RESEARCH ARTICLE

Analysis of ER–mitochondria contacts using correlativefluorescence microscopy and soft X-ray tomography ofmammalian cellsKirstin D. Elgass1, Elizabeth A. Smith2,3, Mark A. LeGros2,3, Carolyn A. Larabell2,3,* and Michael T. Ryan4,*

ABSTRACTMitochondrial fission is important for organelle transport, qualitycontrol and apoptosis. Changes to the fission process can result in awide variety of neurological diseases. In mammals, mitochondrialfission is executed by the GTPase dynamin-related protein 1 (Drp1;encoded by DNM1L), which oligomerizes around mitochondria andconstricts the organelle. The mitochondrial outer membrane proteinsMff, MiD49 (encoded byMIEF2) and MiD51 (encoded by MIEF1) areinvolved in mitochondrial fission by recruiting Drp1 from the cytosol tothe organelle surface. In addition, endoplasmic reticulum (ER) tubuleshave been shown to wrap around and constrict mitochondria before afission event. Up to now, the presence of MiD49 and MiD51 atER–mitochondrial division foci has not been established. Here, wecombine confocal live-cell imaging with correlative cryogenicfluorescence microscopy and soft x-ray tomography to link MiD49and MiD51 to the involvement of the ER in mitochondrial fission. Wegain further insight into this complex process and characterize the 3Dstructure of ER–mitochondria contact sites.

KEY WORDS: Confocal microscopy, Endoplasmic reticulum,Microscopy imaging, Mitochondria, Mitochondrial fission,Soft X-ray tomography, Correlative imaging

INTRODUCTIONMitochondria are highly dynamic organelles that constantly moveand undergo structural changes (Westermann, 2010; Otera andMihara, 2011a; Labbe et al., 2014; Mishra and Chan, 2014). As theycannot be created de novo, individual mitochondria can undergofusion and fission events, thus enabling the proper distributionof mitochondria within the cell (Varadi et al., 2004; Campelloand Scorrano, 2010), as well as the proper distribution of vitalcomponents within the mitochondrial network (Parone et al., 2008).Fission events are crucial for the maintenance of mitochondrial andcellular function by contributing to the proper distribution ofmitochondria in response to the metabolic needs of the cell for ATP(Parone et al., 2008; Otera and Mihara, 2011a). Likewise, duringcell division, the mitochondrial network has to undergo extensivefragmentation to ensure equal distribution of the mitochondria andthe mitochondrial DNA (mtDNA) into the two daughter cells.

One of the main functions of mitochondria is to supply cellularenergy through the process of oxidative phosphorylation.Furthermore, mitochondria are involved in a range of otherprocesses, such as intracellular signaling and apoptosis (Ryanand Hoogenraad, 2007). In accordance with these functions,mitochondrial defects and consequent changes in their morphologyhave been linked to several human diseases. Numerousmitochondrialdiseases and other cell-destructive processes, such as aging andapoptosis, have been linked to a fragmented mitochondrial network,which has resulted from enhanced fission activity (Arduino et al.,2011; Glauser et al., 2011; Nakamura et al., 2011; Song et al., 2011;Elgass et al., 2013).

A number of proteins on the mitochondrial outer membranemediate mitochondrial fission by recruiting the master fissionmediator Drp1 (also known as DNM1L) (Legesse-Miller et al.,2003; Ingerman et al., 2005; Lackner and Nunnari, 2009; Mearset al., 2011) from the cytosol to mitochondria. These include themitochondrial fission factor, Mff (Gandre-Babbe and van der Bliek,2008; Otera et al., 2010; Otera and Mihara, 2011b) and themitochondrial dynamics proteins MiD49 and MiD51 (also knownas MIEF2 and MIEF1, respectively) (Palmer et al., 2011, 2013;Koirala et al., 2013; Loson et al., 2013, 2014; Richter et al., 2014).However, little is known about the complex interplay between theseproteins, the precise mechanisms regulating the fission process orthe involvement of the endoplasmic reticulum (ER) (Friedmanet al., 2011; Friedman and Nunnari, 2014) and actin filaments (DeVos et al., 2005; Korobova et al., 2013).

A connection betweenmitochondria and the ER in Ca2+ signalingand apoptosis has been well established. Contact sites betweenmitochondria and the ER are important for phospholipid synthesisand Ca2+ signaling (Szabadkai et al., 2004; de Brito and Scorrano,2010). A key finding about ER–mitochondrial contacts was madeby de Brito and colleagues, who reported that the fusion proteinMfn2 is also involved in tethering mitochondria to the ER (de Britoand Scorrano, 2008). It has also been discovered recently that theER is involved in regulating mitochondrial dynamics by markingthe prospective sites of mitochondrial division (Friedman et al.,2011). Mitochondrial fission occurs at positions where ER tubulesare wrapped around mitochondria, thus mediating constriction ofthe mitochondrial membranes and reduction of the mitochondrialdiameter by approximately 30% before Drp1 recruitment.Wrappingof the ER around mitochondria is mainly observed at positionsof Mff and Drp1 foci. However, in cells that have been depletedof Drp1 or Mff, mitochondrial constriction at sites of ERcontact are observed, indicating that ER-mediated constriction ofmitochondrial tubules proceeds independently of both Mff andDrp1 (Friedman et al., 2011). Recent studies have provided furtherinsight into the process of ‘ER-associated mitochondrial division’ inyeast (Lackner et al., 2013; Murley et al., 2013; Friedman andReceived 19 January 2015; Accepted 17 June 2015

1Hudson Institute for Medical Research, Monash Micro Imaging, MonashUniversity, Melbourne 3168, Australia. 2Department of Anatomy, School ofMedicine, University of California San Francisco, San Francisco, CA 94158, USA.3National Centre for X-ray Tomography, Advanced Light Source, Berkeley,CA 94720, USA. 4Department of Biochemistry and Molecular Biology, MonashUniversity, Clayton, Melbourne 3800, Australia.

*Authors for correspondence (Carolyn.Larabell@ucsf.edu;Michael.Ryan@monash.edu)

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Nunnari, 2014) and mammalian cells, which include the additionalinvolvement of INF2, actin and myosin II (Korobova et al., 2013,2014; Hatch et al., 2014).Here, we investigate the link between MiD49, MiD51 and the ER

in mitochondrial constriction and fission using a unique approachthat combines confocal live-cell imaging, correlative cryogenicfluorescence microscopy and soft x-ray tomography (CFM–SXT)(Larabell and Nugent, 2010; McDermott et al., 2012; Parkinsonet al., 2013; Smith et al., 2014b).

RESULTSMiD foci combine during constriction of mitochondriaWe have recently reported that mitochondrial fission events areapparent at low expression levels of ectopic MiD proteins (Palmeret al., 2013; Richter et al., 2014), whereas at higher expressionlevels, fission events are blocked owing to Drp1 sequestration on themitochondrial surface, leading to unopposed fusion (Fig. 1A)(Palmer et al., 2011). Indeed, further analysis and quantification ofour data demonstrated that there was an initial increase in fissionevents (Fig. 1B) and mitochondrial number (Fig. 1C), resulting infragmented mitochondria following expression of green fluorescentprotein (GFP)-tagged MiD51 (MiD51–GFP). Longer-termexpression resulted in the fragmented mitochondria moving to amore networked state (moderate MiD levels), followed bymitochondrial elongation (at high MiD levels) (Fig. 1A). Fig. 1Dshows the corresponding timecourse of MiD51–GFP expression, asdetermined by measuring the fluorescence intensity. To verify thatthe changes we saw in mitochondrial number over time are causedbyMiD51–GFP, we also expressed aMiD51 mutant (MiD51R235A–GFP) that is unable to recruit Drp1 (Richter et al., 2014). Asexpected, we did not observe any changes in mitochondrial numberover time with this mutant, whereas the number of mitochondriadecreased over time following MiD51 expression, owing to theblock in fission and unopposed fusion (supplementary materialFig. S1, Table S1).In contrast to what has been reported in the literature for other

fission mediators, namely Mff and Drp1 (Legesse-Miller et al.,2003; Friedman et al., 2011), foci comprising MiD proteins (MiDfoci) were observed not only at mitochondrial constriction sites butwere also frequently observed at other sites of the outermitochondrial membrane (Fig. 2A). In some cases, several MiDfoci appeared to combine during the formation of a constriction site(Fig. 2A,B; supplementary material Movie 1). Constriction of amitochondrion at MiD foci does not necessarily lead to animmediate scission event; observed constrictions appeared to bereversed and/or underwent several constriction–expansion cyclesbefore finally being divided (Fig. 2B; supplementary materialMovie 2). Nevertheless, using live-cell time-lapse imaging, wewereable to show colocalization of MiD51 with Drp1 in the same fissionfoci during fission events (Fig. 2C; supplementary materialMovie 3). Of note, we did not observe colocalization of thedifferent proteins exclusively at fission and/or constriction sites(Fig. 3A–D). However, some foci might represent remnantassemblies following a fission event [e.g. as can be seen in thelast image of the MiD51 and Drp1 fission-event time series(Fig. 2C, far right image)].

Colocalization and interaction of fission proteins in the samefoci at mitochondriaHatch and colleagues have noted the potential existence of severalindependent fission mechanisms in mammalian cells (Hatch et al.,2014). AsMiD49, MiD51 andMff have been separately observed at

mitochondrial fission sites, we asked whether these proteinscolocalize at mitochondria and are therefore part of the samefission machinery. Using confocal microscopy we were indeed ableto observe the presence of MiD51 and the splice isoform 1 of Mff(hereafter always referred to as Mff) (Fig. 3A), as well as MiD49and Mff (Fig. 3B), within the same foci at mitochondria. To gainfurther insight into the presence and interplay of the different fissionproteins in a single fission event, we assessed the colocalization ofdifferent combinations of fission proteins within foci. As can be

Fig. 1. Low levels of MiD-protein overexpression increases the number offission events andmitochondria in a cell. (A) Time-lapse images of a singleCOS-7 cell after transfection with MiD51–GFP (green). Mitochondria werestained with MitoTracker Red. (B) The number of observed fission eventsincreased in the first 15 min after beginning observation (timepoint0=observation start, i.e. several hours after transfection when GFPfluorescence was just detectable); subsequently, with increasing levels ofMiD51–GFP expression, fission was blocked and the number of observedfission events decreased substantially. (C) The number of mitochondriaincreased in the first 15 min after beginning observation; subsequently, withincreasing levels of MiD51–GFP expression, fission was blocked and thenumber of mitochondria decreased accordingly. (D) The intensity of MiD51–GFP fluorescence increased over time following transfection. Fluorescenceintensity was normalized to that at time point 0. Scale bars: 10 µm.

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seen, MiD51 in most cases (90–98%) was found in foci with apartner protein (MiD49, Mff or Drp1; Fig. 3D). Likewise, Drp1almost always colocalized with MiD51 (99%). By contrast, ∼60%of Mff foci had MiD51 as a partner. As an additional indicator ofprotein–protein interactions between different fission proteins,we used Foerster Resonance Energy Transfer (FRET) analysis ofdonor de-quenching upon acceptor photobleaching (Fig. 3E). Weobserved interactions between the investigated fission proteinsMiD51, Mff and Drp1, and the strongest signals of interaction weredetected for MiD51–Drp1, Drp1–Drp1 and MiD51–MiD51. Underthe conditions used here, we did not observe a FRET signal forFis1–Drp1. This is consistent with recent data indicating that Fis1 isnot required for mitochondrial fission (Otera et al., 2010) and that itmight instead be involved in mitophagy (Shen et al., 2014).However, we cannot exclude the possibility of a transient interactionthat is too short-lived to be detected by using FRET.

Mitochondrial fission occurs at MiD foci at mitochondria–ERcontact sitesAlthough MiD49, MiD51 and the ER have been independentlyshown to be involved in mitochondrial fission, details about howthey act together in time and space to drive mitochondrial fission arestill missing. To shed light on the possible interplay between theseproteins during fission, we used live-cell confocal fluorescencemicroscopy to image mitochondrial fission events at MiD foci inrelation to the ER tubules (supplementary material Movies 4, 5).Fig. 2D shows the simultaneous presence of ER tubules (red)and MiD proteins (green) at a mitochondrial constriction site(arrowhead). The same constriction site subsequently underwent

mitochondrial fission (Fig. 4A, arrowhead; supplementary materialMovie 4), and ER tubules were present throughout the completefission process and connected the two daughter organelles after thefission process had been completed. By contrast, Fig. 4B andsupplementary material Movie 6 show a mitochondrion undergoingconstriction at sites of MiD51–ER contacts, which is followed by asubsequent relaxation of the constriction rather than the final fissionevent. The live-cell imaging experiments also showed that ERcontacts at MiD foci were not limited to mitochondrial constrictionsites (Fig. 4B,C, white arrowheads) but that mitochondrialconstriction could subsequently occur at these foci (Fig. 4B,C,yellow arrowheads, supplementary material Movies 6, 7). In fact, lessthan 40% of observed ER–mitochondria contacts at MiD foci(ncells=5) were located at constriction sites. Upon single- anddouble-knockdown of MiD49 and/or MiD51 (Palmer et al., 2011),constriction sites were still observed (Fig. 5A,B; supplementarymaterial Fig. S2); however, the number of constriction sites wasonly significantly reduced upon double-knockdown (Fig. 5B;supplementary material Table S2, P<0.05).

Correlative cryogenic fluorescence microscopy and soft x-ray tomography reveals short ER extensions that contactmitochondria at MiD fociTo characterize the MiD-specific structure of ER–mitochondriacontact sites in three dimensions with high spatial resolution, weused correlated CFM–SXT analyses. SXT analysis of specimensthat had been mounted in thin glass capillaries with a diameter ofless than 15 µm and a wall thickness of <500 nm yields three-dimensional (3D) reconstructions with isotropic resolution(McDermott et al., 2012; Parkinson et al., 2013; Cinquin et al.,2014). Therefore, small suspension cells, such as the viral (v)-Abl-transformed lymphoma mouse B-cell line that we used for theseexperiments, are quite suitable for this approach. In order todemonstrate that MiD proteins remain functional in these cells, weverified that MiD proteins form foci (supplementary materialFig. S3A,B) and recruit Drp1 to mitochondria (supplementarymaterial Fig. S3C,D). In order to perform CFM–SXT analyses,transfected cells were loaded in glass capillaries and thensequentially imaged, first in the brightfield and fluorescencechannels using cryogenic spinning disk microscopy, followed bySXT. A diagram of the workflow is shown in supplementarymaterial Fig. S4. This correlative technique allowed us to identifythe presence of short extensions protruding from ER sheets atER–mitochondria contact sites that contained MiD51–GFP foci.A representative slice generated usingCFM–SXT, the SXT-generatedslice without the fluorescence overlay and a magnification of the areaexhibiting MiD51–GFP fluorescence are shown in Fig. 6. Severalshort ER extensions that protrude from the ER sheet and connect it tothe mitochondrion can be distinguished in two dimensions (Fig. 6C,white arrowheads; Fig. 7D). To characterize these features in threedimensions, SXT tomograms were segmented into differentsubcellular compartments (Fig. 6D–F). A representative segmentedER–mitochondrion contact site also exhibiting MiD51-GFPfluorescence was identified from within the 3D reconstruction(Fig. 6G) and is shown in detail in Fig. 6H. On average, ERextensions exhibited a length of 168±13 nm (mean±s.e.m.) and aretherefore below the resolution limit of conventional confocalfluorescence microscopy, but are resolved well by using SXTanalysis, which has a 50-nm (isotropic) spatial resolution using thex-ray optics and tomographic data collection protocol used in thisstudy (McDermott et al., 2012; Parkinson et al., 2013; Cinquin et al.,2014). Fig. 7 shows four correlative 2D CFT-SXT slices taken from

Fig. 2. Fission events at foci of MiD51–GFP. (A) MiD51–GFP foci (green)were observed at mitochondria before constriction and combined during theevent at the constriction site. Mitochondria were stained with MitoTracker Red(red). (B) Mitochondrion (red) undergoing constriction at MiD51–GFP foci(white arrowhead), followed by subsequent relaxation and reformation of theconstriction. (C) Fission event at MiD51–GFP foci (green) in the presence ofDrp1 (red). Mitochondria were stained with MitoTracker Deep Red and falsecolored in blue. (D) ER tubules (expressing mCherry-KDEL, red) contactmitochondria (blue) at a MiD51–GFP-foci constriction site (white arrowhead).Snapshots from time-lapse imaging of this constriction site are shown inFig. 4A. Scale bars: 1 µm (A–C); 3 µm (D).

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different planes within the cell and an enlargement of regionsshowing a high intensity of local MiD51–GFP fluorescence.Consistent with the data shown in Fig. 6, all the magnified views inFig. 7D show the presence of MiD51–GFP where ER extensionscontact mitochondria.

SXT LAC values of ER and mitochondria reflectmitochondrial morphologySXT analysis provides quantitative information on the density of thebiomolecules present in subcellular structures (Hanssen et al., 2012)in the form of the linear absorption coefficient (LAC) value that isassociated with each voxel. Different LAC values are typicallyrepresented as different grey values in the final SXT reconstruction.Soft x-ray tomograms were segmented for mitochondria and ER(Fig. 8A,B) to assess mitochondrial morphology, as well as

mitochondrial and ER LAC values (see Materials and Methods).We found that the LAC values of mitochondria decreasedupon overexpression of MiD and that the mitochondrial networkunderwent subsequent fragmentation, compared with untransfectedcontrol cells (a two-tailed, unequal variance t-test results inPmito=0.023) (Fig. 8C). The cell shown in Fig. 8A is representativefor all cells within the red-boxed area in Fig. 8D, which showed afragmented mitochondrial network, whereas the cell in Fig. 8B isrepresentative for all the cells within the green-boxed area in Fig. 8D,which showed a normal mitochondrial network.

For CFM–SXT, all cells were sorted for low levels of MiD51–GFP fluorescence. Therefore, SXT experiments were exclusivelyperformed on cells that showed either the fragmented phenotypeat very low expression levels or the normal phenotype observed atslightly higher expression levels (the corresponding timepoints in

Fig. 3. MiD proteins and other fission proteins cancolocalize and interact in the same foci atmitochondria. (A) MiD51–GFP (green) andmCherry–Mff (red) colocalize in foci at mitochondria(blue). An overview image is shown (left panel) alongwith magnifications of the area indicated by the whitedashed square (right panels) as indicated. (B) MiD49–GFP (green) and mCherry–Mff colocalize in foci atmitochondria (blue). Overview image andmagnifications are shown as described in A.(C) MiD51–GFP (green) and RFP–Drp1 colocalize infoci at mitochondria (blue). Overview image andmagnifications are shown as described in A. Outlinesof mitochondria are indicated by white lines in themagnified views. (D) Quantitative analysis ofcolocalizing (light gray) versus individual (dark gray)foci for the GFP and RFP and/or mCherry constructs(as indicated per panel on the x-axis). Total foci countsfor cells expressing the following constructs: 91,MiD51–GFP and MiD49–mCherry; 80, MiD51–GFPand mCherry–Mff; 144, MiD51–mCherry andRFP–Drp1. (E) Fluorescence Resonance EnergyTransfer (FRET) efficiencies between various fissionproteins as detected and analyzed using acceptorphotobleaching (see Materials and Methods).Combinations used were (from left to right):MiD51–GFP and mCherry–Mff; GFP–Mff andmCherry–Mff; MiD51–GFP and MiD51–mCherry;GFP–Mff and RFP–Drp1; MiD51–GFP andRFP–Drp1; GFP–Drp1 and RFP–Drp1; GFP–Fis1and RFP–Drp1. The highest signals were detected forMiD51–MiD51, Drp1–Drp1 and MiD51–Drp1interactions. Error bars represent mean±s.e.m.Scale bars: 10 µm (A–C, overview images); 1 µm(A–C, magnification images).

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Fig. 1A are 10 min and 15 min, respectively). Interestingly, wefound that changes in differential LAC values resulting from theintensity of MiD51–GFP fluorescence also correlated with dramaticchanges in mitochondrial morphology because networks appearedfragmented in cells that exhibited higher differential LAC values(Fig. 8A and red rectangle in Fig. 8D) and normal in cells thatexhibited lower differential LAC values (Fig. 8B and greenrectangle in Fig. 8D). In fragmented cells, MiD–ER contactscould be observed; however, owing to the typically spherical shapeof fragmented mitochondria, no constriction sites were observed.

DISCUSSIONInterplay of fission components at ER–mitochondria divisionfociIn this work, we provide evidence that both Mff and MiD proteinscan associate and colocalize with Drp1 at ER–mitochondriadivision foci. Therefore, we conclude that these proteins are partof the same fission machinery. The results of the colocalization andFRET analyses that we present here demonstrate that interactionsoccur between all of the investigated fission proteins. Bothcolocalization and FRET analyses indicated that the highest levelsof interaction occurred between MiD51 and Drp1, whereas Mffshowed a lower degree of correlation with MiD51. It has beenpreviously shown that Mff can still form foci in cells that have beendepleted of Drp1 (Otera et al., 2010; Friedman et al., 2011),consistent with previous reports that Mff forms mitochondrialconstrictions with the ER independently of Drp1 (Friedman et al.,2011). In contrast to the findings pertaining toMff, we have recently

shown that MiD proteins require the presence of Drp1 in order toassociate within foci (Richter et al., 2014). Our data suggests thatMff functions upstream of MiD proteins, potentially by aiding theformation of constriction sites before recruitment of Drp1 and MiDproteins, which then execute fission. These results are consistentwith a recent study showing that MiD49 enhances the ability ofDrp1 to execute constriction (Koirala et al., 2013). We also foundthat MiD foci can undergo several constriction and relaxation cyclesbefore the final fission event (Fig. 2B; supplementary materialMovie 2). This cycling might be due to partial assembly of thefission machinery at this stage or the requirement of other signalingevents, including post-translational activation of Drp1 (Elgass et al.,2013; Arasaki et al., 2015), the involvement of other components ofthe fission machinery (Hatch et al., 2014) or the binding of co-factors to MiD proteins (Loson et al., 2014; Richter et al., 2014).The exact signal for this awaits further analysis.

The nature of short ER extensionsIn previous studies, both ER and actin have been shown to contactmitochondria at fission sites (Friedman et al., 2011; Korobova et al.,

Fig. 4. Constriction and fission events at MiD51–GFP ER contact sites.(A) Time-lapse imaging of the constriction site (white arrowhead) shown inFig. 2D. The mitochondrion (stained with MitoTracker Deep Red; blue) dividesat theMiD51–GFP foci constriction site. After the scission event, both daughterorganelles remain connected by the ER tubule (expressing mCherry-KDEL,red). (B) MiD51–GFP ER constriction sites do not necessarily undergo fission,but the constriction event (center image, yellow arrowhead) can be reversed(right image, white arrowhead). (C) MiD foci at sites of ER contact are notlimited to mitochondrial constriction sites (white arrowheads) but can thensubsequently lead to mitochondrial constriction (right image, yellowarrowhead). Scale bars: 4 μm (A, left panel of C); 2 μm (B, right panels of C).Times relate to the commencement of observations.

Fig. 5. Mitochondrial constriction sites can be observed upon MiD49single-, and MiD49 and MiD51 double-knockdown but appear to bereduced in MiD49 and MiD51 double-knockdown cells. Mitochondria werestained with MitoTracker Red (MTR; red), and ER was stained with GFP–Sec61.White arrowheads indicatemitochondrial constriction at sites of contactwith the ER. (A) Representative image of a COS-7 cell following knockdown(KD) of MiD49. (B) Representative image of a COS-7 cell following MiD49 andMiD51 double-knockdown (MiD49/51 KD). The number of mitochondrialconstriction sites (arrowheads) was reduced upon double-knockdown ofMiD49 and MiD51 (see supplementary material Table S2). In both panels, theupper row shows overview images, and the bottom row shows magnificationimages. Scale bars: 10 µm (overview images); 2 µm (magnification images).

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2013, 2014). Similar to the electron-dense tethers that have beendescribed by using electron microscopy tomography (Rowland andVoeltz, 2012), by using SXT, we observed short, 80-nm diameterextensions that connected the ER to the mitochondrial membrane.ER sheets and tubules have been reported to exhibit a thickness ofapproximately 60–100 nm (Puhka et al., 2007), suggesting that thefeatures we observed are derived from ER rather than actin.Individual actin fibers have been reported to be much thinner (7–10 nm) (Osborn et al., 1977). However, it cannot be fully excludedthat we observed very thick, or highly absorbent actin bundles,because it has been suggested that actin polymerization and/ormyosin-II-driven condensation of individual fibers into compactring structures have a role in mitochondrial fission (Hatch et al.,2014; Korobova et al., 2014).

Reduced mitochondrial LAC values in cells with fragmentedmitochondrial morphologyCFT-SXT analysis showed that mitochondrial LAC valuesdecreased upon overexpression of MiD proteins. The LAC valuein a SXT-generated reconstruction reflects the amount of soft x-rayabsorption that occurs in that voxel of the specimen, and thereforethe density of carbon and nitrogen (Hanssen et al., 2012). BecauseER and mitochondria comprise lipids and proteins, changes in LACvalues reflect changes to concentrations of either lipids or proteins,or both. The observed reduction of the overall LAC value formitochondria upon low levels of MiD overexpression correlateswith a change in the overall mitochondrial morphology, fromnormal to fragmented; the spherical shape of fragmentedmitochondria indicates the loss of constriction sites and,therewith, the loss of protein-dense areas.Both ER and mitochondrial LAC values decreased upon

mitochondrial fragmentation, but the ER LAC changes were moreprominent. This implies that mitochondrial fragmentation affectsthe connection between the ER and mitochondria not only with

respect to contact sites but, potentially, also with respect tocommunication and component exchange between the twoorganelles. Our results therefore suggest that the role of ER–mitochondria contacts in the regulation of mitochondrialmorphology is not limited to ER-tubule-mediated constriction ofmitochondria before fission events. Either fragmentation ofmitochondria in general or MiD overexpression specificallyappears to decouple or otherwise hinder ER–mitochondriaexchange, which is re-established upon restoration of normalmitochondrial morphology.

Potential use of CFM–SXT to study intracellular changesthroughout the cellCFM–SXT enables the observation of effects that are due to proteinoverexpression throughout the cell, not only with respect tomorphology but also to the composition of various subcellularcompartments, along with variations between those compartments, asdemonstrated by the differential LAC values of ER and mitochondriain our experiments. Compared with confocal fluorescencemicroscopy and the super-resolution techniques that have beendeveloped recently, such as photo-activated localization microscopy(PALM) and stochastic optical reconstruction microscopy (STORM)(Sengupta et al., 2012, 2014; Hensel et al., 2013), CFM–SXTaccesses a variety of subcellular compartments simultaneously withhigher resolution than confocal microscopy, but in the same range asmost super-resolution techniques, and without the need for additionalstaining – apart from that of the protein of interest. Compared withcorrelative electron microscopy, whole cells can be imaged withinseveral minutes without the need to cut thin slices and to subsequentlystitch together the individually recorded images. Additionally, noartificial enhancement of contrast is necessary thanks to the naturallyhigh contrast of soft x-ray imaging in the water window, where waterhas an inherently low x-ray absorption. CFM–SXT thereforerepresents a highly useful technique for intracellular imaging. The

Fig. 6. CFM–SXT analysis reveals short ER extensions thatcontact mitochondria at MiD foci. (A) Two-dimensionalcomputer-generated slice from a reconstruction of a mouselymphoblastoid cell expressing MiD51–GFP (green), generatedusing correlated CFM–SXT. (B) The same computer-generatedslice from the SXT reconstruction as presented in A withoutfluorescence overlay. The orange rectangle outlines the area ofconcentrated MiD51–GFP fluorescence. (C) Magnification of thearea shown in A and B containing a concentration of MiD51–GFP.White arrowheads indicate positions of ER–mitochondria contactsites. (D) Maximum intensity projection of the full 3D SXTreconstruction with the contrast reversed so that features that arelow-absorbing are shaded black and features that are highlyabsorbent are shaded white. (E) ER (green) andmitochondria (red)segmented out and overlaid with the reconstruction. (F) Surface-rendering of segmented cellular features, including the nucleus(orange), lipid droplets (blue), ER (green) and mitochondria (red).(G) Three-dimensional cutaway of the SXT-generatedreconstruction reveals the same 3D location of the MiD51–GFPfluorescence of that shown in A. (H) Detailed view of small ERextensions contacting the mitochondria at the MiD51 foci. Scalebars: 2 µm (A,B,D–F); 400 nm (C); 1 µm (H).

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LAC data from our correlative experiments also demonstrate thepotential use of CFM–SXT to assess – with high accuracy –subcellular changes that result from minor changes in proteinexpression levels, without additional labelling.

MATERIALS AND METHODSPlasmids and reagentsMiD49–GFP, MiD51–GFP, GFP–Drp1 and RNA interference constructshave been described previously (Palmer et al., 2011). GFP–Fis1 has alsobeen described previously (Stojanovski et al., 2004). GFP–Sec61, GFP–Mff(isoform 1), mCherry–Sec61 and mCherry-KDEL were a kind gift from GiaVoeltz (University of Colorado, Boulder, CO). mCherry–Mff, MiD51–mCherry and MiD49–mCherry were cloned from the respective GFPconstructs by removing GFP from the vector and replacing it with themCherry sequence. Red fluorescent protein (RFP)-tagged Drp1 (RFP–Drp1) was cloned using the same approach. Commercial antibodies usedwere against cytochrome c (BD Pharmingen) and suitable secondaryantibodies conjugated to AlexaFluor 647 (Invitrogen).

Cell culture, transfections and treatmentsCOS-7 cells were grown as previously described (Palmer et al., 2011), andtransfections were performed using Lipofectamine 2000 (Invitrogen)according to the manufacturer’s instructions. Cells were incubated with50 nM MitoTracker Red CMXRos (Molecular Probes) or 50 nMMitoTracker Deep Red (Molecular Probes). Single- and double-knockdown was performed as previously described (Palmer et al., 2011).

A female mouse v-Abl-transformed lymphoma B-cell linewas provided byBarbara Panning (University of California, San Francisco, CA) (D’Andreaet al., 1987). The cell line was maintained in RPMI 1640 medium (GIBCO,Invitrogen, Life Technologies, Grand Island, NY) that had been supplemented

with 10% fetal bovine serum (FBS; American Type Culture Collection,Manassas, VA). Cells were grown in vented polystyrene tissue-culture flasks(Corning Incorporated Life Sciences, Tewksbury, MA) in a humidifiedcell culture incubator at 37°C under 5% CO2. For transfection of thelymphoblastoid cell line, 107 cells were spun down and resuspended in 360 µl‘intracellular’ electroporation buffer (ICEB) (Harkin and Hay, 1996). MiD51-GFP plasmid (20 µg) was added, followed by incubation for 10 min at roomtemperature. Then cells were electroporated (BioRad Gene Pulser IIElectroporation System, BioRad Life Sciences, Hercules, CA) with a singlepulse (300 V, 975 µF, τ=25-30 ms). Subsequently cells were resuspended in20 ml of fresh medium (37°C) and grown overnight before further processing(see section Cell preparation for SXT).

Live-cell microscopyConfocal microscopy was performed with a Zeiss confocal microscopeequipped with a ConfoCor 3 system containing avalanche photodiodedetectors using a 40× oil immersion objective or a Zeiss AxioObserverSpinning Disk microscope using a 63× objective. Cells were sustained inDulbecco’s modified Eagle’s medium (DMEM; GIBCO, Invitrogen, LifeTechnologies) with 5% FBS at 37°C and 5% CO2.

FRET analysisFRET detection and analysis was performed using acceptor photobleachingexperiments (Shrestha et al., 2015). GFP and RFP and/or mCherryconstructs of the respective fission proteins were co-transfected into cells.Fluorescence images of the donor and acceptor were recorded before andafter photobleaching of the acceptor. FRET efficiency, E, was calculatedfrom the degree of donor de-quenching according to E=1−(IDA/ID)×100with IDA being the intensity of fluorescence before photobleaching, and IDbeing the intensity of fluorescence after photobleaching.

Fig. 7. CFM–SXT reveals foci of MiD51–GFP fluorescenceat several ER–mitochondria contact sites.(A) Fluorescence confocal slice, (B) virtual section of theSXT-generated reconstruction, and (C) correlative CFM–SXToverlay are shown at four different planes, or virtual sections,within the cell volume. (D) The rightmost panels showmagnifications of the boxed areas in C, which indicateER–mitochondria contact sites that overlap with MiD51–GFPfluorescence foci (white arrowheads). Scale bars: 2 µm (A–C).

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Cell preparation for soft X-ray tomographyElectroporated cells were pelleted by centrifuging (400 g, 5 min), resuspendedin Leibovitz’s L-15 growth medium (GIBCO) that had been supplementedwith 10% FBS. Cells exhibiting MiD51–GFP foci were separated from non-transfected cells, cellular debris and overexpressing cells using fluorescence-activated cell sorting (FACS; BD FACSAria, BD Biosciences, San Jose, CA)by gating for cells with low levels of fluorescence (Palmer et al., 2011). Cellswere sorted directly into L-15 growth medium, which limited their residencetime in FACS sheath fluid (PBS). Cells that had been sorted were pelleted bycentrifuging (400 g, 5 min) and most of the supernatant was removed in orderto increase the cell density. Cells were then pipetted into custom-made glasscapillaries (Parkinson et al., 2013; Smith et al., 2014a) before vitrification byplunging the tip of the specimen capillary into a ∼90-K reservoir of liquidpropane at∼2 m s−1 using a device that had beenmade in-house, as describedpreviously (Smith et al., 2014a).

Cryogenic confocal fluorescence microscopyVitrified lymphoblastoids were imaged using a cryogenic brightfield andconfocal fluorescence microscope. The body of the microscopewas custom-

built to image specimens mounted in the tip of a glass capillary through a∼90-K reservoir of propane. The microscope uses a commercially-availablespinning disk confocal head (CSU-X1, Yokogawa, Tokyo, Japan) andacousto-optical tuneable filter-controlled laser system (Andor LaserCombiner, Model LC-501A). A more detailed description of thisinstrument can be found in Smith et al. (2014a). GFP was excited with alaser at 491 nm and imaged onto an EMCCD camera (iXon DV887ECS-BV, Andor Technologies, Belfast, UK) using a 525/50 emission filter(Chroma Technology Corp., Bellows Falls, VT). Confocal ‘z-stacks’ of thespecimen were taken as the precision-encoded piezo flexure specimen stage(Physik Instrumente, Irvine, CA) was translated through the focal plane ofthe microscope in 0.75-µm steps.

Soft X-ray tomographyThe cryogenic soft x-ray microscope XM-2, located at the National Centerfor X-ray Tomography (http://ncxt.lbl.gov) at the Advanced Light Source(Berkeley, CA) was used to collect the SXT data. During data collection,specimens were kept in a stream of liquid-nitrogen-cooled helium gas tomaintain their cryopreservation and to mitigate radiation damage. Cells ofinterest were chosen from the fluorescence images recorded in the previousstep; these were cells showing the formation of MiD51–GFP foci. A fullangular tilt series was taken of each specimen of interest (180 projectionimages with 1° of rotation between each image). Each image used 150- to200-ms exposure times, depending on the thickness of the specimen. Three-dimensional reconstructions were calculated from the tomographic tilt seriesafter manual alignment of the projection images by tracking fiducial markersusing the IMOD software (Kremer et al., 1996). Iterative tomographicreconstructions were calculated using methods published previously(Stayman and Fessler, 2004a,b; Mastronarde, 2005; Parkinson et al.,2012); LAC values were determined as in described previously (Weiss et al.,2000).

Image processing and analysisAll images were processed using Fiji, Zeiss ZEN lite 2011 (blue edition),Amira (FEI) or Imaris imaging software (Bitplane AG). Soft x-rayreconstructions were segmented into different subcellular compartments(lipid droplets, mitochondria and ER) using the Imaris software (BitplaneAG) ‘Surface’ module to choose different gray level ranges, each of whichrepresents a different subcellular compartment. Segmentation of the nucleuswas performed manually. LAC values for mitochondria and ER,respectively, are defined as the mean LAC inside the respective surfaces.The two 3D datasets – a fluorescence confocal z-stack and an SXTreconstruction – from the same vitrified cell were overlayed in silico, givingthe distribution of MiD51–GFP within the context of the complete 3Dcellular ultrastructure of the cells. Alignment of the cryogenic fluorescenceand SXT datasets was performed manually using Amira’s Multi PlanarViewer module (Version 5.3, Amira, Visage Imaging, San Diego, CA). ER–mitochondria contact sites inMiD single- and double-knockdown cells weredetected using the Imaris ‘Colocalization’ module to build an independentcolocalization channel (supplementary material Fig. S4). ER–mitochondriacontact sites and constriction sites were counted on images of five individualcells per condition. Constriction sites were defined as positions of lowerlocal mitochondrial diameter (Fig. 5A,B, magnification panels, whitearrows).

Fission events were counted on individual 5-min subsets from thecomplete long-term time-lapse series. From the recorded 2D image, wholecells were chosen as the region of interest. The number of mitochondria percell was obtained using the Imaris ‘Surface’ module by thresholding forMitoTracker Red or cytochrome c signal and counting the number ofdetected surfaces in the selected region of interest.

AcknowledgementsWe thank Professor Barbara Panning (University of California, San FranciscoBiochemistry and Biophysics, San Francisco, CA) for providing the mousev-Abl-transformed lymphoma cell line; Gia Voeltz for providing the ER markerplasmids; and Laura Osellame, Abeer Singh and Catherine Palmer for advice andreagents.

Fig. 8. SXT LAC values change upon MiD51-GFP overexpression.(A,B) Three-dimensional surface-rendering of SXT segmentations showing afragmented (A) and a normal (B) mitochondrial network. (C) The relative SXTLAC values of mitochondria decrease upon MiD51–GFP overexpression. Forcalculation of the relative LAC values, the absolute LAC mean values of ERand mitochondria in control cells were set to 100% and used for normalization.Error bars represent mean±s.e.m. (D) The difference between the LAC valuesof the ER and mitochondria changed with the level of MiD51–GFP expression,concomitant with mitochondrial morphology changes. Differential LAC valuesincreased at very low expression levels and consequent fragmentation of themitochondrial network (red-boxed area and panel A) and decreased uponslightly higher expression levels, as the mitochondrial network returns tonormal (green-boxed area and panel B). The fluorescence intensity in Drepresents the mean fluorescence intensity of MiD51–GFP as determined ineach foci and averaged per cell. The fluorescence intensity per foci waschosen rather than per cell to account for differently sized cells and the differentnumbers of foci per cell. Scale bars: 3 µm (A,B).

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Competing interestsThe authors declare no competing or financial interests.

Author contributionsK.D.E. prepared samples for and performed all confocal experiments. K.D.E. andE.A.S. prepared samples for and performed all CFM–SXT experiments. All authorsinterpreted the results and wrote the manuscript.

FundingThis work was supported with funds from the ARCCentre of Excellence for CoherentX-ray Science (CXS) and the National Health and Medical Research Council [grantnumber 1049968 toM.T.R.]. The National Center for X-ray Tomography is supportedby the National Institute of General Medical Sciences of the National Institutes ofHealth [grant number P41GM103445 to C.A.L.]; and the US Department of Energy,Office of Biological and Environmental Research [grant number DE-AC02-05CH11231 to C.A.L.]. C.A.L., M.A.L. and E.A.S. are supported by the Gordon andBetty Moore Foundation [grant number 3497 to C.A.L.]. Deposited in PMC forrelease after 12 months.

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.169136/-/DC1

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