Dephosphorylation by calcineurin regulates
translocation of Drp1 to mitochondria
G. M. Cereghetti*†‡, A. Stangherlin*†, O. Martins de Brito*†, C. R. Chang§, C. Blackstone§, P. Bernardi†¶,
and L. Scorrano*†?
*Dulbecco-Telethon Institute;†Venetian Institute of Molecular Medicine, Via Orus 2, 35129 Padua, Italy;§Cellular Neurology Unit, National Institute of
Neurological Disorders and Stroke, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892; and¶Department of Biomedical Sciences,
University of Padua, Via G. Colombo 3, 35121 Padua, Italy
Communicated by Tullio Pozzan, University of Padua, Padua, Italy, August 21, 2008 (received for review April 24, 2008)
Changes in mitochondrial morphology that occur during cell cycle,
differentiation, and death are tightly regulated by the balance
be caused by inhibition of the fusion machinery and is a common
consequence of dysfunction of the organelle. Here, we show a role
for calcineurin-dependent translocation of the profission dynamin
related protein 1 (Drp1) to mitochondria in dysfunction-induced
fragmentation. When mitochondrial depolarization is associated
with sustained cytosolic Ca2?rise, it activates the cytosolic phos-
phatase calcineurin that normally interacts with Drp1. Calcineurin-
dependent dephosphorylation of Drp1, and in particular of its
depolarized mitochondria depends on a loop involving sustained
Ca2?rise, activation of calcineurin, and dephosphorylation of Drp1
and its translocation to the organelle.
fission ? phosphatase ? subcellular localization ? calcium ? cyclosporine A
equilibrium is tightly regulated to ensure proper mitochondrial
content in daughter cells and is impaired during apoptosis, when
mitochondria fragment and cluster in the perinuclear region (2).
Apoptotic fission is associated with remodeling of the cristae,
characterized by opening of their tubular junction. This causes
the complete release of proapoptotic factors such as cytochrome
c, required in the cytosol for the activation of downstream
effector caspases (3).
Several proteins, including evolutionarily conserved, dy-
namin-related GTPases, regulate mitochondrial fusion and fis-
sion. Fusion relies on the inner membrane protein optic atrophy
1 (Opa1) and on the outer membrane proteins mitofusin (Mfn)
1 and 2. Fission requires the additional step of translocation of
dynamin-related protein 1 (Drp1) from the cytosol, where it
associates with microtubules, to mitochondria where it presum-
ably docks on hFis1, its adaptor in the outer membrane. Oli-
to constrict mitochondrial membranes and to fragment the
organelle (1, 4).
Current understanding of the regulation of mitochondrial
fusion and fission is limited. Fusion of the inner membrane
requires the electrical component of the proton electrochemical
gradient, sustained by proton pumping at complexes of the
respiratory chain. Dissipation of the electrochemical gradient
results in blockage of fusion in vitro and in situ (1). The small
GTPase Rab32 functions as a protein kinase A anchoring
protein (AKAP) on mitochondria and coordinates mitochon-
drial fission, suggesting a role for cAMP and protein phosphor-
ylation (5). The inner membrane rhomboid protease Parl, which
participates in the production of the soluble intermembrane
space form of Opa1 that regulates apoptosis (6, 7), is specifically
phosphorylated on its vertebrate-specific P? domain to block its
self-cleavage and mitochondrial fragmentation (8). Additionally,
itochondria are complex organelles that can exist in a
network, shaped by continuous fusion and fission (1). This
cyclin dependent kinase (Cdk) 5 has been reported to promote
mitochondrial fragmentation in neurons, albeit the molecular
nature of the mediator of its effects on mitochondrial shape is
unknown (9). A step forward in our understanding of the
with the discovery that Drp1-dependent mitochondrial fragmen-
tation is controlled by phosphorylation at two different con-
served sites, serine 616 and 637, by Cdk1 and protein kinase A
(PKA) (10–12). The two sites seem to have opposing effects on
mitochondrial shape, because phosphorylation on S616 by Cdk1
promotes mitochondrial fission during mitosis, while dephos-
phorylation of S637 by the Ca2?-dependent phosphatase cal-
cineurin promotes mitochondrial fission and is involved in the
propagation of apoptosis (13). Interestingly, phosphorylation of
these two residues does not affect the GTPase activity of Drp1,
or it has marginal effects on it, raising the question of the link
between phosphorylation and Drp1-dependent changes in mi-
tochondrial shape. Finally, sumoylation stabilizes the mitochon-
drial pool of Drp1 and enhances fission, further substantiating
the importance of posttranslational modification in regulating
morphology of the organelle (14).
Mitochondrial fragmentation is a common feature of several
pathological conditions where they are dysfunctional. Inhibition
of the fusion machinery has been suggested as the underlying
cause of these morphological changes (15, 16). However, it is
unclear whether inhibition of mitochondrial fusion is the only
mechanism that leads to fragmentation of the dysfunctional
organelle or if it is accompanied by a controlled increase in
fission. Along this line, the relative role of Drp1 in this process
and how it is recruited on dysfunctional mitochondria are
To address these questions, we investigated the relationship
between mitochondrial function and shape. We show that in-
ducers of mitochondrial depolarization trigger Ca2?, cal-
cineurin-dependent dephosphorylation of Drp1, driving its
translocation to mitochondria and fission of the organelle.
Depolarization Causes Drp1-Dependent Mitochondrial Fragmenta-
tion. We addressed the relationship between mitochondrial
depolarization and shape in HeLa cells. We induced depolar-
Author contributions: G.M.C. and L.S. designed research; G.M.C., A.S., and O.M.d.B. per-
O.M.d.B. analyzed data; and G.M.C., P.B., and L.S. wrote the paper.
The authors declare no conflict of interest.
?To whom correspondence should be sent at the present address: Department of Cell
Physiology and Metabolism, University of Geneva Medical School, 1 Rue M. Servet, 1205
Geneva, Switzerland. E-mail: firstname.lastname@example.org.
‡Present address: Department of Cell Physiology and Metabolism, University of Geneva
Medical School, 1 Rue M. Servet, 1205 Geneva, Switzerland.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
October 14, 2008 ?
vol. 105 ?
no. 41 ?
ization by two means: (i) triggering the permeability transition
(PT), which depends on the opening of an inner membrane
channel permeant to solutes with a molecular mass ?1500 Da
(17); (ii) treating cells with a protonophore that dissipates the
electrochemical gradient. The calcein staining/Co2?-quenching
technique allows direct imaging of PT in situ, while changes in
mitochondrial fluorescence of tetramethyl rhodamine methyl-
ester (TMRM) permit to measure mitochondrial membrane
potential (18). The lipid second messenger arachidonic acid
(ArA) induced the expected decrease in mitochondrial fluores-
cence of both calcein and TMRM (Fig. 1A), documenting the
occurrence of PT accompanied by long-lasting mitochondrial
A (CsA), a calcineurin inhibitor that also desensitizes the PT
pore via its mitochondrial receptor cyclophilin D (17) (Fig. 1A).
Conversely, the protonophore carbonylcyanide-p-trifluorome-
thoxy phenylhydrazone (FCCP) induced complete mitochon-
lack of calcein efflux from mitochondria and by the insensitivity
to CsA of the drop in mitochondrial fluorescence of TMRM
We next addressed the relationship between mitochondrial
depolarization and shape. Real time confocal imaging of a
mitochondrially targeted dsRed fluorescent protein (mtRFP)
showed that ArA and FCCP caused complete fragmentation of
the mitochondrial network within 15 min. In the case of FCCP
this was preceded by the appearance of doughnut-like mitochon-
drial rings (Fig. 1 C and D). Of note, fragmentation relied on
active Drp1 since it was completely blocked by expression of a
Lys 38 to Ala mutant of Drp1 that acts as dominant negative
(Drp1DN, Fig. 1 C and D) (19). Thus, fragmentation induced by
mitochondrial depolarization requires Drp1.
To address whether fragmentation was a consequence of
depolarization per se or whether it required additional mito-
chondrial changes, such as PT, we decided to measure the effect
of the PT inhibitor CsA on fragmentation by ArA and FCCP. As
expected, CsA prevented fragmentation by the PTP inducer
ArA (Fig. 1C), but unexpectedly also by FCCP (Fig. 1D), which
does not induce PT and whose effects on mitochondrial mem-
brane potential are insensitive to CsA (Fig. 1B).
Mitochondrial Fragmentation by Depolarization Depends on the Ca2?-
Dependent Phosphatase Calcineurin. These results suggested a role
for CsA-sensitive pathways other than the PT in Drp1-
dependent fragmentation of mitochondria induced by depolar-
ization of the organelle and directed our attention to the
Ca2?-dependent cytosolic phosphatase calcineurin, inhibited by
the CsA-cyclophilin A complex (20). Calcineurin is a het-
erodimeric protein composed of a catalytic subunit (CnA) that
binds calmodulin and a regulatory subunit (CnB) that binds
Ca2?. It is involved in the transduction of a variety of Ca2?-
mediated signals, ranging from proliferation to death, to secre-
tion of vesicles (20). We therefore reasoned that mitochondrial
depolarization induced by ArA and FCCP could be associated
with changes in cytosolic Ca2?levels and therefore with the
activation of calcineurin. The ratiometric dye Fura-2 showed a
rapid increase in cytosolic Ca2?after treatment with ArA or
FCCP, followed by a sustained plateau (Fig. 2A). Emptying of
ER Ca2?stores completely abolished FCCP-induced Ca2?tran-
sients and markedly inhibited those elicited by ArA (data not
shown). Moreover, we found that TG-releasable Ca2?peak was
significantly reduced from 58 ? 4 to 27 ? 8 nM or to 39 ? 6 nM
in cells treated with ArA or FCCP, respectively. Thus, mito-
chondrial depolarization by ArA and FCCP is associated with
ER Ca2?release. Together with the likely inhibition of the ATP
dependent efflux pathways of the plasma membrane, this release
dependent fission. (A and B) Changes in mitochon-
drial fluorescence of TMRM or calcein. HeLa cells
FCCP (B). Fluorescence intensities were normalized
to the initial value for comparative reasons. Data
represent mean ? SE of four independent experi-
ments. Where indicated, cells were pretreated with
CsA or with the inactive analogue CsH (18). (C and D)
Representative confocal images of mtRFP fluores-
indicated, cells were pretreated with CsA or cotrans-
fected with K38A Drp1 (Drp1DN) (Scale bar, 20 ?m).
Mitochondrial depolarization causes Drp1-
www.pnas.org?cgi?doi?10.1073?pnas.0808249105Cereghetti et al.
results in a sustained cytosolic Ca2?rise that is required for the
activation of calcineurin (21). Accordingly, we recorded a 2- to
3-fold increase in calcineurin activity, which was, as expected,
inhibited by CsA (Fig. 2D). This suggests that the effects of CsA
on mitochondrial fragmentation could be related to the inhibi-
tion of calcineurin. To investigate the relative role of calcineurin
in mitochondrial fragmentation, we turned to specific inhibitors
of PT or calcineurin: the CsA analogue N-MethylVal-4-CsA
(MeValCsA), a non-immunosuppressive analogue of CsA that
binds to cyclophilins but not to calcineurin (22) and FK506, an
immunosuppressive agent that blocks calcineurin via its receptor
FKBP12 and has no effect on the PTP (23). FK506 greatly
delayed fragmentation induced by both ArA and FCCP, while
MeValCsA prevented fission only when caused by ArA (Fig. 2
A–C). Accordingly, MeValCsA but not FK506 inhibited depo-
larization by ArA [supporting information (SI) Fig. S1]. We next
turned to a genetic approach to test the relationship between
calcineurin and mitochondrial fission by ArA/FCCP. Expression
of a constitutively active mutant of calcineurin lacking the CaM
binding domain due to the introduction of a stop codon at
position 392 (?CnA) increased fragmentation per se. It also
accelerated fragmentation induced by both ArA and FCCP (Fig.
2 E and F). Conversely, a dominant negative mutant of cal-
cineurin devoid of the CaM binding and the autoinhibitory
domains and harboring an inactivating His-151 to Gln point
mutation (?CnAH151Q) (24) efficiently blocked depolarization-
induced fragmentation (Fig. 2 E and F). Since calcineurin is
activated in a Ca2?-calmodulin dependent fashion, we explored
whether fission by FCCP and ArA was dependent on Ca2?by
incubating cells with the membrane-permeant acetoxymethyl-
ester of the Ca2?chelating agent 1,2-bis (o-aminophe-
noxy)ethane-N,N,N?,N?-tetraacetic acid (BAPTA). Chelation
of intracellular Ca2?completely blocked cytosolic Ca2?rise
(data not shown) and fission by ArA and FCCP (Fig. 2 E and F).
In conclusion, these experiments show that mitochondrial de-
calcineurin; and that inhibition of this phosphatase or chelation
of intracellular Ca2?blocks Drp1-dependent fission of the
Drp1 Is Dephosphorylated by Calcineurin in Response to Inducers of
Mitochondrial Depolarization. How does calcineurin regulate
Drp1-dependent mitochondrial fission? We reasoned that Drp1,
similarly to dynamin I (25), could physically interact with cal-
cineurin. Immunoprecipitation under mild detergent conditions
in Ca2?-free buffers showed that CnA indeed associates with
Drp1 (Fig. 3A). Of note, a significant fraction of cyclophilin A
was also immunoprecipitated by Drp1, suggesting that Drp1
could serve as a scaffold for both calcineurin and cyclophilin A.
This possibility was confirmed when we found that CnA directly
immunoprecipitated Drp1, but not cyclophilin A, as expected
(Fig. 3A). The interaction between Drp1 and CnA was stimu-
lated in cells treated with ArA (Fig. 3B) and FCCP (data not
shown) and inhibited by the calcineurin inhibitors CsA and
drial fission by ArA and FCCP. (A) Representative
traces of cytosolic Ca2?([Ca2?]i) as measured by
Fura-2 in HeLa cells treated where indicated with 20
?M ArA or 2 ?M FCCP. (B) Calcineurin activity mea-
indicated for 15 min with 20 ?M ArA or 2 ?M FCCP.
Where indicated, cells were pretreated with 2 ?M
CsA for 30 min. Data are compared with the activity
SE of four independent experiments. (C) Represen-
tative frames acquired at indicated times from real
time confocal imaging of HeLa cells transfected with
mtRFP. At t ? 3 min, cells were treated as indicated
were pretreated for 30 min with 2 ?M mMeValCsA,
0.6 ?M FK506 or 40 ?M BAPTA-AM, or cotransfected
with CnB plus ?CnA or ?CnAH151Q. Bar, 20 ?m. (D)
Quantitative analysis of mitochondrial shape
changes. Experiments were performed exactly as in
(C). Morphometry was performed as described. Data
represent mean ? SE of 8 different experiments. In
each experiment, 20 cells were scored. In the case of
BAPTA-AM, no fragmentation could be detected in
the timeframe of the experiment.
Inhibition of calcineurin blocks mitochon-
Cereghetti et al.
October 14, 2008 ?
vol. 105 ?
no. 41 ?
FK506 (Fig. 3B). This suggests that the interaction between
by mitochondrial dysfunction, and is sensitive to the inhibition of
calcineurin, similar to what was observed in the case of the
dynamin I-calcineurin complex (25).
Previous reports showed that Drp1 can be reversibly phos-
phorylated, but whether this is associated with sustained changes
in its GTPase activity is unclear (10, 13). A rate-limiting step in
could be in principle modulated by calcineurin. To verify this
of Drp1 associated with mitochondria. ArA (Fig. 3C) or FCCP
(data not shown) induced an increase in mitochondrial levels of
Drp1. Real time imaging experiments of Drp1-YFP transloca-
tion further confirmed that both FCCP (Fig. S2) and ArA (data
before the occurrence of organellar fragmentation. We next
evaluated whether ArA induced dephosphorylation of Drp1 and
translocation of the dephosphorylated form to mitochondria.
Immunoblotting of total lysates with an anti phospho-Serine 637
antibody indicated that after ArA treatment this residue be-
comes dephosphorylated (Fig. S3). We extended our analysis to
the total phosphorylation status of the protein. A specific resin
that selectively binds phosphorylated proteins allows quantifi-
cation of the relative amount of dephosphorylated vs. phosphor-
ylated Drp1 in subcellular fractions. We could identify discrete
amounts of phosphorylated Drp1 in the cytosol of unstimulated
cells, but no Drp1 bound to resin was retrieved in the cytosol of
cells treated with ArA. Accordingly, mitochondrial Drp1 re-
sulted, completely dephosphorylated, and its levels increased
when organelles were isolated from cells treated with ArA (Fig.
3D). Immunofluorescence showed that a significant fraction of
Drp1-YFP remained associated with microtubules (MT) after
addition of ArA to cells treated with FK506 (Fig. S4). Moreover,
real-time imaging confirmed that translocation of Drp1-YFP to
mitochondria in response to ArA and FCCP is blocked by FK506
(data not shown). In conclusion, mitochondrial dysfunction is
associated with dephosphorylation of Drp1 and the dephospho-
rylated form of Drp1 is retrieved in mitochondria. Translocation
is prevented by blockage of calcineurin, suggesting that dephos-
phorylation is a key factor in controlling subcellular distribution
Subcellular Distribution of Drp1 Is Controlled by Phosphorylation at
Ser-637. To verify the role of phosphorylation in subcellular
distribution of Drp1 we generated mutants of a Drp1-YFP
chimera mimicking constitutive phosphorylation (S3D) or de-
phosphorylation (S3A) of the two recently identified Drp1
phosphorylation sites, S616 and S637. While Drp1S616-YFP
mutants behaved in an almost superimposable manner to that of
wt Drp1-YFP, mutagenesis of S637 had a major impact on
subcellular localization and mitochondrial shape: Drp1S637D-
YFP was almost completely cytosolic, while the Drp1S637A-YFP
localized on mitochondria (Fig. 4 A and B). Accordingly, the
Drp1S616-YFP mutants displayed only marginal effects on
steady-state mitochondrial morphology. We next investigated
the relative role of each site on subcellular localization of Drp1
and on mitochondrial morphology by expressing Drp1-YFP
chimeras mutated at both sites. This analysis confirmed that the
double S616A, S637D mutant is mainly cytosolic and does not
cause extensive mitochondrial fragmentation, while the double
S616D, S637A mutant behaves exactly the opposite (Fig. 4 A and
B). The effect on mitochondrial morphology of these mutants
could reflect a differential ability to assemble into active oli-
gomers. We therefore tested whether they retained normal
not shown). Thus, the observed mitochondrial fragmentation
seems to reflect the differential ability of the mutants to
associate with mitochondria rather than their activity (10, 13) or
their ability to oligomerize. We next measured translocation of
the Drp1-YFP mutants to mitochondria induced by depolariza-
tion of the organelle. Real time confocal imaging experiments
showed that while Drp1S637A-YFP was already mostly mitochon-
drial, Drp1S637D-YFP was retained in the cytoplasm, even when
most of the wt and S616 mutants of Drp1 had translocated to
mitochondria (Fig. S2). Accordingly, when we quantified the
effect of overexpressed Ser mutants of Drp1 on FCCP and ArA
induced mitochondrial fission, we found that the S637D mutant
delayed fission in response to both, while S616D displayed no
effect as compared with cells expressing wt Drp1. Accordingly,
mitochondrial fragmentation was delayed in cells expressing the
double S616A, S637D mutant (Fig. 4 C and D). It should be
noted that residual fragmentation was observed also in cells
expressing the S637D mutant as a consequence of the endoge-
nous levels of Drp1. In conclusion, phosphorylation of residue
S637 regulates translocation of Drp1 to mitochondria and there-
fore its ability to fragment the organelle.
We have shown that two different inducers of mitochondrial
depolarization cause Drp1-dependent fragmentation of the or-
ganelle. The cytosolic phosphatase calcineurin mediates a cru-
cial step in this process, as shown by multiple lines of evidence.
amounts of HeLa cell proteins (100 ?g) dissolved in CPBS were immunopre-
cipitated with the indicated antibodies and proteins in the lysates (Input, 1:5
dilution) and coprecipitated proteins were separated by SDS/PAGE and im-
munoblotted with the indicated antibodies. (B) Lysates from HeLa cells
treated where indicated with 20 ?M ArA or 2 ?M FCCP for 15 min were
immunoprecipitated with an anti-Drp1 antibody. Coprecipitated proteins
ies. Where indicated, cells were pretreated for 30 min with 2 ?M mMeValCsA
or 0.6 ?M FK506. Input represents a 1:5 dilution of the total lysates. (C)
15 min were prepared and equal amounts (40 ?g) of protein from total,
cytosolic and mitochondrial fractions were separated by SDS/PAGE and im-
munoblotted using the indicated antibodies. LDH, lactate dehydrogenase;
MnSOD, Mn-dependent superoxide dismutase. (D) Experiments were pre-
formed as in (C), except that cytosolic and mitochondrial fractions were
loaded on a column binding phosphorylated proteins to separate phosphor-
ylated and unphosphorylated proteins. Thirty ?g of total (tot), phosphory-
lated (P) and unphosphorylated (nP) proteins from each fraction were sepa-
rated by SDS/PAGE and immunoblotted using an anti-Drp1 antibody.
Dephosphorylated Drp1 accumulates on mitochondria. (A) Equal
www.pnas.org?cgi?doi?10.1073?pnas.0808249105Cereghetti et al.
First, mitochondrial depolarization is associated with a sustained
rise in intracellular Ca2?levels, which in turn activate cal-
cineurin. Second, fragmentation is sensitive to genetic and
pharmacological inhibition of calcineurin. Third, Drp1 is re-
trieved in a complex with calcineurin and cyclophilin A. Fourth,
depolarization results in calcineurin-dependent dephosphoryla-
tion of Drp1 and association of the dephosphorylated form with
mitochondria. Fifth, site-specific mutagenesis shows that de-
phosphorylation of S637 regulates the translocation of Drp1 to
The overall shape of the mitochondrial network likely results
from the integration of organellar and cytosolic cues. This is
essential to integrate the remodeling of the reticulum during
different phases of the cell cycle and to control the transport of
the organelle (2). However, changes in mitochondrial bioener-
getic parameters impinge on morphology of the organelle (3).
Here, we link mitochondrial dysfunction to the recruitment of
Drp1 on the organelle, mediated by its Ca2?/calcineurin depen-
dent dephosphorylation. The rise in cytoplasmic Ca2?occurs
independently of whether the depolarizing stimulus triggers PT.
Thus, the determining factor is probably the severe depletion of
ATP aggravated when the ATPase works in reverse as an
attempt to maintain membrane potential of dysfunctional mito-
chondria (26). ATP depletion impairs plasma membrane and
intracellular Ca2?extrusion mechanisms to sustain the cytoplas-
mic Ca2?rise. The ATPase inhibitor oligomycin, which does not
induce severe ATP depletion or mitochondrial depolarization,
does not increase cytoplasmic Ca2?(data not shown). The
reported effect of oligomycin on mitochondrial shape is there-
fore the likely consequence of Opa1 degradation (27): accord-
ingly, overexpressed Opa1 inhibits it (data not shown). Mito-
chondrial fragmentation observed in several mitochondrial
diseases can therefore result from inhibition of Opa1-dependent
fusion (15), or from calcineurin/Drp1-dependent fission, when
ATP levels drop to sustain the cytosolic Ca2?plateau. For
example, in NARP the ATPase is mutated and cannot hydrolyze
ATP, while in MERRF the mutation affects the respiratory
chain and the ATPase can work as an ATP consumer, trying to
maintain membrane potential. Accordingly, dysregulation of
Ca2?signaling occurs only in MERRF (28) and MERRF
mitochondria appear fragmented (15).
Our results suggest that calcineurin-dependent dephosphor-
ylation of Drp1 primarily controls its association with mitochon-
dria. Regulation of intracellular localization by reversible phos-
phorylation is a common theme in cell biology and calcineurin
has been reported to be a key player in these processes, con-
trolling translocation of NFaT to the nucleus and of the proapo-
ptotic Bcl-2 family member Bad to mitochondria. During exo-
cytosis, calcineurin couples the rise in presynaptic Ca2?levels to
What is the relative role of S616 and S637 in controlling
subcellular localization and function of Drp1? At least in cells in
interphase (likely the main population in unsynchronized cul-
tured cells) S637D dominates over S616A. Intriguingly, we found
that the S616A mutant, mimicking dephosphorylation, promotes
mitochondrial fission rather than fusion. It appears that the
profusion effect of this mutation observed by Mihara and
GTPase activity of recombinant Drp1 is greatly inhibited by
PKA, yet the activity of Drp1 carrying the phosphomimetic
S637D mutation is only slightly reduced (10). Thus, additional
mechanisms should support the dramatic effect in mitochondrial
morphology observed in cells expressing the S637 mutants. We
show that Drp1 with phosphomimetic mutations on S637 is
effects on shape. Furthermore, the opposing effects of PKA and
calcineurin on the same site likely serve as a switch to translate
signals associated with Ca2?changes of different strength,
duration, and tone into different mitochondrial morphologies.
During physiological (i.e., agonist evoked) Ca2?signaling, acti-
vation of PKA can prevail over calcineurin mediated dephos-
phorylation of Drp1. Moreover, compartmentalization of both
Ca2?and cAMP signals could play a role in the local regulation
of mitochondrial shape. Conversely, long lasting Ca2?plateaus
in the cytosol linked to full activation of calcineurin and to
generalized fragmentation could for example account for the
apoptotic mitochondrial fission.
Drp1 also participates in fission of endoplasmic reticulum and
of peroxisomes (31), implying regulatory events that determine
organelle selectivity. This could be achieved by posttranslational
modifications orchestrated in the cytosol. Drp1 could be tar-
geted to the organelle of interest by different spatiotemporal
patterns of Ca2?rises that are coupled to local calcineurin
activation, an appealing possibility that deserves future
Representative confocal images of HeLa cells cotransfected with mtRFP and
the indicated Drp1-YFP mutants. Bar, 20 ?m. (B) Analysis of subcellular local-
ization of Drp1-YFP. Experiments were exactly as in (A). Classification of
subcellular distribution of Drp1-YFP and mitochondrial morphometric analy-
sis was performed as described in SI Experimental Procedures. Sixty to eighty
cells were scored in each condition. (C) Representative frames acquired at
indicated times from real time confocal imaging of HeLa cells cotransfected
with mtRFP and the indicated mutants of Drp1-YFP. At t ? 3 min, cells were
treated with 2 ?M FCCP. (D) Quantitative analysis of mitochondrial shape
changes. Experiments were exactly as in C, except that after 3 min cells were
performed as described. Data represent mean ? SE of four different experi-
ments. In each experiment, 10 cells were scored.
Cereghetti et al.
October 14, 2008 ?
vol. 105 ?
no. 41 ?
Materials and Methods Download full-text
Imaging. Imaging of PTP opening and mitochondrial membrane potential in
HeLa cells was performed as previously described (18). Data are graphed as
percentage of the initial value for comparative reasons.
For confocal imaging, 105cells seeded onto 24 mm-round glass coverslips
were transfected as indicated and after 24 h were incubated in HBSS supple-
mented with 10 mM Hepes and placed on the stage of a Nikon Eclipse TE300
confocal system, a piezoelectric z-axis motorized stage (Pifoc, Physik Instru-
mente), and a Orca ER 12-bit CCD camera (Hamamatsu Photonics). Cells
expressing mtRFP and/or Drp-1-YFP were excited using the 568-nm or the
440-nm line of the He-Ne laser (Perkin–Elmer) respectively with exposure
times of 100 msec using a 60 ? 1.4 NA Plan Apo objective (Nikon). Cells
immunostained with TRITC- conjugated anti-tubulin antibody were excited
with the 568-nm laser line.
For immunofluorescence, cells grown on 13-mm coverslips were fixed in
3.7% paraformaldehyde in PBS (20 min, 4 °C), permeabilized with 0.01%
Nonidet-P40 (Gibco) and incubated with anti-tubulin (Santa Cruz Biotechnol-
ogy) antibody (1:200). Cells were then stained with an isotype matched TRITC
secondary antibody (Amersham, 1:200).
In time-course experiments, images were acquired every 10 sec for 40 min.
Quantitative analysis of mitochondrial shape changes was performed by
evaluating the time at which cells displayed fragmented mitochondria after
of the total cellular mitochondria displayed a major axis ?5 ?m.
as mitochondrial if ?50% of YFP fluorescence colocalized with mtRFP
Ca2?Measurements. Cytosolic Ca2?measurements were performed exactly as
previously described (32).
Calcineurin Activity Assay. Cells grown on 24-mm wells incubated in HBSS
Tris-Cl, pH 7.4, 150 mM NaCl), resuspended in TBS, and lysed in CTBS buffer (6
mM CHAPS in TBS, pH 7.4). calcineurin activity was determined using an in
vitro assay kit and following manufacturer instructions (Calbiochem). For com-
parative reasons, basal calcineurin activity in untreated cells was set to 100%.
Subcellular Fractionation, Immunoprecipitation, and Immunoblotting. Subcel-
lular fractionation was performed as described in ref. 33.
For immunoprecipitation experiments, cells were lysed in CPBS buffer (6
(1:50, 14 h, 4 °C) and the protein-antibody complex was precipitated by
2 h, 4 °C). The immunoprecipitated material was washed twice in CPBS and
resuspended in SDS/PAGE loading buffer (NuPAGE), boiled, and loaded on
4–12% gels (NuPAGE).
For phosphorylation studies, cytosolic and mitochondrial fractions were
loaded on a phosphoprotein binding column (Qiagen). Flow-through (un-
phosphorylated) and eluted (phosphorylated proteins) proteins were col-
lected and concentrated and 30 ?g of proteins were separated by 4–12%
For immunoblotting, proteins were transferred onto polyvinylidene fluoride
(Millipore) membranes and probed with the following antibodies: ?-Drp1 (BD,
land, 1:2000), ?-MnSOD (Stressgen, 1:3000), ?-phosphoSer637Drp1 (10) (1:750).
Isotype-matched, horseradish peroxidase-conjugated secondary abodies (Amer-
sham) were used, followed by detection by chemiluminescence (Amersham).
ACKNOWLEDGMENTS. This study was supported by Telethon Italy, AIRC Italy,
Swiss National Science Foundation (to L.S.); and by Telethon Italy and AIRC
Italy (to P.B.). G.M.C. was supported by a grant for prospective researchers of
the Swiss National Science Foundation.
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