Atypical mitochondrial fission upon bacterial infection
Fabrizia Stavrua,b,c,1, Amy E. Palmera,b,c,2, Chunxin Wangd, Richard J. Youled, and Pascale Cossarta,b,c,1
aUnité des Interactions Bactéries-Cellules, Institut Pasteur, 75015 Paris, France;bInstitut National de la Santé et de la Recherche Médicale, Unité 604, 75015
Paris, France;cInstitut National de la Recherche Agronomique Unité Sous Contrat 2020, 75015 Paris, France; anddBiochemistry Section, Surgical
Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892
Contributed by Pascale Cossart, August 21, 2013 (sent for review July 16, 2013)
We recently showed that infection by Listeria monocytogenes
causes mitochondrial network fragmentation through the se-
creted pore-forming toxin listeriolysin O (LLO). Here, we examine
factors involved in canonical fusion and fission. Strikingly, LLO-
induced mitochondrial fragmentation does not require the traditional
fission machinery, as Drp1 oligomers are absent from fragmented
mitochondria following Listeria infection or LLO treatment, as the
dynamin-like protein 1 (Drp1) receptor Mff is rapidly degraded, and
as fragmentation proceeds efficiently in cells with impaired Drp1
function. LLO does not cause processing of the fusion protein optic
atrophy protein 1 (Opa1), despite inducing a decrease in the mito-
chondrial membrane potential, suggesting a unique Drp1- and
Opa1-independent fission mechanism distinct from that triggered
by uncouplers or the apoptosis inducer staurosporine. We show
that the ER marks LLO-induced mitochondrial fragmentation sites
even in the absence of functional Drp1, demonstrating that the
ER activity in regulating mitochondrial fission can be induced by
exogenous agents and that the ER appears to regulate fission by
a mechanism independent of the canonical mitochondrial fission
mitochondrial dynamics|live cell imaging|actin
thetic intermediates and energy to innate immune signaling and
cellular calcium buffering or the storage of proapoptotic com-
ponents (1). To perform these diverse functions, mitochondria
respond to cellular cues and display a highly variable and dynamic
morphology, constantly undergoing fusion and fission. It is be-
coming increasingly clear that mitochondrial dynamics and func-
tion are deeply interconnected, and mitochondrial dysfunction is
associated with a range of diseases.
Wild-type mitochondrial morphology and function are main-
tained by a balance between mitochondrial fusion and fission.
Fusion allows exchange of genetic material between single mi-
tochondria and is mediated by two large guanosine triphosphate
phosphohydrolases (GTPases) embedded in the outer membrane
(mitofusin 1 and 2) and an inner membrane GTPase, Opa1 (2).
Deletion mutants affecting these three proteins accumulate dys-
functional mitochondria, leading to neurodegenerative phenotypes
and different forms of myopathy (1, 3).
Mitochondrial fusion is balanced by fission, which is essential
to ensure proper distribution of mitochondria and energy supply
to daughter cells in mitosis or within a single cell. This necessity
is particularly evident in neurons, where fission defects prevent
efficient mitochondrial transport to synapses, the crucial sites of
energy consumption (4, 5). The physiological importance of
mitochondrial fission is further highlighted by its essential role in
embryonic development in mice and nematodes (6–8).
Mitochondrial fission is thought to be accomplished by the
dynamin-like protein Drp1, a mainly cytosolic protein that is
recruited to future fission sites, where it oligomerizes to form
at several levels: by initial ER- and actin-mediated mitochondrial
constriction (9, 10), leading to the accumulation of the membrane-
bound Drp1 receptor Mff and by several posttranslational mod-
ifications of Drp1, which modulate its activity (11).
itochondria are essential organelles that perform a multi-
tude of functions, ranging from the production of biosyn-
Listeria monocytogenes is a foodborne pathogen capable of
invading nonphagocytic cells, where it can replicate and spread.
The pathogenic potential of L. monocytogenes correlates with the
expression of several virulence genes (12). One of the most im-
portant virulence factors is listeriolysin O (LLO), a highly reg-
ulated secreted pore-forming toxin (reviewed in ref. 13). LLO
belongs to the family of cholesterol-dependent cytolysins (CDCs),
most of which are produced by extracellular bacteria such as
Streptococci or Clostridia. CDCs oligomerize on cholesterol-con-
taining membranes to form nonselective ion-permeable pores of
variable sizes (14) that act in concert with bacterial phospholipases
to allow bacterial escape from the phagosome. More recently, LLO
has been found to have several intracellular and extracellular roles
that extend beyond phagosomal escape. For example, we have
shown that infection with L. monocytogenes causes fragmentation of
the host mitochondrial network by action of its pore-forming toxin
LLO before bacterial entry (15).
In this study, we demonstrate that LLO-induced mitochondrial
fragmentation does not follow canonical pathways, because it is
independent of key fusion and fission components, such as Opa1
and Drp1. We demonstrate that the ER marks mitochondrial
fragmentation sites even in the absence of functional Drp1, and
that the actin cytoskeleton also facilitates fragmentation. LLO-
(CCCP)] and apoptosis inducers (such as staurosporine), revealing
a unique pathway for mitochondrial fragmentation that can be in-
duced by an exogenous agent.
Mitochondria are dynamic organelles that constantly fuse and
fragment while acting as central hubs of energy production,
apoptosis regulation, and Ca++signaling, therefore emerging
as potential targets of pathogens. We previously showed that
the foodborne bacterial pathogen Listeria monocytogenes
interferes with the dynamics and function of the host cell mi-
tochondrial network via the bacterial toxin listeriolysin O (LLO).
In this study, we analyze the effects of LLO on key players
known to be involved in mitochondrial dynamics and show
that the fission protein dynamin-like protein 1 (Drp1) is not
essential for LLO-induced fragmentation of the mitochondrial
network, whereas the endoplasmic reticulum (ER) plays an
important role, suggesting a unique Drp1-independent and
ER-dependent mechanism that is different from the canonical
Author contributions: F.S., A.E.P., and P.C. designed research; F.S. and A.E.P. performed
research; C.W. and R.Y. contributed new reagents/analytic tools; F.S., A.E.P., and P.C.
analyzed data; and F.S., A.E.P., R.Y., and P.C. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or fabrizia.
2Permanent address: Department of Chemistry and Biochemistry and BioFrontiers Insti-
tute, University of Colorado, Boulder, CO 80309.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| October 1, 2013
| vol. 110
| no. 40
Results and Discussion
Opa1 Is Not Processed upon LLO-Induced Mitochondrial Fragmentation.
Mitochondrial fragmentation can result either from an increase in
fission activity, an inhibition of fusion, or both. One possible
pathway for mitochondrial fragmentation is rapid proteolytic
processing of the inner membrane GTPase Opa1, as observed
upon uncoupler [CCCP or trifluorocarbonyl cyanidephenylhy-
drazone (FCCP)]-induced mitochondrial fragmentation, which
results in a fusion block and an ensuing mitochondrial frag-
mentation due to unopposed fission events (16–18). In contrast
to CCCP, Opa1 is not processed upon LLO treatment (Fig. 1A),
indicating that LLO-induced mitochondrial fragmentation is not
caused by a block in inner membrane fusion. This finding is con-
sistent with the rapid fission kinetics that we have described, which
suggests an active fission mechanism (15). We therefore addressed
whether LLO induces fragmentation by recruiting the key fission
Mitochondrial Drp1 Oligomers Dissociate upon Infection or LLO
Treatment. Fission-inducing agents such as uncouplers or the
apoptosis inducer staurosporine appear to act by recruiting Drp1
to fragmenting mitochondria (Fig. 1C; refs. 19–21). In contrast,
LLO treatment and L. monocytogenes infection were character-
ized by a decrease in Drp1 puncta associated with mitochondria
(Fig. 1 C and D and Fig. S1A). This phenomenon was unique to
LLO and L. monocytogenes infection because a number of other
fission-inducing agents (the protonophore FCCP, the potassium
ionophore valinomycin, and the detergent digitonin) did not
show a decrease in mitochondria-associated Drp1 (Fig. S1B). In-
terestingly, potassium accumulation into mitochondria was
invoked by Dimmer et al. to explain Drp1-independent frag-
mentation upon silencing of the inner membrane protein
LETM1, because the potassium ionophore nigericin could re-
store tubular morphology of mitochondria in these cells (22). LLO
may cause an intramitochondrial potassium imbalance that could
impinge on mitochondrial morphology, although it does not ex-
plain the dissociation of Drp1 from mitochondria. Western blot
analysis of cell extracts showed no decrease in total Drp1 levels in
LLO-treated (Fig. 1B and Fig. S1C) cells. Use of two different
antibodies against endogenous Drp1 on fixed samples (Fig. S1A)
and live cell imaging of overexpressed Drp1-GFP (23) (Fig. S1D)
confirmed a strong decrease in mitochondria-associated Drp1
staining upon LLO treatment.
LLO Effect on the Drp1 Receptors Fis1 and Mff. Drp1 requires several
membrane-anchored receptor proteins to bind to the mitochondrial
surface. We thus investigated whether the LLO-induced decrease
in mitochondria-associated Drp1 staining was due to modulation of
its receptors Fis1 (24–26) and Mff (27) at the mitochondrial
Upon LLO treatment or infection with L. monocytogenes, there
was no detectable decrease in the association of Fis1 with mito-
chondria by immunofluorescence or total Fis1 protein levels by
Western blotting (Fig. S2 A and B), suggesting that loss of mi-
tochondrial Drp1 does not depend on a decrease in Fis1. In
contrast, LLO treatment and L. monocytogenes infection induced
a decrease in Mff protein as observed in total cell extracts by
Western blotting and at the single-cell level by immunofluores-
cence and live-cell imaging (Fig. 2). The level of Mff mRNA was
unaltered, suggesting posttranscriptional modulation of Mff (Fig.
S2C). Mff did not decrease upon uncoupling (Fig. 2 A and B) nor
when LLO treatment was performed in the absence of extracel-
lular calcium (Fig. S2D). However, ionophore-induced calcium
influx was not sufficient to induce Mff degradation (Fig. S2D),
suggesting that calcium influx alone is not sufficient to cause Mff
degradation upon LLO treatment. LLO-induced degradation of
Mff appeared proteasome independent, because it was not pre-
vented by treatment with MG132 and lactacystin (Fig. S2E),
mentation. (A) Western blot of HeLa cells treated for 1, 5, or 10 min with
6 nM LLO or for 10 min with 10 μM CCCP and probed for Opa1, which is
readily processed upon CCCP, but not LLO treatment. Tubulin was used as
a loading control. (B) HeLa cells were treated for 30 min with 10 μM CCCP,
for 1.5 h with 1.2 μM staurosporine (sts), or with 6 nM LLO for the indicated
timepoints, and total cell lysates are analyzed by Western blotting, showing
no apparent decrease in total Drp1 levels upon LLO or drug treatment.
GAPDH was used as a loading control. (C) Immunofluorescence labeling of
the same cells analyzed in B. Drp1 is shown in green (Drp1NT), mitochondria
in red (Tom20), and nuclei in blue (DAPI). Staining reveals that mitochondrial
Drp1 oligomers are lost upon LLO treatment (decrease in green puncta) but
are retained upon CCCP or staurosporine treatment. (D) HeLa cells were
infected for 1 h with L. monocytogenes or L.monocytogenes Δhly (LLO−) at
an MOI of 50 and stained for Drp1 (green), mitochondria (CoxIV; red), and
nuclei/bacteria with DAPI (blue). Wild-type L. monocytogenes induces mi-
tochondrial fragmentation and loss of mitochondria-associated Drp1,
whereas infection with the Δhly mutant does not overtly affect Drp1. For C
and D, the white box indicates a 2× enlarged region, which is shown below.
(Scale bars: 10 μm.)
Analysis of Opa1 and Drp1 upon LLO-induced mitochondrial frag-
| www.pnas.org/cgi/doi/10.1073/pnas.1315784110 Stavru et al.
consistent with previous reports suggesting that LLO induces pro-
tein degradation through an as-yet-unknown pathway (28, 29).
Timelapse studies of Mff-GFP in HeLa cells treated with LLO in-
dicate that Mff degradation begins almost immediately after LLO
addition (Fig. 2D and Fig. S2F) and before mitochondrial frag-
mentation. Indeed, LLO induced mitochondrial fragmentation in
primary receptors for anchoring Drp1 at mitochondria, this result
further suggesting that Drp1 is not required at fragmentation sites.
LLO-Induced Mitochondrial Fragmentation Is Drp1 Independent. Drp1
is described as the key and essential factor mediating mitochon-
drial fission. Given that we observed a decrease rather than an
augmentation of the Drp1 signal at mitochondria upon infection
or LLO treatment, we tested whether Drp1 is essential for LLO-
induced mitochondrial fragmentation by using dominant-negative
K38A Drp1 in HeLa cells, Drp1−/−mouse embryonic fibroblasts
(MEFs) (6), and HCT116 Drp1−/−cells. Strikingly, there was
no difference in mitochondrial fragmentation compared with
controls in cells transfected with K38A Drp1, as well as both
Drp1 knockout cell lines (Fig. 3), indicating that LLO- and
L. monocytogenes-induced mitochondrial fragmentation is truly
Drp1 independent. In contrast, CCCP-induced mitochondrial
fragmentation was substantially reduced in the Drp1 knockout cell
lines. Staining of fixed cells, and live cell imaging with fluorescent
protein-tagged markers, with both matrix and outer-membrane
markers confirmed complete abscission of mitochondria upon
LLO treatment (Fig. 3C and Fig. S3 A and B). Furthermore, upon
incubation of wild-type and knockout cells with LLO for different
amounts of time, similar percentages of cells with fragmented
mitochondria were scored, indicating that the loss of Drp1 did not
delay the kinetics of LLO-induced mitochondrial fragmentation
The Drp1 dependence of mitochondrial fission induced by
uncouplers or activators of apoptosis is a matter of debate. For
example, it has been demonstrated that transfection of K38A
Drp1 does not block uncoupling-induced formation of disk-
shaped mitochondria (30), and deletion of Drp1 does not affect
the number of cells with fragmented mitochondria upon in-
duction of apoptosis (8). However, several other groups found
that overexpression of K38A Drp1 or Drp1 depletion prevented
or diminished apoptosis- or CCCP-induced mitochondrial frag-
mentation (6, 31–33). It has been suggested that this discrepancy
may arise because fragmentation depends both on the cell type
and the physiological cue used to induce apoptosis (6). In con-
trast, we see no measureable perturbation of fragmentation upon
knock out of Drp1 or expression of dominant-negative Drp1,
indicating that fragmentation induced by LLO is truly independent
of functional Drp1. Moreover, LLO-induced fragmentation is not
associated with cytochrome c release and activation of canonical
apoptosis (15), suggesting that this mechanism of fragmentation
is distinct from the one that activates uncouplers or apoptosis
inducers. Opening of the mitochondrial transition pore (MTP)
may be invoked to explain LLO-induced mitochondrial frag-
mentation. We do not favor this hypothesis because of previous
experiments confirming that the mitochondrial inner membrane
remains intact upon LLO treatment, as respiration can resume
(15). Furthermore, we could not inhibit the fragmentation
phenotype with the MTP inhibitor cyclosporine A (15).
The ER Marks Sites of Mitochondrial Fragmentation Even in the
Absence of Functional Drp1. It has recently been shown that mi-
tochondrial fission occurs at contact sites with the ER, and that
mitochondria are constricted at such contact sites (9). Although
the mechanism of how the ER regulates fission was not identi-
fied, it was suggested that ER proteins may participate in fission
and/or that mitochondrial constriction by the ER facilitates re-
cruitment of the mitochondrial fission machinery (i.e., Drp1).
We thus explored whether LLO-induced and Drp1-independent
mitochondrial fragmentation was marked by ER-mitochondrial
contact sites. Timelapse studies of U2OS epithelial cells expressing
the ER marker Sec61β-GFP (34) upon treatment with LLO
revealed that the ER marked sites of mitochondrial fragmenta-
tion in 87 ± 8% of events (Fig. 4). Transfection of U2OS cells
with K38A Drp1 or siRNA to knockdown Drp1 did not prevent
LLO-induced mitochondrial fragmentation. Intriguingly, there
was no change in the percent of fragmentations marked by ER-
mitochondria contact sites (96 ± 6% for K38A; 82 ± 6% for
siRNA), demonstrating that the ER plays an active role in reg-
ulating mitochondria fission even in the absence of Drp1.
Actin Is Involved in LLO-Induced Mitochondrial Fragmentation. Actin
has been shown to play a role in drug-induced mitochondrial
fission in mammalian cells, although the effect appears to be
stimulus dependent because oligomycin and cyclosporine A, but
not CCCP, depended on F-actin for efficient mitochondrial
network fragmentation (30). We tested whether the action of
the Drp1 receptor Mff. (A) HeLa cells were treated for 10 min with 6 nM LLO,
30 min with 10 μM CCCP, or infected at an MOI of 50 with the indicated
strains for 1 h. Representative Western blot of Mff and GAPDH (loading
control) on total cell lysates shows a decrease in Mff levels upon LLO treat-
ment or infection with wild-type L. monocytogenes. (B) Quantification of
Immunofluorescence of HeLa cells treated for 10 min with LLO and stained for
Mff (green), mitochondria (Tom20; red), and DNA (DAPI; blue). Although
there is heterogeneity in staining of endogenous Mff, there is a significant
decrease in Mff staining upon LLO treatment. White box indicates a 4× en-
larged region that is shown below. (D) Heterogeneous behavior of mito-
chondria-associated Mff puncta observed by live cell imaging. Timelapse
spinning disk confocal images of HeLa cells expressing Mff-GFP (green) and
TagBFP-mito (red). Data were collected every 2 s 2 nM LLO was added at time
t = 50 s White arrowheads mark Mff-GFP puncta that are rapidly lost from
mitochondria. Yellow arrowhead marks an Mff dot that disappears before
fragmentation and green arrowhead marks one that remains associated with
mitochondria over the time course. (Scale bars: C, 10 μm; D, 2.6 μm.)
LLO treatment and L. monocytogenes infection cause a decrease in
Stavru et al. PNAS
| October 1, 2013
| vol. 110
| no. 40
LLO on mitochondria required the presence of F-actin and
found that actin depolymerization by cytochalasin D reduces the
number of cells that display a fragmented mitochondrial network
upon LLO treatment (Fig. 5). Given that Drp1 is dispensable for
LLO-induced mitochondrial fragmentation, we hypothesize that,
in addition to contributing to Drp1 recruitment to mitochondria
as observed by ref. 30, actin may also facilitate mitochondrial
fragmentation by preconstricting or physically stretching mito-
chondria independently of Drp1.
In summary, we have shown that in contrast to uncouplers and
apoptosis-inducing agents, mitochondrial fragmentation caused by
Listeria via its toxin LLO proceeds in a Drp1-independent manner.
LLO-induced mitochondrial fragmentation concurs with deg-
radation of the Drp1-receptor Mff, causing a loss of Drp1 at
mitochondria. Drp1 has been proposed to be a multifunctional
protein that contributes to peroxisomal and possibly also to
ER fission (35, 36), as well as possessing a putative GTPase-
independent role in cell death (37). Its modulation by bacterial
infection might therefore represent a means to act on several
organelles and cellular functions in a coordinated fashion.
The data presented in this study suggests that actin and the ER
play an active role in regulating LLO-induced mitochondrial fis-
sion. Recently, the ER-localized formin INF2 has been proposed as
a link between the ER and actin at mitochondrial constriction sites
(10), but it remains to be clarified whether this protein plays a role
in LLO-induced mitochondrial fragmentation. Although LLO acts
on mitochondrial dynamics before bacterial invasion and we could
not detect overt LLO accumulation on intracellular structures by
confocal imaging (15), we cannot exclude that a fraction of LLO
inserts into mitochondria-associated membranes enriched in cho-
lesterol (38), where it could modulate mitochondria-ER contact
sites or their protein components, such as Mfn2 (39). Upon LLO
treatment, ER-mediated mitochondrial fission occurs even in the
absence of Drp1, indicating that either ER proteins may contribute
to mitochondrial abscission or that the ER recruits another as-yet-
unidentified mitochondrial fission machinery that exists in mam-
Given that among the exogenous agents that induce mitochon-
drial fragmentation LLO is unique in its ability to act rapidly,
without activating canonical apoptosis, and in a Drp1-independent
manner, we suggest it could serve as an unprecedented tool to
understand the regulation and function of mitochondrial dynamics
proteins. Indeed, efforts in this field should aim toward the
identification of the molecule(s) that facilitate mitochondrial
fragmentation upon LLO treatment in the absence of Drp1
and the signaling cascade that activates such fragmentation.
Materials and Methods
Reagent references, detailed experimental protocols, image and statistical
analysis, and nonstandard abbreviations used in this study are provided in
SI Materials and Methods.
chondrial-fragmentation in Drp1−/−cells. (A) In-
fection of HeLa cells transfected with K38A-HA
Drp1 with L. monocytogenes for 1 h or treatment
with 6 nM LLO for 10 min induces mitochondrial
fragmentation. Immunofluorescence images show
mitochondria (CoxIV; red) and K38A-HA Drp1 (anti-
HA; green). Asterisk marks transfected cells, as
identified by immufluorescence. The white box
indicates the portion of the image enlarged 4× and
shown below. (Scale bars: 20 μm.) (B) Immunofluo-
rescence of mitochondria (CoxIV; red) and nuclei
(DAPI; blue) upon treatment of WT or Drp1−/−MEFs
with either 6 nM LLO (10 min) or 10 μM CCCP
(30 min). (C) The percentage of cells displaying a frag-
mented mitochondrial network upon CCCP or LLO
treatment was quantified in five or six randomly
chosen fields of view (n > 150 cells) per experiment.
Pooled data from three independent experiments is
shown (**P < 0.003, one-tailed Student t test). (D)
Immunofluorescence of mitochondrial matrix (cyto-
chrome c; red), mitochondrial outer membrane
(Tom20; green), and nuclei (DAPI; blue) upon treat-
ment of WT or Drp1−/−HCT116 cells with either 6 nM
LLO (10 min) or 10 μM CCCP (30 min). White box
indicates 2× enlarged region shown below. (Scale bars:
LLO and L. monocytogenes induce mito-
Drp1. (A) U2OS Sec61β-GFP cells were treatedwith 2 nMLLO and examinedlive
by confocal microscopy. Representative images of mitochondria (DsRed2-mito;
red)and ER(Sec61β-GFP;green)areshown. Arrowheads indicate mitochondrial
fragmentation sites marked by ER-mitochondria contact sites. Experiments
were repeated a minimum of three times. (Scale bars: 2.6 μm.) (B) Quantifica-
tion of mitochondrial fragmentations marked by ER-mitochondrial contact
sites. Analysis was carried out as described in Materials and Methods.
The ER marks sites of mitochondrial fragmentation independent of
| www.pnas.org/cgi/doi/10.1073/pnas.1315784110Stavru et al.
Bacterial Infections. For infection experiments, bacteria were grown over-
night in brain-heart infusion medium (Difco; BD Biosciences) at 37 °C, sub-
cultured at a 1:10 dilution until an optical density of 0.8–1 was reached,
washed three times in medium without serum, and added to cells for 1 h at
a multiplicity of infection (MOI) of 50. The strains used were as follows: wild-
type L. monocytogenes (EGD, BUG600), L. monocytogenes Δhly (EGD,
BUG2132), and L. innocua overexpressing InlB (BUG1642).
Cell Culture, Drug Treatments, and Transfection. HeLa CCL2 cells were cultured
according to American Type Culture Collection guidelines. MEF (kind gift
from K. Mihara, Kyushu University, Japan) and U2OS Sec61β-GFP cells (kind
gift from Yoko Shibata and Tom Rapoport, Harvard Medical School, Boston;
for the Sec61β-GFP plasmid, see ref. 34) were maintained in DMEM supple-
mented with 10% (vol/vol) FCS. HCT116 cells were maintained in McCoys 5A
medium supplemented with 10% FCS, nonessential amino acids, and gluat-
amine. All cells were maintained under 10% (vol/vol) CO2. Media/additives
were purchased from Invitrogen. Drugs were purchased from Sigma-Aldrich
and incubated in full medium unless otherwise stated. Drugs were incubated
at the following concentrations and times: 10 μM CCCP for 30 min, 0.1 μM
valinomycin for 30 min, 1.2 μM staurosporine for 90 min, 0.05% (wt/vol)
digitonin for 5 min, 20 μM MG132 and 20 μM lactacystine for 90 min, 2.5μM
cytochalasin D for 30 min (HeLa) and 1 μM for 20 min (MEF), and 2–6 nM LLO
for 10 min (in absence of FCS). DNA transfection (3.5-cm wells) was performed
with FuGENE HD (Roche) according to the manufacturer’s instructions. The
following plasmids were used for live-cell imaging: pDsRed2-mito (Clontech;
0.2 μg), Mff-GFP (ref. 9; 0.125 μg), mCherry-Drp1 (ref. 9; 0.1 μg), Drp1-GFP (ref.
23; 0.5 μg), BFP-mito (ref. 9; 0.5 μg), OM-GFP (“Mito-GFP”, ref. 40; 1 μg), and
K38A-HA Drp1 (ref. 23; 4 μg). siRNA transfection was performed with Oligo-
fectamine (Invitrogen) according to the manufacturer’s instructions, using
Drp1-specific sequences described in ref. 15.
After treatment, cells were either analyzed with a spinning disk micro-
scope for live-cell imaging or processed for immunofluorescence with the
antibodies indicated in the respective figure legends (see SI Materials and
Methods for antibody catalog numbers and for detailed live cell imaging
Quantitative RT-PCR. Total RNA was extracted using the RNeasy kit (Qiagen)
and treated with TurboDNase (Ambion). cDNA was synthetized from 500 ng
of RNA with the RT2cDNA synthesis kit (Qiagen). Standard quantitative RT-
PCR was performed with the EvaGreen kit (Bio-Rad) on a Bio-Rad CFX ma-
chine, and gene expression was calculated by the 2^(-ΔΔ Ct) method. Two
independent primer sets were used for MFF. Data represent the mean of
two biological replicates and three technical replicates.
Western Blotting. Cells were lysed with 2× Laemmli loading buffer (124 mM
Tris·HCl at pH 6.8, 4% (vol/vol) SDS, 20% (wt/vol) glycerol, 0.02% bromo-
phenol blue, and 0.03% DTT), and genomic DNA was digested with Ben-
zonase (Novagen). Proteins were separated by SDS/PAGE (Bio-Rad),
transferred by wet transfer to a nitrocellulose membrane (GE Healthcare)
that was blocked in 10% (wt/vol) milk; antibodies were incubated in milk at
dilutions indicated by the manufacturers. Quantification of Western blots
was accomplished by using the Gel Analyzer plugin in ImageJ. The Mff signal
was normalized to GAPDH signal, and ≥3 independent experiments were
ACKNOWLEDGMENTS. We thank Dr. K. Mihara for Drp1 knockout MEFs,
Drs. Yoko Shibata and Tom Rapoport for U2OS-Sec61βGFP cells, and Drs. Gia
Voeltz and A. van der Bliek for constructs; Dr. Tham Tho-Nam for excellent
technical support and the Plate-Forme Imagerie Dynamique staff at Institut
Pasteur for support; Dr. Ascel Samba-Louaka for critical reading of the man-
uscript; and Drs. Janet Shaw and Aurélien Roux for insightful discussions.
This work received financial support from the Institut Pasteur, Institut Na-
tional de la Santé et de la Recherche Médicale Unité 604, Institut National de
la Recherche Agronomique Unité Sous Contrat 2020, Fondation Louis Jeantet,
European Research Council Advanced Grant 233348 MODELIST, and Agence
Nationale pour la Recherche (ERANET Pathogenomics Grant LISTRESS and
Grant Blanc MITOPATHO). F.S. was supported by postdoctoral fellowships
from European Molecular Biology Organization and the Fondation pour la
Recherche Médicale. C.W. and R.Y. are supported in part by the Intramural
Research Program of the National Institutes of Neurological Disorders and
Stroke, National Institutes of Health. P.C. is a Howard Hughes Medical In-
stitute Senior International Research Scholar.
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