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
19. Frank S, et al. (2001) The role of dynamin-related protein 1, a mediator of mito-
chondrial fission, in apoptosis. Dev Cell 1(4):515–525.
20. Cereghetti GM, et al. (2008) Dephosphorylation by calcineurin regulates translocation
of Drp1 to mitochondria. Proc Natl Acad Sci USA 105(41):15803–15808.
21. Cereghetti GM, Costa V, Scorrano L (2010) Inhibition of Drp1-dependent mitochon-
drial fragmentation and apoptosis by a polypeptide antagonist of calcineurin. Cell
Death Differ 17(11):1785–1794.
22. Dimmer KS, et al. (2008) LETM1, deleted in Wolf-Hirschhorn syndrome is required for
normal mitochondrial morphology and cellular viability. Hum Mol Genet 17(2):201–214.
23. Smirnova E, Griparic L, Shurland DL, van der Bliek AM (2001) Dynamin-related protein
Drp1 is required for mitochondrial division in mammalian cells. Mol Biol Cell 12(8):
24. Mozdy AD, McCaffery JM, Shaw JM (2000) Dnm1p GTPase-mediated mitochondrial
fission is a multi-step process requiring the novel integral membrane component
Fis1p. J Cell Biol 151(2):367–380.
25. Yoon Y, Krueger EW, Oswald BJ, McNiven MA (2003) The mitochondrial protein hFis1
regulates mitochondrial fission in mammalian cells through an interaction with the
dynamin-like protein DLP1. Mol Cell Biol 23(15):5409–5420.
26. Yu T, Fox RJ,Burwell LS,Yoon Y (2005) Regulationofmitochondrial fission and apoptosis
by the mitochondrial outer membrane protein hFis1. J Cell Sci 118(Pt 18):4141–4151.
27. Otera H, et al. (2010) Mff is an essential factor for mitochondrial recruitment of Drp1
during mitochondrial fission in mammalian cells. J Cell Biol 191(6):1141–1158.
28. Ribet D, et al. (2010) Listeria monocytogenes impairs SUMOylation for efficient in-
fection. Nature 464(7292):1192–1195.
29. Samba-Louaka A, Stavru F, Cossart P (2012) Role for telomerase in Listeria mono-
cytogenes infection. Infect Immun 80(12):4257–4263.
30. De Vos KJ, Allan VJ, Grierson AJ, Sheetz MP (2005) Mitochondrial function and actin
regulate dynamin-related protein 1-dependent mitochondrial fission. Curr Biol 15(7):
31. Legros F, Lombès A, Frachon P, Rojo M (2002) Mitochondrial fusion in human cells is
efficient, requires the inner membrane potential, and is mediated by mitofusins. Mol
Biol Cell 13(12):4343–4354.
32. Ishihara N, Jofuku A, Eura Y, Mihara K (2003) Regulation of mitochondrial mor-
phology by membrane potential, and DRP1-dependent division and FZO1-dependent
fusion reaction in mammalian cells. Biochem Biophys Res Commun 301(4):891–898.
33. Malka F, et al. (2005) Separate fusion of outer and inner mitochondrial membranes.
EMBO Rep 6(9):853–859.
34. Shibata Y, et al. (2008) The reticulon and DP1/Yop1p proteins form immobile
oligomers in the tubular endoplasmic reticulum. J Biol Chem 283(27):18892–18904.
35. Koch A, et al. (2003) Dynamin-like protein 1 is involved in peroxisomal fission. J Biol
36. Pitts KR, Yoon Y, Krueger EW, McNiven MA (1999) The dynamin-like protein DLP1 is
essential for normal distribution and morphology of the endoplasmic reticulum and
mitochondria in mammalian cells. Mol Biol Cell 10(12):4403–4417.
37. Bras M, et al. (2007) Drp1 mediates caspase-independent type III cell death in normal
and leukemic cells. Mol Cell Biol 27(20):7073–7088.
38. Area-Gomez E, et al. (2012) Upregulated function of mitochondria-associated ER
membranes in Alzheimer disease. EMBO J 31(21):4106–4123.
39. de Brito OM, Scorrano L (2008) Mitofusin 2 tethers endoplasmic reticulum to mito-
chondria. Nature 456(7222):605–610.
40. Stavru F, Nautrup-Pedersen G, Cordes VC, Görlich D (2006) Nuclear pore complex
assembly and maintenance in POM121- and gp210-deficient cells. J Cell Biol 173(4):
| www.pnas.org/cgi/doi/10.1073/pnas.1315784110Stavru et al.