FORUM ORIGINAL RESEARCH COMMUNICATION
Mitophagy Selectively Degrades Individual Damaged
Mitochondria After Photoirradiation
Insil Kim1,2,* and John J. Lemasters1
Damaged and dysfunctional mitochondria are proposed to be removed by autophagy. However, selective
degradation of damaged mitochondria by autophagy (mitophagy) has yet to be experimentally verified. In this
study, we investigated the cellular fate of individual mitochondria damaged by photoirradiation in hepatocytes
isolated from transgenic mice expressing green fluorescent protein fused to microtubule-associated protein 1
light chain 3, a marker of forming and newly formed autophagosomes. Photoirradiation with 488-nm light
induced mitochondrial depolarization (release of tetramethylrhodamine methylester [TMRM]) in a dose-
dependent fashion. At lower doses of light, mitochondria depolarized transiently with re-polarization within
3min. With greater light, mitochondrial depolarization became irreversible. Irreversible, but not reversible,
photodamage induced autophagosome formation after 32–5min. Photodamage-induced mitophagy was in-
dependent of TMRM, as photodamage also induced mitophagy in the absence of TMRM. Photoirradiation with
543-nm light did not induce mitophagy. As revealed by uptake of LysoTracker Red, mitochondria weakly
acidified after photodamage before a much stronger acidification after autophagosome formation. Photo-
damage-induced mitophagy was not blocked by phosphatidylinositol 3-kinase inhibition with 3-methyladenine
(10mM) or wortmannin (100 nM). In conclusion, individual damaged mitochondria become selectively degraded
by mitophagy, but photodamage-induced mitophagic sequestration occurs independently of the phosphatidyli-
nositol 3-kinase signaling pathway, the classical upstream signaling pathway of nutrient deprivation-induced
autophagy. Antioxid. Redox Signal. 14, 1919–1928.
removal of damaged and superfluous cellular constituents.
Proteasomal degradation typically eliminates short-lived
proteins that are damaged or misfolded; whereas autophagy
removes long-lived proteins, protein aggregates, and whole
organelles, including endoplasmic reticulum, peroxisomes,
and mitochondria (9, 32). Autophagy is now categorized into
chaperone-mediated autophagy, microautophagy, and macro-
autophagy (9). Autophagy as originally defined corresponds
to macroautophagy whereby whole organelles and pieces of
cytoplasm are first sequestered into vesicles called autopha-
gosomes that then fuse with lysosomes for digestion. Thus,
the terms autophagy and macroautophagy are often used
ellular homeostasis is maintained by a fine balance
between anabolic and catabolic pathways. Together,
interchangeably, a practice followed here unless otherwise
In general, insulin and other growth factors inhibit autop-
hagy, whereas nutrient deprivation and glucagon promote
autophagy (4,41).Inthenormallife ofcells, autophagy occurs
more or less continuously to remove damaged and superflu-
ous organelles, including dysfunctional mitochondria that
couldbe detrimental tocells. Severalstudies suggest thatboth
inadequate and excess autophagy promote cell injury and
death (25, 28). Therefore, proper regulation of autophagy is
fundamental to cellular well being.
During autophagy, a novel membranous structure called
a phagophore elongates and encloses cellular components
to form a double membrane vesicle called an autophago-
some (42). Lysosomes then fuse with autophagosomes
to form autolysosomes in which lysosomal hydrolases
degrade the sequestered contents. Autophagosomes can
This work is in partial fulfillment of the requirements for a Ph.D. to I. Kim from the University of North Carolina at Chapel Hill.
1Center for Cell Death, Injury, and Regeneration, Departments of Pharmaceutical and Biomedical Sciences and Biochemistry and Mole-
cular Biology, Medical University of South Carolina, Charleston, South Carolina.
2Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
*Current affiliation: Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania.
ANTIOXIDANTS & REDOX SIGNALING
Volume 14, Number 10, 2011
ª Mary Ann Liebert, Inc.
contain virtually any cytoplasmic element, including cyto-
solic proteins and various membranous organelles, includ-
ing endoplasmic reticulum, peroxisomes, and mitochondria
In yeast, genetic screens identified a series of evolutionarily
conserved autophagy (ATG) genes that are required for au-
tophagy. The protein products of ATG genes are called Atg
proteins and are categorized according to function. For ex-
ample, a mammalian homologue of yeast Atg6, Beclin 1 in-
teracts with phosphatidylinositol 3-kinase (PI3K) to form a
complex that is involved in an initial step in autophagosome
formation (11). Microtubule-associated protein 1 light chain 3
(LC3) was then identified as a mammalian ortholog of yeast
Atg8 (15). In mammalian cells, cleavage of a 22 amino-acid
fragment from newly synthesized pro-LC3 produces LC3-I.
The concerted actions of Atg7 (an E1-like activity), Atg3 (an
E2-like conjugating activity), and Atg12–Atg5-Atg16L com-
plexes (E3-like ligase activity) conjugate LC3-I with phos-
phatidylethanolamine to produce LC3-II (5, 46). So activated,
LC3-II localizes selectively to forming and newly formed
autophagosomes even after other Atg proteins dissociate.
Thus, LC3-II is a marker of ongoing autophagy. After se-
questration, some LC3-II become entrapped on the inner
surfaces of the double-membrane autophagosomes. After
fusion with lysosomes, this LC3-II is degraded. LC3-II on the
outer surface also disappears, most likely by cleavage of the
phospholipid conjugate, and the LC3 is reutilized. A trans-
genic mouse strain was created that expresses a green fluo-
rescent protein (GFP)-LC3 fusion protein. In cells and tissues
of GFP-LC3 transgenic mice, GFP fluorescence selectively
identifies the membranes of forming and newly formed
Whether or not autophagy selectively targets specific or-
ganelles has been controversial. After withdrawal of peroxi-
some proliferators in the presence of protease inhibitors,
peroxisomes selectively accumulate in autophagosomes (51).
The removal of peroxisomes by autophagy is referred to as
pexophagy and occurs in yeast when methanol-containing
glycogen, but not mitochondria and other organelles, occurs
in the early postnatal period (23). Mitochondria of non-
proliferating tissues such as heart, brain, liver, and kidney
constantly turn over witha half-life of10–25days(29,36),and
recent evidence supports selective autophagic removal of
mitochondria, a process of mitophagy (16, 26, 35, 45). In
staurosporine-treated cells where apoptosis is inhibited by
caspase inhibitors, mitochondria are eliminated by mito-
phagy in a specific and regulated manner (50). In yeast, the
mitochondrial outer membrane protein Uth1 is required for
efficient mitophagy in a nutrient-poor medium, but a corre-
sponding mammalian protein has yet to be identified (21).
Also, a yeast protein phosphatase homologue, Aup1, in the
mitochondrial intermembrane space is required for mito-
phagy (45). In a recent study, parkin was recruited to mito-
chondria depolarized by treatment with carbonyl cyanide
m-chlorophenyl hydrazone, an uncoupler, to promote au-
tophagy (33). Although these data support mitophagy as a
distinctive pathway from the autophagy of other cytoplasmic
mitochondria in normal cellular physiology has not been di-
To that end, we used 488-nm light to photodamage indi-
vidual mitochondria of primary hepatocytes from GFP-LC3
transgenic mice. Visible light of 400- to 500-nm excites and
damages flavin-containing proteins in mitochondria, result-
ing in mitochondrial alterations and damage by reactive
oxygen species (ROS) production (1, 2). Here, such photo-
damage depolarized and permeabilized mitochondria, which
led to PI3K-independent sequestration of individual mito-
chondria into GFP-LC3-labeled autophagosomes.
Materials and Methods
3-Methyladenine (3MA; cat. no. 08592) and wortmannin
Louis, MO). Calcein acetoxymethyl ester (AM) (cat. no.
C3100MP), LysoTracker Red (LTR; cat. no. L7528), Mito-
FluorFar Red (MFFR; cat. no M22423), and tetramethy-
lrhodamine methylester (TMRM) (cat. no. T668) were
obtained from Invitrogen/Molecular Probes (Carlsbad, CA).
Collagenase A (cat. no. 11088793001) was obtained from
Roche (Penzberg, Germany).
Hepatocyte isolation and culture
Hepatocytes from GFP-LC3 transgenic C57BL/6 mice or
C57/BL6 wild-type mice were isolated by collagenase per-
fusion and cultured overnight in 5% CO2/95% air at 37?C on
of 300,000 cells per plate in Waymouth’s MB-752/1 growth
medium supplemented with 27mM NaHCO3,10% fetal bo-
vine serum, 100 nM insulin, and 100nM dexamethasone
(WM) (13). For nutrient deprivation-induced autophagy, he-
patocytes were placed in Krebs-Ringer-HEPES buffer (in mM:
115NaCl, 5KCl, 1CaCl2, 1KH2PO4, 1.2MgSO4, and 25Na-
HEPES buffer, pH 7.4) plus 1 (M glucagon (KRH/G).
Loading of fluorophores
Hepatocytes isolated from GFP-LC3 transgenic mice were
loaded with red-fluorescing TMRM (200nM) or far red-
fluorescing MFFR (200nM) for 30min in the absence or
supplemented with 25mM Na-HEPES buffer, pH 7.4. TMRM
and MFFR are membrane-permeable monovalent cations that
accumulate electrogenically into mitochondria (52). In other
experiments, acidic compartments were labeled with LTR
(500nM) under identical conditions. After TMRM, MFFR, and
LTR loading, one-third of the initial loading concentration was
maintained in the medium to maintain a steady-state. Hepa-
tocytes isolated from wild-type mice were loaded with LTR as
just described. Subsequently, the cells were photoirradiated
and loaded with 1mM calcein AM for 10min at 37?C to assess
mitochondrial inner membrane permeabilization. After LTR
and calcein loading, one-third of the initial LTR loading con-
centration was maintained in the medium.
Photodamage and confocal microscopy
Laser-induced photodamage and confocal microscopy
were performed with a Zeiss LSM 510 Meta NLO laser scan-
ning confocal microscope (Carl Zeiss, Thornwood, NY) using
a 63·N.A. 1.4 oil-immersion planapochromat objective lens.
1920 KIM AND LEMASTERS
To induce photodamage, selected areas of individual cells
containing 5–10 mitochondria were exposed to 488-nm argon
laser light at 100% power for times of 80, 160, 320, 640, and
1280ms per pixel. Green (GFP-LC3), red (TMRM and LTR),
and far red (MFFR) fluorescence was excited by the 488, 543,
and 633-nm laser lines, respectively, of argon and helium-
neon lasers in the multitracking mode. To image green and
red fluorescence simultaneously, emitted light was separated
by a 545-nm dichroic mirror and directed to different photo-
nm (red) long-pass filters. To image green, red, and far-red
fluorescence simultaneously, emitted red fluorescence was
additionally separated by a 635-nm dichroic mirror and di-
rected to different photomultipliers through 565- to 615-nm
(red) and 650- to 710-nm (far red) band-pass filters. For serial
imaging at about one frame a minute, laser illumination was
attenuated to less than 0.1% transmission power for pixel
dwell times of 3.2ms. Temperature on the microscope stage
was maintained at 37?C.
Photodamaged mitochondria are selectively
removed by mitophagy
To investigate possible mitophagy after photodamage to
mice were first loaded with TMRM, a red-fluorescing fluor-
ophore that labels polarized mitochondria and is released on
mitochondrial depolarization. Selected areas of hepatocytes
containing 5–10 TMRM-loaded mitochondria were exposed
to 488-nm argon laser light at 100% transmission for 80, 160,
320, 640, and 1280ms. This illumination was, respectively, 2.5,
5, 10, 20, and 40·104times greater than the power of illumi-
nation used for imaging. These pixel powers were designated
area photoirradiation, confocal images of red TMRM and
green GFP-LC3 fluorescence were collected every minute for
up to 120min. After lower laser photoirradiation (at or below
2·), mitochondria released TMRM, indicating depolariza-
tion, but subsequently recovered TMRM fluorescence (Fig. 1,
double arrow). This transient depolarization signified re-
versible photodamage. At higher laser power (above 2·),
mitochondria became irreversibly depolarized, indicating
permanent mitochondrial damage (Fig. 1). At 16·power,
adjacent mitochondria surrounding the illuminated mito-
chondria also became depolarized (Fig. 1, arrow). Pre-
sumably, this bystander injury was due to free radical
generation by regions of the hepatocytes actually exposed to
light (1). Overall, these data showed that photoirradiation
with 488-nm laser light caused sustained mitochondrial de-
polarization in a dose-dependent fashion. Nonetheless, the
photoirradiated hepatocytes remained viable and healthy
with no morphological evidence of injury, necrosis, or apo-
ptosis (e.g., cell surface blebbing, chromatin condensation,
nuclear lobulation) over the time course of the experiments.
At 32–5min after photoirradiation (4·and up), GFP-LC3
fluorescence began to associate with depolarized mitochon-
ring structures (autophagosomes) (Fig. 2, bottom right panel).
However, GFP-LC3 did not localize to mitochondria that
in a dose-dependent manner after expo-
sure to 488-nm light. Hepatocytes iso-
lated from GFP-LC3 transgenic mice were
loaded with 200nM TMRM for 30min.
Regions of the TMRM (red)-loaded hepa-
tocytes were exposed to different amounts
of 488-nm light at 100% power at 2.7, 5.5,
11, 22, and 44·104times greater than the
pixel illumination used for imaging. These
illumination powers were labeled in as-
cending order as 1·, 2·, 4·, 8·, and
16·. The circles represent areas of photo-
irradiation. Images of red TMRM and
green GFP-LC3 were collected before
(baseline) and every minute after photo-
irradiation. Loss of TMRM fluorescence
indicated depolarization of mitochondria.
TMRM loss after photodamage at 1·.
The arrow illustrates TMRM loss from the
mitochondria just outside the zone of
illumination at 16·. GFP-LC3, green
fluorescence protein fused to microtubule-
associated protein1 light chain 3; TMRM,
tetramethyl rhodamine methyl ester. (To
see this illustration in color the reader is
referred to the web version of this article
Mitochondrial damage occurs
transiently depolarized after low light exposure. Unlike nu-
trient deprivation-induced mitophagy (19), GFP-LC3-labeled
cup-shaped phagophores did not grow around and sequester
individual mitochondria; rather, GFP-LC3 began to decorate
the surface of the photodamaged mitochondria (Fig. 2).
Photodamage-induced mitophagy is independent
of TMRM loading
To determine whether light-induced mitophagy is depen-
dent on the presence of TMRM as a photosensitizer, small
regions of GFP-LC3 hepatocytes were exposed to 488-nm il-
lumination at 16·laser power in the absence of TMRM. Si-
milar to observations in the presence of TMRM, the region
exposed to 16·laser power became decorated by GFP-LC3
after about 27min (Fig. 3). The GFP-LC3 fluorescence subse-
quently coalesced into green rings indistinguishable from the
GFP-LC3 rings that formed after photodamage in the pres-
ence of TMRM. Thus, mitophagy after photoirradiation with
488-nm light occurred independently of TMRM, which is
consistent with only weak absorbance of 488-nm light by
TMRM (Fig. 3). In another experiment, hepatocytes were
loaded with TMRM and subjected to photodamage with dif-
ferent doses of 543-nm laser light, a wavelength that is ab-
sorbed by TMRM. Green excitation light at 543-nm caused
photobleaching of TMRM (Fig. 4, top right panel). However,
GFP-LC3 did not localize to the mitochondria that were ex-
posed to 543-nm light (Fig. 4, bottom panels). Taken together,
these observations indicate that 488-nm light induces mito-
chondrial photodamage that leads to mitophagy. The pres-
ence of TMRM did not sensitize the process.
Photodamages induces the mitochondrial
Photoirradiation generates ROS that can induce the mito-
chondrial permeability transition (MPT) in mitochondria (14).
During the MPT, mitochondria become permeable to mole-
cules up to 1.5kDa, which causes mitochondrial depolar-
ization. To investigate a role for the MPT in initiating
photodamage-induced mitophagy, hepatocytes from wild-
type mice were loaded with TMRM, exposed to 488-nm light,
and then loaded with calcein AM. Calcein AM enters the cy-
tosol (and nucleus) where esterases release green-fluorescing
calcein free acid, which outlines mitochondria as round dark
voids. When mitochondria undergo the MPT, calcein fluores-
cence fills the voids (27, 37,38). After exposure to 488-nm light,
mitochondria released TMRM transiently at lower doses and
permanently at higher light levels (Fig. 5, compare top and
middle left panels). Subsequently, after loading with calcein
AM, polarized mitochondria excluded the green-fluorescing
probe. By contrast, depolarized mitochondria that had been
exposed to high power light (16·) filled with calcein imme-
diately (Fig. 5, lower panels), but mitochondria exposed to in-
mitophagy. A TMRM-loaded GFP-LC-3 hepatocyte was ex-
posed to 16·illumination, as described in Figure 1. The circle
indicates the area of photoirradiation. The arrow indicates
first localization of GFP-LC3. The double arrow illustrates
GFP-LC3 ring formation. (To see this illustration in color the
reader is referred to the web version of this article at
Photodamaged mitochondria are degraded by
of TMRM. GFP-LC3 hepatocytes were exposed to 488-nm
light at 16·relative power. The circles indicate the area of
photoirradiation. (To see this illustration in color the reader
is referred to the web version of this article at www
Photodamage-induced mitophagy is independent
1922 KIM AND LEMASTERS
termediate power (8·) released TMRM within about a minute
but only took up calcein after several minutes more (data not
shown). Over time, however, all mitochondria irreversibly re-
leasing TMRM became filled with calcein fluorescence (Fig. 5).
At 1·power, mitochondria recovered TMRM fluorescence
calcein (Fig. 5, bottom panel). Thus, photoirradiation with
higher doses of 488-nm light led to inner membrane permea-
bilization, which is the hallmark feature of the MPT.
In general after autophagic sequestration, autophagosomes
fuse with lysosomes and acidify. To investigate acidification
of mitophagosomes (autophagosomes containing mitochon-
dria), GFP-LC3 transgenic hepatocytes were loaded with
MFFR, a far red-fluorescing fluorophore that accumulates
electrophoretically into polarized mitochondria like TMRM
(40). MFFR-labeled mitochondria were photodamaged with
488-nm light at 16·power, and then red-fluorescing LTR, a
weakly basic fluorophore that accumulates into acidic com-
partments, was loaded. After photodamage, mitochondria
released their MFFR fluorescence signifying depolarization
(Fig. 6, top panels). After 20min, LTR started accumulating
panel), and GFP-LC3 (green) particles began to decorate the
photodamaged mitochondria (Fig. 6, middle right panel, ar-
row). Red LTR fluorescence progressively intensified after-
ward (Fig. 6, bottom panels). Thus, after photodamage,
the structures strongly acidified.
Photodamage-induced mitophagy activates
downstream of PI3K signaling
3MA,the classical inhibitor of autophagy, and wortmannin
block autophagy after nutrient deprivation by inhibition of
class III PI3K (8, 43). To illustrate this effect of 3MA, GFP-LC3
hepatocytes labeled with TMRM were incubated in complete
growth medium (WM) or KRH/G with and without 3MA. In
WM, GFP-LC3-labeled rings and disks were rarely observed
(Fig. 7A, left panel). By contrast, after incubation of hepato-
cytes for 120min in KRH/G, GFP-LC3-labeled structures
phagy. TMRM (red)-loaded GFP-LC3 hepatocytes were
exposed to different amounts of 543-nm light at 100% power–
5.5, 11, 22, and 44·104times higher than the power used for
imaging. These pixel powers were labeled in ascending or-
der as 2·, 4·, 8·, and 16·. The circles represent the areas
of irradiation. Images of red TMRM and green GFP-LC3 were
collected every minute before (baseline) and after photo-
damage. (To see this illustration in color the reader is re-
ferred to the web version of this article at www.liebert
Exposure to 543-nm light does not induce mito-
photodamage. Hepatocytes from a wild-type mouse were
loaded with TMRM and exposed to 488-nm laser light at
relative powers of 1·, 2·, 4·, 8·, and 16·, as described in
Figure 1. Images were collected before (baseline) and after
photoirradiation. After exposure, hepatocytes were loaded
with 1mM calcein acetoxymethyl ester for 10min, and images
of red TMRM and green calcein fluorescence were collected.
The circles indicate areas of photoirradiation. (To see this il-
lustration in color the reader is referred to the web version of
this article at www.liebertonline.com/ars).
Inner membrane permeabilization occurs after
PHOTODAMAGE-INDUCED MITOPHAGY 1923
LC3-labeled rings and disks during incubation in KRH/G
was virtually completely blocked by 3MA (Fig. 7A, right
panel). A similar inhibition of nutrient deprivation-induced
autophagosome proliferation in hepatocytes under identical
conditions occurs after treatment with wortmannin (39). To
determine the role of PI3K on mitophagy after photodamage,
GFP-LC3 hepatocytes were loaded with TMRM and treated
group of mitochondria with 488-nm laser light at 16·laser
power. Photodamaged mitochondria again irreversibly lost
TMRM fluorescence, indicating loss of membrane potential
(Fig. 7B, C, top panels). In the presence of 3MA, green ring
structures again started to form around photodamaged mi-
tochondria after about 30min (Fig. 7B). To further confirm
that photodamage-induced mitophagy occurred after inhibi-
tion of PI3K, GFP-LC3 hepatocytes were treated with 100nM
wortmannin for 30min before photoirradiation. Wortmannin
did not inhibit photodamage-induced mitophagy (Fig. 7C). In
the presence of either 3MA or wortmannin, the number of
GFP-LC3 rings and the strength of labeling were greater than
in their absence (Fig. 7).
Our results provide direct evidence that mitophagy selec-
tively removes and degrades damaged mitochondria. In he-
patocytes incubated in nutritionally replete growth medium,
only photoirradiated mitochondria that depolarized were
sequestered into autophagosomes. When mitochondria were
photoirradiated at lower laser power, mitochondria initially
depolarized but then recovered polarization as indicated by
reuptake of TMRM. Such mitochondria did not undergo
mitophagy. Thus, photodamage leading to sustained mito-
chondrial depolarization was required to initiate sequestra-
tion into autophagosomes.
dependent manner after photoirradiation with 488-nm light
(Fig. 1). The lowest light exposure, namely that used to image
hepatocytes, did not cause mitochondrial depolarization or
induce mitophagy. Initially, after a light exposure about
within about 3min. At greater photoirradiation, mitochondria
depolarized in a sustained fashion. At highest illumination, a
bystander effect occurred in which depolarization not only
occurred in mitochondria under the light beam but also in
adjacent mitochondria outside the illuminated region (Fig. 2).
Bystander photoirradiation suggests that a toxic agent formed
in illuminated regions diffused into immediately adjacent ar-
eas.This toxic agent is mostlikelyROS, suchas singlet oxygen,
which is produced during exposure to strong light. Photo-
xicity-dependent mitophagy occurred in the absence ofTMRM
or other added fluorophore and, thus, did not require photo-
sensitization of an exogenous absorber (Fig. 3). In addition,
mitophagy did not occur with 543-nm light (Fig. 4). Previous
work shows that photoirradiation of 400- to 500-nm light
causes oxygen-dependent inactivation of flavoproteins and
succinate dehydrogenase that is mediated by production of
ROS (1). Thus, photodamage to mitochondria in our experi-
ments is likely via photoexcitation of succinate dehydrogenase
and other mitochondrial flavoproteins.
ROS induce the MPT in mitochondria, leading to depolar-
ization, uncoupling, and more ROS formation (34). After
photodamage, sustained mitochondrial depolarization ap-
peared to be a prerequisite for subsequent mitophagy. Mi-
tochondria that depolarized transiently after light exposure
did not undergo subsequent mitophagy, whereas photo-
damaged mitochondria that underwent sustained depolar-
ization were reproducibly sequestered into autophagosomes
(Fig. 2). Moreover, this latter group of mitochondria became
permeable to calcein, indicative of the inner membrane per-
meabilization of the MPT (Fig. 5). Thus, sustained mitochon-
drial depolarization andassociated
permeabilization seemed to be required for autophagy sig-
naling. These results are consistent with involvement of
the MPT in photodamage-induced mitophagy, as previ-
acidify. GFP-LC3 hepatocytes were loaded with 300nM
MFFR and 500nM LTR for 30min. Circles show the area of
photoirradiation with 488-nm light at a relative intensity
of 16 ·. After photoirradiation, mitochondria lost MFFR
fluorescence (pseudocolored blue) and took up red LTR
fluorescence, which signified acidication. Arrow indicates
localization of green GFP-LC3 to damaged mitochondria.
Double arrow indicates GFP-LC3 rings (autophagosomes)
around LTR-labeled photodamaged mitochondria. LTR, Ly-
sotracker Red. (To see this illustration in color the reader is
referred to the web version of this article at www.lie-
Mitophagosomes formed after photodamage
1924KIM AND LEMASTERS
ously proposed for autophagy stimulated by nutritional
deprivation (10). Moreover, when cells are treated with
m-chlorophenylhydrazone, depolarized mitochondria are re-
Photodamage-induced mitophagy, however, differed from
nutrient deprivation-induced mitophagy in several ways. In
nutrient deprivation-induced mitophagy, small (0.2–0.3mm)
pre-autophagosomal structures associate with polarized mi-
tochondria and grow into crescent-shaped phagophores that
envelope and enclose individual polarized mitochondria
into mitophagosomes (19). Mitochondrial depolarization
only occurs at or after formation of mitophagosomes. Subse-
quently, as themitophagosomal vesicles acidify andfuse with
lysosomes, GFP-LC3 is released and/or degraded (20).
However, when the MPT is inhibited, autophagy also be-
comes inhibited, indicating that the MPT likely plays a role in
the nutrient deprivation-induced mitophagy, possibly in co-
ordination with autophagosomal sequestration.
By contrast, in mitophagy induced by photodamage with
488-nm light, only depolarized mitochondria were targeted
for autophagic degradation. Moreover, instead of being en-
veloped by a crescent-shaped phagophore, the periphery of
photodamaged mitochondria became decorated with small
enveloping the entire mitochondrion (Figs. 2 and 6). Ad-
ditionally, mild acidification of photodamaged mitochondria
occurred before assembly of continuous GFP-LC3-decorated
studies will be needed to determine whether depolarized
mitochondria themselves undergo mild acidification or
whether a sequestration membrane encloses photodamaged
mitochondria before recruitment of GFP-LC3 (Fig. 6).
Mitophagy stimulated by 488-nm light also occurred in
the absence of TMRM, indicating that TMRM was not acting
as a photosensitizer (Fig. 3). Photoirradiation with 543-nm
did not induce mitophagy (Fig. 4). Unexpectedly, photo-
irradiation with 543-nm light did lead to loss of mitochon-
drial TMRM fluorescence (Fig. 4). This may reflect direct
photobleaching of TMRM rather than photodamage and
depolarization of mitochondria. However, if mitochondria
remained polarized, reaccumulation of TMRM from the cy-
tosol and extracellular medium into mitochondria would be
were loaded with TMRM. In (A), hepatocytes were incubated 120min in WM (left panel), KRH/G (middle panel) or KRH/G
plus 10mM 3MA (right panel). In (B) and (C), hepatocytes were pretreated with 10mM 3MA (B) or 100nM wortmannin (C)
for 30min. Photoirradiation with a 488-nm laser at a relative intensity of 16·(circles) was then performed. Images were
collected every minute. WM, Waymouth’s medium/10% fetal bovine serum/insulin/dexamethasone; KRH/G, Krebs-
Ringer-HEPES plus glucagons; 3MA, 3-methyladenine. (To see this illustration in color the reader is referred to the web
version of this article at www.liebertonline.com/ars).
3-Methyladenine and wortmanin do not prevent mitophagy after photodamage. GFP-LC3 transgenic hepatocytes
expected to occur over time, and such reuptake did not oc-
cur. Alternatively, mitophagy after photoirradiation may
depend on the nature of the mitochondrial injury, and
damage to flavoproteins by 488-nm irradiation may be the
more potent mitophagy inducer. Additional studies will be
needed to characterize what specific mitochondrial injuries
induce mitophagy the most.
A particularly noteworthy difference between nutrient
deprivation-induced mitophagy and photodamage-induced
mitophagy is that the latter was not blocked by PI3K inhibi-
tion with 3MA (10mM) or wortmannin (100nM) (Fig. 7) (18).
Class III PI3K/p150 interacts with Beclin 1, a mammalian
homologue of Atg6 that is required for an early stage of au-
tophagosome formation during nutrient deprivation (47).
Rather, in our study, PI3K inhibition appeared to augment
GFP-LC3 association with photodamaged mitochondria (Fig.
7). These findings indicate that activation of mitophagy after
photodamage occurs independently of PI3K signaling, which
is in striking contrast to autophagy after other stimuli, such as
nutritent deprivation, that is blocked by PI3K inhibitors (see
Fig. 7A). Indeed, photodamage-induced GFP-LC3 ring
formation was more robust after PI3K inhibition, which sug-
gests that subsequent processing of mitophagosomes may
require PI3K, as shown for the processing of mitophagosomes
in nutrient deprivation-induced autophagy (19, 31, 47).
Mitochondria of nonproliferating tissues such as heart,
brain, liver, and kidney have a half-life of 10 to 25 days (29,
36). In this normal turnover, old and presumably dysfunc-
tional mitochondria are removed by mitophagy and replaced
by biogenesis of new mitochondria. Such mitophagy serves
the physiological function of segregating and degrading
dysfunctional mitochondria that might otherwise release
ROS, pro-apoptotic proteins, and other toxic mediators. Since
mitochondria are a primary site of ROS generation, mito-
chondrial DNA (mtDNA) is prone to oxidative damage. Due
to limited mtDNA repair mechanisms, damaged mtDNA
likely accumulates with time. Since mtDNA is nearly 100%
active in transcription (compared to 2% or 3% for nuclear
DNA), damage to mtDNA will lead quickly to mitochondrial
dysfunction. Decreased mitophagy may promote accumula-
caloric restriction and rapamycin, inducers of autophagy, in-
crease longevity in rodents (7, 12). Moreover, mitochondria
with mutant mtDNA are selectively removed in hetero-
plasmic cells (44).
In conclusion, photoirradiation by 488-nm light caused
mitochondrial depolarization, inner membrane permeabili-
zation, and subsequent selective mitophagy, consistent with
previous reports of photodynamic induction of the MPT and
involvement of the MPT in mitophagy (10, 24, 53). However,
upstream signaling for photodamage-induced mitophagy
bypassed the classical PI3K signaling pathway of nutrient
to be needed for downstream processing of newly formed
mitophagosomes. Thus, mitophagy is an important mecha-
nism to sequester and degrade damaged mitochondria in
otherwise viable and healthy cells.
The authors thank Dr. Noboru Mizushima at Tokyo
Medical and Dental University for GFP-LC3 mice. This
work was supported, in part, by Grants 5 R01 CA119079,
2-R01 DK37034, 1 P01 DK59340, 1 R01 DK073336, and C06
RR015455 from the National Institutes of Health.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Dr. John J. Lemasters
Center for Cell Death, Injury, and Regeneration
Department of Pharmaceutical & Biomedical Sciences
Medical University of South Carolina
QF308 Quadrangle Building
280 Calhoun St.
P.O. Box 250140
Charleston, SC 29425
Date of first submission to ARS Central, November 08, 2010;
date of acceptance, December 02, 2010.
GFP¼green fluorescence protein
KRH/G¼Krebs-Ringer-HEPES plus glucagon
light chain 3
MFFR¼MitoFluor Far Red
MPT¼mitochondrial permeability transition
ROS¼reactive oxygen species
TMRM¼tetramethyl rhodamine methyl ester
WM¼Waymouth’s medium/10% fetal bovine
1928 KIM AND LEMASTERS