Atypical mitochondrial fission upon bacterial infection

Article (PDF Available)inProceedings of the National Academy of Sciences 110(40) · September 2013with27 Reads
DOI: 10.1073/pnas.1315784110 · Source: PubMed
We recently showed that infection by Listeria monocytogenes causes mitochondrial network fragmentation through the secreted 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 mitochondrial 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 machinery.
Atypical mitochondrial ssion upon bacterial infection
Fabrizia Stavru
, Amy E. Palmer
, Chunxin Wang
, Richard J. Youle
, and Pascale Cossart
Unité des Interactions Bactéries-Cellules, Institut Pasteur, 75015 Paris, France;
Institut National de la Santé et de la Recherche Médicale, Unité 604, 75015
Paris, France;
Institut National de la Recherche Agronomique Unité Sous Contrat 2020, 75015 Paris, France; and
Biochemistry Section, Surgical
Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892
Contributed by Pasca le 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 in volved in canonical fusion and ssion.Strikingly,LLO-
induced mitochondrial fragmentation does not require the traditional
ssion 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 efciently 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, sugg esting a unique Drp1- and
Opa1-independent ssion mechanism distinct from that triggered
by uncouplers o r t he ap opto sis inducer stauros pori ne . We sh ow
that the ER marks LLO-induced mitochondrial fragmentation sites
even in the absence of functional Drp1, demonstrating that the
ER activity in regulating mitochondrial ssion can be induce d by
exogenous agents and that the ER appears t o regulate ssion by
a mechanism independent of the canonical mitochondrial ssion
mitochondrial dynamics
live cell imaging
itochondria are essential organelles that perform a multi-
tude of functions, ranging from the production of biosyn-
thetic intermediates and energy to innate immune signaling and
cellular calcium buffering or the sto rage of proapoptotic com-
ponents (1). To perform these di verse functions, mitochondria
respond to cellular cues and display a highly variable and dynamic
morphology, constantly undergoing fusion and ssion. 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 ssion.
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 ssion, 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 ssion defects prevent
efcient mitochondrial transport to synapses, the crucial sites of
energy consumption (4, 5). The physiological importance of
mitochondrial ssion is further highlighted by its essential role in
embryonic development in mice and nematodes (68).
Mitochondrial ssion is thought to be accomplished by the
dynamin-like prote in Drp1, a mainly cytosolic protei n that is
recruitedtofuturession sites, where it oligomerizes to form
spirals that constrict mitochondria. Mitochondrial ssion is regulated
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-
ications of Drp1, which modulate its activity (11).
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 thephagosome.Morerecently,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 ssion 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-
induced fragmentation is distinct from that observed upon treatment
with uncouplers [such as carbonyl cyanide m-chlorophenylhydrazone
(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 ssion 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
ssion machinery.
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 conict of interest.
Freely available online through the PNAS open access option.
To whom correspondence may be addressed. E-mail: or fabrizia.
Permanent address: Department of Chemistry and Biochemistry and BioFrontiers Insti-
tute, University of Colorado, Boulder, CO 80309.
This article contains supporting information online at
1073/pnas.1315784110/-/DCSupplemental. PNAS
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
ssion 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 triuorocarbonyl cyanidephenylhy-
drazone ( FCCP)]-induced mitochondrial fragmentation, which
results in a fusion block and an ensuing mitochondrial frag-
mentation due to unopposed ssion 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 nding is con-
sisten t with the rapid ssion kinetics that we have described, which
suggests an active ssion mechanism (15). We therefore addressed
whether LLO induces fragmentation by recruiting the key ssion
protein Drp1.
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. 1921). 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
ssion-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 xed samples (Fig. S1A)
and live cell imaging of overexpressed Drp1-GFP (23) (Fig. S1D)
conrmed 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 (2426) and Mff (27) at the mitochondrial
outer membrane.
Upon LLO treatment or infection with L. monocytogenes, there
was no detectable decrease in the association of Fis1 with mito-
chondria by immunouorescence 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 immunouores-
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
inux was not sufcient to induce Mff degradation (Fig. S2D),
suggesting that calcium inux alone is not sufcient 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),
Fig. 1. Analysis of Opa1 and Drp1 upon LLO-induced mitochondrial frag-
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) Immunouorescence 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
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.)
| Stavru et al.
consistent with previous reports suggesting that LLO induces pro-
tein d egradation 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
HCT116 cells (Fig. S2G). Given that Mff serves as one of the
primary receptors for anchoring Drp1 at mitochondria, this result
indicates Drp1 may be lost from mitochondria before fragmentation,
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 ssion. 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 broblasts
(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 xed cells, and live cell imaging with uorescent
protein-tagged markers, with both matrix and outer-membrane
markers conrmed 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
(Fig. S3C).
The Drp1 dependence of mitochondrial ssion 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, 3133). 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
induc ers. Opening of the mitochondrial transition pore (MTP)
may be in voked to explain LLO-induced mitochondrial frag-
menta tion. We do not favor this hypothesis because of previous
experiments conrming that the mitochondrial inner membrane
remains intact upon LLO treatment, as respiration can resume
(15). Furthermor e, we could not inhib it 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 ssion 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 ssion was not identi-
ed, it was suggested that ER proteins may participate in ssion
and/or that mitochondrial constriction by the ER facilitates re-
cruitment of the mitochondrial ssion 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 ssion 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
ssion in mammalian cells, although the effect appears to be
stimulus dependent because oligomycin and cyclosporine A, but
not CCCP, depended on F-actin for efcient mitochondrial
network fragmentation (30). We tested whether the action of
Fig. 2. LLO treatment and L. monocytogenes infection cause a decrease in
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. mono cytogenes.(B) Quantication of
Western blots from >3 independent experiments (***P < 0.001; *P < 0.05). (C)
Immunouorescence 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 signicant
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,10μm; D,2.6μm.)
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 ssion (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
organ elles and cellular functi ons 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 s-
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 claried 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 mitochon dria-ER contact
sites or their protein components, such as Mfn2 (39). Upon LLO
treatment, ER-mediated mitochondria l ssionoccurseveninthe
absence of Drp1, indicating that either ER proteins may contribute
to mitochondrial abscission or that the ER recruits another as-yet-
unidentied mitochondrial ssion machinery that exists in mam-
malian cells.
Given that among the exogenous agents that induce mitochon-
drial f ragmentation 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 eld should aim toward t he
identication of the molecule(s) that facilitate mitochond rial
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.
Fig. 3. LLO and L. monocytogenes induce mito-
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. Immunouorescence images show
mitochondria (CoxIV; red) and K38A-HA Drp1 (anti-
HA; green). Asterisk marks transfected cells, as
identied by immuuorescence. The white box
indicates the portion of the image enlar ged 4× and
shown below. (Scale bars: 20 μm.) (B) Immunouo-
rescence of mitochondria (CoxIV; red) and nuclei
(DAPI; blue) upon treatment of WT or Drp1
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 quantied in ve or six randomly
chosen elds of view (n > 150 cells) per experiment.
Pooled data from three independent experiments is
shown (**P < 0.003, one-tailed Student t test). (D)
Immuno uorescence 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:
10 μm.)
Fig. 4. The ER marks sites of mitochondrial fragmentation independent of
Drp1. (A) U2OS Sec61β-GFP cells were treated with 2 nM LLO and examined live
by confocal microscopy. Representative images of mitochondria (DsRed2-mito;
red) and ER (Sec61β-GFP; green) are shown. 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)Quantica-
tion of mitochondrial fragmentations marked by ER-mitochondrial contact
sites. Analysis was carried out as described in Materials and Methods.
| Stavru 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 0.81 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, Harvar d Medical School, Bosto n;
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) CO
. 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 26nMLLO
for 10 min (in absence of FCS). DNA transfection (3.5-cm wells) was performed
with FuGENE HD (Roche) according to the manufacturers 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;1μg), and
K38A-HA Drp1 (ref. 23; 4 μg). siRNA transfection was performed with Oligo-
fectamine (Invi trog en) accor din g to th e man ufactur ers instructions, using
Drp1-specic sequences described i n ref. 15.
After treatm ent, cells were either analyzed with a spinning disk micro-
scope for live-cell imaging or processed for immunouorescence with the
antibodies indicated in the respective gure legends (see SI Materials and
Methods for antibody catalog numbers and for detailed live cell imaging
immunouorescence procedures).
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 RT
cDNA 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 geno mic DNA was digested with Ben-
zonase (Novagen). Proteins were separated by SDS/PAGE (Bio-Rad),
transferred by wet transf er 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. Quantication 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 nancial 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 G rant 233348 MODELIST, and Agence
Nationale pour la Recherche (E RANET Pathogenomics Grant LISTRESS and
Grant Blanc MITOPATHO). F.S. was supported by postdoctoral fellowships
from European Molecular Biology Organization and the Fondation pour l a
Recherche dicale. C.W. and R.Y. are supported in part by the Intramural
Research Program of the National I nstitutes of Neurological Disorders and
Strok e, National Institutes of Hea lth. P.C. is a Howard Hughes Medic al In-
stitute Senior International Research Scholar.
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Stavru et al. PNAS
October 1, 2013
vol. 110
no. 40
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| Stavru et al.
    • "Immunofluorescence and live cell imaging were performed essentially as described in [48,72]. For super-resolution microscopy, cells were seeded onto high-precision coverslips (Marienfeld), and after immunofluorescence, samples were mounted in Slow- Fade Gold (Invitrogen) or Fluoromount G. Super-resolution structured illumination (SR-SIM), providing an expected resolution of about 140 nm, was performed on a Zeiss LSM780 Elyra PS1 (Carl Zeiss, Germany) using 63×/1.4 "
    [Show abstract] [Hide abstract] ABSTRACT: Mitochondria are essential eukaryotic organelles often forming intricate networks. The overall network morphology is determined by mitochondrial fusion and fission. Among the multiple mechanisms that appear to regulate mitochondrial fission, the ER and actin have recently been shown to play an important role by mediating mitochondrial constriction and promoting the action of a key fission factor, the dynamin-like protein Drp1. Here, we report that the cytoskeletal component septin 2 is involved in Drp1-dependent mitochondrial fission in mammalian cells. Septin 2 localizes to a subset of mitochondrial constrictions and directly binds Drp1, as shown by immunoprecipitation of the endogenous proteins and by pulldown assays with recombinant proteins. Depletion of septin 2 reduces Drp1 recruitment to mitochondria and results in hyperfused mitochondria and delayed FCCP-induced fission. Strikingly, septin depletion also affects mitochondrial morphology in Caenorhabditis elegans, strongly suggesting that the role of septins in mitochondrial dynamics is evolutionarily conserved.
    Article · May 2016
    • "The link between mitochondrial dynamics and the efficiency of the L. monocytogenes infection has been demonstrated. Indeed, the inhibition of the mitochondrial fusion has been shown to decrease the efficiency of L. monocytogenes infection [72]. Similarly, inducing mitochondrial fusion the legS2 mutant resulted in a more efficient infection than in the WT. "
    [Show abstract] [Hide abstract] ABSTRACT: L. pneumophila is the causative agent of Legionnaires' disease, a human illness characterized by severe pneumonia. In contrast to those derived from humans, macrophages derived from most mouse strains restrict L. pneumophila replication. The restriction of L. pneumophila replication has been shown to require bacterial flagellin, a component of the type IV secretion system as well as the cytosolic NOD-like receptor (NLR) Nlrc4/ Ipaf. These events lead to caspase-1 activation which, in turn, activates caspase-7. Following caspase-7 activation, the phagosome-containing L. pneumophila fuses with the lysosome, resulting in the restriction of L. pneumophila growth. The LegS2 effector is injected by the type IV secretion system and functions as a sphingosine 1-phosphate lyase. It is homologous to the eukaryotic sphingosine lyase (SPL), an enzyme required in the terminal steps of sphingolipid metabolism. Herein, we show that mice Bone Marrow-Derived Macrophages (BMDMs) and human Monocyte-Derived Macrophages (hMDMs) are more permissive to L. pneumophila legS2 mutants than wild-type (WT) strains. This permissiveness to L. pneumophila legS2 is neither attributed to abolished caspase-1, caspase-7 or caspase-3 activation, nor due to the impairment of phagosome-lysosome fusion. Instead, an infection with the legS2 mutant resulted in the reduction of some inflammatory cytokines and their corresponding mRNA; this effect is mediated by the inhibition of the nuclear transcription factor kappa-B (NF-κB). Moreover, BMDMs infected with L. pneumophila legS2 mutant showed elongated mitochondria that resembles mitochondrial fusion. Therefore, the absence of LegS2 effector is associated with reduced NF-κB activation and atypical morphology of mitochondria.
    Full-text · Article · Jan 2016
    • "Regulation of Drp1 binding also occurs at the level of its posttranslational modifications. The uncoupler FCCP induces mitochondrial fragmentation in wilddtype cells but not in cells expressing nonfunctional mutant variant of Drp1 K38A [51, 52]. It turned out that the uncoupler causes a decrease in Ser637 phosphorylation levels of Drp1, thus activating Drp1 and preventing its binding to the receptors Mid49 and Mid51 [53]. "
    [Show abstract] [Hide abstract] ABSTRACT: Dissipation of transmembrane potential inhibits mitochondrial fusion and thus prevents reintegration of damaged mitochondria into the mitochondrial network. Consequently, damaged mitochondria are removed by autophagy. Does transmembrane potential directly regulate the mitochondrial fusion machinery? It was shown that inhibition of ATP-synthase induces fragmentation of mitochondria while preserving transmembrane potential. Moreover, mitochondria of the yeast Saccharomyces cerevisiae retain the ability to fuse even in the absence of transmembrane potential. Metazoan mitochondria in some cases retain ability to fuse for a short period even in a depolarized state. It also seems unlikely that transmembrane potential-based regulation of mitochondrial fusion would prevent reintegration of mitochondria with damaged ATP-synthase into the mitochondrial network. Such reintegration could lead to clonal expansion of mtDNAs harboring deleterious mutations in ATP synthase. We speculate that transmembrane potential is not directly involved in regulation of mitochondrial fusion but affects mitochondrial NTP/NDP ratio, which in turn regulates their fusion.
    Article · May 2015
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