment isobvious, an univocal assignment of molecules by MSD analysisto different localizations (IBM, CM) is limited due to some

inaccuracy deriving from the 2D projection of 3D trajectories aswell as the imperfect structure of mitochondria (e.g. also bendedcristae or regions with no cristae exist).

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Finally, we compared the diffusion coefficients of CII and CV

(Fig. 6F). For CII and CV, a three fraction fit best matched the

distribution of jump sizes respectively in the different

experiments (n$300 mitochondria from 42 cells in n$3

independent preparations and at least 30,000 trajectories). It

clearly evolved that complex II overall is more mobile than

complex V: the mobile fraction of CII had an apparent diffusion

constant of Dapp50.14060.035 mm2/s (6s.d.; s.e.m.50.005 mm/

s), while the mobile fraction of complex V was characterized by

Dapp50.08260.014 mm2/s (s.d.; s.e.m.50.002 mm2/s). The

Fig. 6. Mobility of OXPHOS complexes II and V in microcompartments of the inner mitochondrial membrane. Complex II subunit SDHB and complex V

subunit-c were fused to HaloTag7H protein and covalently labeled with HTL-TMR for tracking (Appelhans et al., 2012). (A) Trajectory map of single SDH

complexes in three adjacent mitochondria. The trajectory map was superimposed on an averaged wide-field fluorescent image of the same molecules from 1000

subsequent frames recorded at 60 Hz. (B) Distribution of step lengths for individual CII molecules (1000 frames, recorded at 60 Hz, exposure time 15 ms).

Diffusion constants were determined by grading the obtained jump size histograms by a mixture model of increasing complexity. The final complexity was

validated by applying a x2-test resulting in optimal P-values and minimal residuals. (C) Trajectory map of CV-Halo/TMR in mitochondria with a significant

proportion of track courses orthogonal to the longitudinal axis of mitochondria (superimposed on an averaged image from raw data of 3000 consecutive frames

recorded at 60 Hz). (D) Step length analysis reveals three subpopulations of CV-TMR molecules: mobile (dark blue curve), less mobile (light blue curve) and

immobile molecules (red curve). (E) Selected trajectories of CV-Halo/TMR to show preferential confinement in single cristae (red, yellow). The typical shape of a

crista is depicted as a gray dotted line. The dark blue trajectory indicates free diffusion in the IBM, whereas the light blue trajectories indicate molecules that

apparently transit between CM and IBM. The mean precision for the localization of single molecules in each frame was 12 nm. (F) Apparent diffusion constants

for mobile fractions of complex II and complex V determined by analysis of jump size distributions. Mean values 6 s.e.m. n$300 mitochondria from 42 cells in

three independent preparations, and at least 30,000 trajectories. Statistical significance was proved by a two-samples t-test for null-hypothesis – means are equal

and alternative hypothesis – difference of means unequal zero.

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diffusion coefficient of the less mobile fraction of CII was

Dapp50.03960.013 mm2/s (s.e.m.50.002 mm2/s), while it

was Dapp50.01960.007 mm2/s (s.e.m.50.001 mm2/s) for CV.

A small immobile fraction (#15%) with Dapp50.00660.001

mm2/s (CII) and 0.00460 (CV), respectively was also found.

Dual-color single molecule fluorescence microscopy

reveals piecewise composition of fused mitochondria

Single molecule fluorescence microscopy allows the localization

of single molecules with a precision of 15 nm in living cells

(Appelhans et al., 2012). We combined FPALM (Hess et al.,

2006) in combination with TALM (Appelhans et al., 2012) to

perform dual-color super-resolution imaging of OXPHOS

complexes in fused mitochondria (Fig. 7A). One OXPHOS

complex was tagged with paGFP, the second with the Halo-tag,

which was posttranslational labeled with HTL-TMR. Rendered

images (from 3000 single processed frames) show the

distribution of fluorescence-tagged OXPHOS complexes with

high resolution. Complex II-halo/TMR distribution shows a

broader signal in the cross section, which is visible as a red

border in mixed parts. These are probably the CII molecules

located in the IBM microcompartment. The green Complex I

signal is smaller in the cross section, because CI almost

exclusively is found in the cristae membranes (Fig. 2B).

Different states of fused mitochondria were found with respect

to their OXPHOS complex distribution pattern. State I

comprises mitochondria that clearly show areas in the size of

several hundreds of nanometers harboring one type of labeled

OXPHOS only, alternated by areas with OXPHOS complexes of

different origin (Fig. 7A). Right at the border between these

alternating red- and green areas, small yellow areas are visible,

indicating overlap of red- and green fluorescence signals (Fig. 7A,

yellow arrowheads). This strongly indicates colocalization of

OXPHOS complexes, probably in the same CM. In state II,

mitochondria possessed larger areas with colocalizing complexes,

sometimes in the middle of a long mitochondrion, sometimes at one

Fig. 7. States of fused mitochondria showing consecutive mixing of OXPHOS complexes and progressive homogenization is dependent on fusion and fission

cycles. Illustrated is the plausible sequence of fusion/fission events that first generate patchy mitochondria with heterogeneous distribution of OXPHOS complexes

but with time result in more and more amalgamation. (A) Fusion-and-fission-dependent redistribution of OXPHOS complexes in mitochondria in a syncytium of

fused CI-paGFP- and CII-TMR-expressing cells revealed by means of dual color super-resolution FPALM/TALM. Different states of mixing are depicted (I–III).

(B) Proposed model describing the dependence of homogenization of inner membrane OXPHOS complexes on ongoing fusion and fission events. In the early state,

mitochondria from different origins fuse successfully and completely. Because cristae are maintained and diffusion of OXPHOS complexes is restricted, exchange

OXPHOS complexes (yellow area) between cristae occurs only at the fusion sites. In an intermediate state – after few fusion and fission events – the

mitochondria appear patchy, with alternating conserved areas and areas with mixed OXPHOS composition (green, yellow and red parts). The mixing improves with

each fusion and fission cycle. On the ultrastructural level, the mixing of OXPHOS complexes progresses to the cristae level (TEM micrograph), resulting in

mitochondria consisting of cristae that retained their original state and hybrid cristae with a mixed composition. In the late state, the outcome of multiple fusion and

fission cycles is a homogeneous distribution of the OXPHOS complexes throughout the mitochondrion. Scale bar: 300 nm (A), 100 nm (B).

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of the ends (Fig. 7A, state II). Mitochondria with extensive areas of

homogeneous distribution of both OXPHOS complexes were alsofound, in some cases with a red or green part at one end (Fig. 7A,state III). We attribute this to a recent fusion of an original

mitochondrion to an already well mixed mitochondrion. Altogetherdual color super-resolution image analysis completed the modelobtained by distribution analysis of OXPHOS in IEM micrographsof fused cells. This method provides a higher set of single molecule

data than IEM but does not allow the direct visualization ofunderlying structures of microcompartments. Thus, single moleculefluorescence analysis has to be seen as a complementary method to

IEM.

DiscussionMitochondria fuse and divide continuously, but little is known

about the reformation of the inner mitochondrial membrane inthese processes. Heterogeneous distribution of IMM complexesin recently fused mitochondria indicates that spatiotemporal

constraints exist retarding homogeneous remixing. Here, wedissected this observation on the molecular and ultrastructurallevel by IEM and super-resolution tracking and localizationmicroscopy. First, a distribution profile for all five OXPHOS-

complexes in the different IMM compartments IBM, CJ and CMof mammalian HeLa cells was compiled. Complex I (NADH-ubiquinone-oxidoreductase), complex III (cytochrome c

reductase) and complex IV (cytochrome c oxidase) showed asimilar distribution profile along the cristae membrane. This is inline with previous observations derived from activity staining and

immunolabeling of EM sections of mitochondria (Perotti et al.,1983; Gilkerson et al., 2003; Vogel et al., 2006), whichdesignated the cristae membrane as the site of oxidative

phosphorylation.

F1FO ATP synthase was homogeneously distributed along thecristae membranes. The enrichment of F1FO ATP synthase inpositively curved membrane parts of cristae was observed before

(Parsons, 1963; Strauss et al., 2008; Bieling et al., 2010; Truta-Feles et al., 2010), and ATP synthase dimerization probably amain determinant of cristae shaping (Paumard et al., 2002; Gavin

et al., 2004; Minauro-Sanmiguel et al., 2005; Strauss et al., 2008;Davies et al., 2012). Unfortunately, the limited degree ofimmunogold labeling does not render the explicit confirmationof F1FO ATP synthase dimers possible. Beside its known

localization in positively curved membranes, the presence ofF1FO ATP synthase in negatively curved membranes such as theCJ are was rather new. Presumably, F1FO ATP synthase

complexes in the negatively curved CJ are not dimerized butexist as single, less rigid F1FO ATP synthase complexes (Rablet al., 2009). Of all five OXPHOS complexes, only F1FO ATP

synthase and complex II (succinate dehydrogenase) would fit intoa negatively curved membrane, since the intramembrane parts ofCI, CIII and CIV, are too large to be integrated in a strongly

negatively curved membrane of roughly 40 nm, especially whenassembled in supercomplexes (Althoff et al., 2011). Indeed,hardly any complex I was found in the CJ region.

After ensuring the localization of OXPHOS complexes in the

cristae microcompartment we used them as markers to resolvethe cristae reorganization during mitochondrial fusion and fissiondynamics. Distribution and localization analysis of OXPHOS by

IEM showed that fused mitochondria possess roughly one thirdtrue hybrid cristae with remixed OXPHOS composition. Themajority of cristae displayed a preserved OPXHOS composition,

though. In elongated SIMH mitochondria, the number ofidentified hybrid cristae was lower. Dual color super-resolution

images of fused mitochondria confirmed these results andunderscore a piecewise or mosaic configuration of fusedmitochondria with respect to the inner membrane composition.Together, our data suggest that cristae are maintained during

fusion and fission and hybrid cristae with mixed OXPHOScomplex distribution are only successively formed. We proposethe following working model (Fig. 7B): first, mitochondria of

different origin fuse. Whether cristae reform at the fusion sites isstill unreadable, but they are maintained in the original arms.Restricted diffusion of OXPHOS complexes supports this

preservation. Hardly, OXPHOS complexes move betweenexisting cristae. As a consequence, hybrid cristae with mixedcomposition predominantly exist at the fusion sites flanked bycristae with a preserved composition of OXPHOS complexes

(Fig. 7B, early state). After several rounds of fusion and fission amosaic mitochondrion comprising alternating preserved zones(red and green parts) and parts with mixed composition (yellow)

at the former fusion sites evolves (Fig. 7B, intermediate state). Inthe light microscope this generates the patchy appearance ofmitochondria. By time and corresponding to repetitive fusion/

fission cycles, mitochondrial heterogeneity decreases. ElongatedSIMH mitochondria possess larger patches of conserved regionsand less hybrid cristae. This can be explained when a relative

increase in fusion results in the addition of further mitochondrialmaterial without true mixing and underlines the significance ofintermediate fission events for the merging process. Only byundergoing balanced fusion/fission mitochondria progressively

reach a well-mixed state with respect to inner membrane proteindistribution (Fig. 7B, late state).

A possible alternative to explain the mosaic composition of

mitochondria is while the OMM is fused completely, the innermembrane is not (Liu et al., 2009). This would result in singularmitoplasts surrounded by a common OMM (Skulachev et al.,

2004). Our EM micrographs gave no evidence for transientfusion or mitoplasts, though. The mitochondria we investigateddisplayed normal shape and matrix appearance, and a regulararrangement of cristae with no segmentation of the IMM. We

thus exclude that incomplete inner membrane fusion is the reasonfor the mosaic appearance.

The explanation we provide here is that diffusion and thus

spreading of OXPHOS complexes in the IMM microcompartmentsis restricted. Single particle trajectory analysis revealed thatcomplex V particles showed confined diffusion in cristae

membranes and punctual immobility (supplementary materialFig. S6B, box), while transitions between the CM and IBM or CMand CM compartments were only rarely observed. It was for longer

suggested that cristae junctions are diffusion barriers (Sukhorukovand Bereiter-Hahn, 2009), a discussion that recently found supportby the finding that a large protein complex exists in the CJ region(Harner et al., 2011; Hoppins et al., 2011; von der Malsburg et al.,

2011; Alkhaja et al., 2012). It is likely that this MINOS/MitOScomplex with a size of ,1200 kDa constitutes a diffusion barrierfor the transition of proteins between the CM and IBM

microcompartment.

How supercomplex formation influences diffusibility ofOXPHOS complexes, is not clear, yet. OXPHOS complexes I,

III and IV assemble in supercomplexes of different stoichiometry(Schagger and Pfeiffer, 2000; Eubel et al., 2004; Dudkina et al.,2005; Schafer et al., 2006; Wittig et al., 2006; Althoff et al.,

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2011), while dimers of F1F0 ATP synthase often organize in row

like structures in cristae membranes (Davies et al., 2011).

According to the Saffman-Delbruck model, the translational

mobility is independent of the amount by which the particle

sticks out of the membrane sheet (Saffman and Delbruck, 1975)

and diffusion coefficients of membrane domains are only

logarithmically related to domain radii suggesting little

influence (Petrov and Schwille, 2008). In the future, diffusion

analysis of single membrane proteins and complexes hopefully

will help to elucidate these relations further.

Localization and mobility analysis of succinate dehydrogenase

(complex II) revealed its exceptional role within the OXPHOS

complex ensemble. Complex II has a dual role in mitochondria: it

is part of the OXPHOS system, but is also a key membrane

complex of the citric acid cycle. Complex II does not translocate

protons and thus not necessarily need to be confined to cristae.

Indeed, a substantial amount of molecules were found within the

IBM microcompartment in line with previous results showing a

strong activity staining in this membrane part (Kalina et al., 1969;

Bertoni-Freddari et al., 2001). Mobility of CII was significantly

higher than of complex V and thus the mixing of OXPHOS

complexes with complex II was best. This finding endorses our

model that the homogeneity of dynamic mitochondria is

determined and limited by the diffusion ability of inner

membrane proteins.

Following this, the described heterogeneous spatiotemporal

organization of OXPHOS complexes – trapped in the cristae

microcompartment – provides an explanation for mitochondrial

heterogeneity. Therefore, zones of different membrane potential

within single mitochondria (Bereiter-Hahn and Voeth, 1998;

Collins et al., 2002; Distelmaier et al., 2008; Wikstrom et al.,

2009) could be assigned to local accumulation of functional

compromised OXPHOS complexes that are not or only slowly

exchanged by remixing. For the future, it would be exciting to

identify and characterize these zones more precisely and correlate

local membrane potential with the actual distribution and

functionality of the present OXPHOS complexes.

Materials and MethodsGeneration of fluorescent proteins

The different fluorescent fusion proteins were generated using plasmid cassettes

obtained by substitutions of eGFPm or DsRed–HA into pSEMS-26 m vector fromNEB biosciencesH. Indicated subunits of OXPHOS complexes were N-terminallyfused to fluorescent proteins with monomeric DsRed–HA and monomeric eGFP

(DsRed amplified from pDsRed-Monomer-N1 vector from HBD biosciencesH;modified monomeric eGFP, a gift from J. Sieber, Gottingen), as described by

Muster et al. (Muster et al., 2010). For genetic selection of double transfected cellsthe neomycin resistance was exchanged for a puromycin resistance in one type ofvector. For tracking experiments, respective subunits were cloned N-terminally of

the HaloTag7 in the pSEMS-26m-HaloTag7 vector (Lisse et al., 2011). CII-Haloand CV-Halo were then covalently labeled in situ with 1 nM HTL-TMR (HaloTag

conjugated with tetramethylrhodamine ligand) for 20 min. Before measurements,the labeling medium was replaced by medium without HTL-TMR.

Cell culture

HeLa cells were cultured in Minimal Essential Medium with Earle’s salts (PAA

Lab GmbH) containing 10% (v/v) fetal calf serum (FCS, Biochrom AG), 1% MEMnonessential amino acids (PAA lab GmbH) and 1% 4-(2-hydroxyethyl)piperazine-

1-ethanesulfonic acid (HEPES, PAA Lab GmbH) (MEM++) at 37 C with 5% CO2.The cells were transfected with the plasmids described in the text using the methodof Muster et al. (Muster et al., 2010). Neomycin (800 mg/ml G418) and puromycin

(0.5 mg/ml) resistance were used for selection of stable cell lines. Hyperfusion wasinduced by starvation stress 30 min after PEG-mediated fusion. After incubation

for 2 h in PBS, cells were prepared for IEM. Longer incubation times with PBSturned out to be too stressful for cells.

Cell fusion and co-transfection

For the fusion assays, stable expressing cells (CI-eGFPm and CIII-DsRed; CI-eGFPm and CV-DsRed; CI-DsRed–HA and CIII-eGFPm) were sown on 6 cmdiameter Petri dishes and co-cultured for 24 h. For fusion, the cells were incubatedwith prewarmed 40% PEG in PBS (37 C) for 1 min. Afterwards, cells werecultured in medium for 4–7 h before microscopic analysis.

For co-transfection assays, cells stable expressing CI-DsRed–HA and CIII-eGFPm were generated. Cells stable expressing CI-DsRed–HA were transientlytransfected with CII-eGFPm and cells stable expressing CI-pa-eGFPm weretransiently transfected with CIV-DsRed–HA.

Confocal laser scanning microscopy

Mixing of OXPHOS in fused cells was followed using an Olympus Fluoview FV1000cLSM equipped with a 606 objective (UPLSAPO oil, NA 1.35) and two spectraldetectors. DsRed was excited with a 559 nm laser diode and emission was recorded inthe range of 580–630 nm, eGFP was excited with the 488 nm wavelength of an argonion laser and emission was collected in the range of 500–560 nm. Measurements wereperformed in a sequential excitation mode to avoid cross-talk. Z-stacks ofmitochondria were taken in certain region of interests (12612 mm2), with a pixelsize corresponding to 30 nm.

Immunoelectron microscopy

Cells were prepared according to previously described methods (Tokuyasu, 1978;Tokuyasu, 1973; Oorschot et al., 2002). In short, cells were fixed with 2%paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) atroom temperature, embedded as monolayer or pellet in 10% gelatin, mounted onpins, frozen in liquid nitrogen and cut at 2120 C (Leica UCT). Grids were washedwith PBS, 20 mM glycine in PBS, blocked with 1% BSA in PBS, incubated withthe primary antibodies against eGFPm (rabbit polyclonal to eGFPm, Abcam,ab6556, 1:100) and HA (mouse monoclonal to HA.11, Covance, BIOT-101L,1:100) for 1 h at room temperature, washed with 0.1% BSA in PBS and incubatedwith the secondary antibody-gold-complexes (6 nm goat anti-mouse and 12 nmgoat anti-rabbit, Jackson Immuno Research, 1:50, no. 115-195-146 and 111-205-144; 20 nm goat anti-mouse, Abcam, ab27242, 1:50). Cells were stained in amixture of 1.6% methylcellulose/0.4% uranyl acetate, and analyzed usingtransmission electron microscopy (TEM; Zeiss 902A).

Image processing

Images were processed with HBitplane Imaris and ImageJH (NIH, Bethesda, MD,USA). The datasets were deconvolved using Autoquant software to reduce noiseand improve image quality. A stack of 1000 images recording single moleculeswas collected and averaged to create the equivalent of an epi-fluorescence image.Single trajectories were projected on this pseudo-average image.

TEM micrographs were recorded using iTEM software (SIS). The images wereprocessed afterwards in Adobe Photoshop CS4, making linear adjustments tocontrast and brightness. Adobe Photoshop CS4 was also used to color the originalmicrographs subsequently. To outline single cristae we used a Wacom IntuosH4drawing tablet. All figures were assembled in Adobe Illustrator CS4 and convertedinto TIFF using LZW compression.

Quantitative analysis

To dissect the distribution of OXPHOS complexes in the IMM a representativemodel of a crista was generated as described (Vogel et al., 2006). The dimensionsof this model were obtained by averaging 141 cristae in cryosectioned HeLa cells.The cristae dimensions and the distances of the gold particles to the membraneswere measured with iTEM (SIS) and superimposed to the immunogold-labeledcristae (digitalized by Adobe Photoshop CS4 and Adobe Illustrator CS4). Themeasured distances represent the absolute values of 160 gold particles of eachantigen that were plotted to this model. Particles close to badly preservedmembranes, those bound to large cristae more than 450 nm distal from the cristaejunction or particles which could not be assigned to a membrane within a space of60 nm radius around a membrane were not counted. The specific, labeled subunitsof the different OXPHOS complexes required the adaptation of a radius oftolerance in which a gold particle was assigned to the membrane. This wasconsidered for plotting the distribution ratio diagram of localization between OM/IBM, CM and matrix space. The specific distances of the labeled subunits to themembrane for each of the five complexes were determined according to formerpublished structures (Efremov et al., 2010; Yoshikawa et al., 1998; Zhang et al.,1998; Sun et al., 2005; Watt et al., 2010). Box-and-whisker plot analyses wereperformed for each of the complexes using Microsoft Office Excel software(Microsoft Corporation, Redmond, WA, USA) and Microcal Origin 6.0 software(OriginLab Corporation, Northampton, MA, USA). Statistical significance wasperformed on original data and determined by one-way-ANOVA with MicrocalOrigin 6.0 software (OriginLab Corporation, Northampton, MA, USA). Analysisof the mixing degree in fused and coexpressing cells was performed by thedetermination of the distances of differently sized antibodies in single hybridcristae. To compare the mixing in normally fused and SIMH mitochondria, a

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nearest neighbor analysis was performed because although the degree of labelingin the normal and SIMH specimen was comparable the overall number of hybridcristae in SIMH cells was low. Thus, an analysis of distances between differentlabels within one hybrid cristae was limited by the number of hybrid cristae. Forthe analysis of the relative and absolute distribution of CV alongside cristae,approximately the same set of raw images was used. Starting from the IBM region,the true positions (in shorter and longer cristae) were recalculated to thecorresponding position in an average crista (20% distal from CJ in actual cristae isequal to 20% distal from CJ in the average crista).

Single molecule tracking and localization microscopy

For single molecule (SM) recording, an inverse microscope (IX71, Olympus)equipped with a TIRF condenser (Olympus), and a back-illuminated EMCCDcamera (Andor iXON 897) was used. For excitation of TMR, a solid state laser(561 nm, 200 mW; CrystaLaser) was focused onto 700 mm multi-mode-opticalpolarization maintaining monomode fiber (KineFlex, Pointsource) and transmittedvia the rear illumination port of the microscope. For excitation, the HiLo mode(illumination with a highly inclined and thin beam) was used (Tokunaga et al.,2008). By this, the signal to background ratio can be significantly improved. Theestimated intensity in the thin light sheet with a thickness of 9 mm was 2568 kW/cm2 (561 nm, 200 mW, CrystaLaser). A digitally synchronized mechanical shuttercontrolled exposure times. Laser light reflected from a dichroic mirror (OBS-U-M3TIR 405/488/561, Semrock) passed through a high-numerical apertureobjective (1506 TIRF objective NA 1.45, Olympus, UAPO). Fluorescenceemission from TMR was passed through a bandpass filter (Semrock BrightLineFF01-523/610-25), and was projected on top a back-illuminated EMCCD camerayielding an image pixel size of 107 nm. Equivalent fiber and condenser equipmentwas used for coupling the 488 nm laser line of a Saphire laser (200 mW, Coherent)for paGFP visualization into the system. The photoactivation of paGFP by therecording 488 nm laser line was sufficient and activation by 405 nm pre-excitationwas not necessary. The camera was operated continuously using the FrameTransfer Mode of the CCD allowing full-frame acquisition at 60 Hz. Forlocalization of single emitters, a modified 2D Gaussian mask for approximationwas used (Thompson et al., 2002; Gould et al., 2009). Single molecules weretracked using the multiple-target tracing algorithm (Serge et al., 2008). Multipledeflation loops were performed to ensure identification of all particles. Particletrajectories were recovered based on maximum likelihood estimators (Serge et al.,2008). For more details see Appelhans et al. (Appelhans et al., 2012).

In dual color super-resolution experiments a combination of fluorescent paGFPand Halo/TMR was used as described recently (Appelhans et al., 2012; Wilmeset al., 2012). paGFP and TMR were excited in parallel allowing a recording rate of30 Hz. Up to 3000 frames were recorded. Fluorescence emissions from paGFP andTMR were passed through a bandpass filter (Semrock BrightLine FF01-523/610-25). The dual view (Photometrics) was equipped with an according dichroid mirrorand filters (Chroma 585 DCXR; HQ 525/50; brightline HC 620/52). Possiblecrosstalk signals of paGFP signals in the TMR channel were erased by a renderingprocedure after single molecule localization allowing only signal intensities abovea certain threshold (usually 100 counts). The count to photon conversion factor was20.5 under our conditions (gain 300, 1506objective/oil, 1.45 NA, 16). For singlemolecule localization a 2D fitting algorithm (kindly provided by Sam T. Hess,University of Maine, Orono, ME) was used (Gould et al., 2009).

AcknowledgementsWe thank Achim Paululat and Jacob Piehler for continuous support,Dirk Wenzel for initial technical advice and Christian Ungermannfor critically reading the manuscript.

FundingThe study was supported by the Deutsche Forschungsgemeinschaft[grant number Bu 2288/1 to K.B.B.] and a SFB 944 grant to K.B.B.

Supplementary material available online at

http://jcs.biologists.org/lookup/doi:10.1242/jcs.108852/-/DC1

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