[Autophagy 1:1, 23-36; January 2005]; ©2005 Landes Bioscience

The Dynamics of Autophagy Visualized in Live Cells

Research Paper

From Autophagosome Formation to Fusion with Endo/Lysosomes

Edward T. W. Bampton1 ABSTRACT

Christoph G. Goemans1 Autophagy has been implicated in a range of disorders and hence is of major interest.

Dhevahi Niranjan1
However, imaging autophagy in real time has been hampered by lack of suitable markers.

.
Noboru Mizushima2
We have compared the potential of monodansylcadaverine, widely used as an autophago-

E
somal marker, and the Atg8 homologue LC3, to follow autophagy by fluorescence

UT
Aviva M. Tolkovsky1,*
microscopy whilst labelling late endosomes and lysosomes simultaneously using EGFP-
CD63. Monodansylcadaverine labelled only acidic CD63-positive compartments in

RIB
1Department of Biochemistry; University of Cambridge; Cambridge UK
response to a range of autophagic inducers in various live or post-fixed cells, staining
being identical in atg5+/+ and atg5-/- MEFs in which autophagosome formation is disabled.
2Department of Bioregulation and Metabolism; The Tokyo Metropolitan Institute of
Monodansylcadaverine staining was essentially indistinguishable from that of LysoTracker
Medical Science; Bunkyo-ku, Tokyo, Japan

IST
Red, LAMP-1 or LAMP-2. In contrast, 60–90% of EGFP-LC3-positive punctate organelles
*Correspondence to: A.M. Tolkovsky; Department of Biochemistry; University of did not colocalise with LAMP-1/LAMP-2/CD63 and were monodansylcadaverine-nega-
Cambridge; Building 0, Downing Site; Cambridge CB2 1QW UK; Tel: 01223.

D
tive while EGFP-LC3 puncta that did colocalise with LAMP-1/LAMP-2/CD63 were also
339319; Fax 01223.333345; Email: amt@mole.bio.cam.ac.uk
monodansylcadaverine-positive. Hence monodansylcadaverine is no different from other
Received 08/24/04; Accepted 12/21/04

Previously published online as an Autophagy E-publication:
http://www.landesbioscience.com/journals/autophagy/abstract.php?id=1495
KEY WORDS
apoptosis, LC3, mitochondria, monodansylca-
daverine (MDC), rapamycin
OT
markers of acidic compartments and it cannot be used to follow autophagosome forma-
tion. In contrast, fusion of mRFP-LC3-labelled autophagosomes with EGFP-CD63-positive
ON
endosomes and lysosomes and sequestration of dsRed-labelled mitochondria by
EGFP-LC3- and EGFP-CD63-positive compartments could be visualized in real time.
Moreover, transition of EGFP-LC3-I (45 kDa) to EGFP-LC3-II (43 kDa)—traced by
immunoblotting and verified by [3H]ethanolamine labelling—revealed novel insights into
.D
the dynamics of autophagosome homeostasis, including the rapid activation of
autophagy by the apoptotic inducer staurosporine prior to apoptosis proper. Use of
CE

ABBREVIATIONS
fluorescent LC3 and a counter-fluorescent endosomal/lysosomal protein clearly allows the
entire autophagic process to be followed by live cell imaging with high fidelity.
IEN

LAMP-1/2 lysosomal associated membrane

protein -1/2
INTRODUCTION
SC

LBPA lysobisphosphatidic acid

LC3 light chain 3
BIO

LTR lysotracker red Autophagy is a process by which long lived proteins and larger cytoplasmic components
MDC monodansylcadaverine
are degraded.1-7 Upon induction of autophagy a double membraned crescent, the
MDH monodansylpentane
PtdEth phosphatidylethanolamine
so-called isolation membrane, is formed which then surrounds a region of cytoplasm and
ES

forms a double membraned vesicle termed the autophagosome (the term initial autophagic
ACKNOWLEDGEMENTS
vacuole, Avi, is sometimes used). The autophagosome goes on to fuse with the vacuole in
ND

yeast or with endosomes to form amphisomes and lysosomes to form autolysosomes (also
We thank Helen Bye for help with the tissue referred to as the degradative autophagic vacuole Avd) in mammalian cells.8
culture, Molly Sheldon for preparing the stable Autophagy has been described at the electron microscopic level for many years,9,10 one
LA

dsRed-mito HeLa cell line and Patricia Boya for of the first examples being the engulfment of mitochondria in livers of glucagon-treated
critical reading of the manuscript. We are also rats,11 but direct cellular and biochemical markers have been sorely lacking. The majority
indebted to J. Paul Luzio for providing the pEGFP-
06

of studies of autophagy have relied on indirect biochemical tracers and electron microscopic
CD63 plasmid and for helpful advice, Tamotsu
Yoshimori for providing the pEGFP-LC3 plasmid, studies to determine whether and to what extent autophagy is occurring. However,
20

Roger Tsien for the mRFP construct, Jean interpreting electron microscopic data is subjective, especially distinguishing autophago-
Gruenberg for providing the anti-LBPA antibody somes and autolysosomes, and even differentiating autophagic vacuoles from endocytic/
©

and Yasuo Uchiyama for providing the anti-LC3 phagocytic compartments.12 Another drawback of electron microscopy is its static nature,
antibody. making it very difficult to follow what is in fact a highly dynamic process. Other methods
This work was supported by a programme grant have included biochemical correlates such as the degradation of long-lived proteins.13
to AMT from the Wellcome Trust. However, starvation induces an increased degradation of long-lived proteins even in
Atg5-/- ES cells, which are incapable of initiating macroautophagy,14 presumably because
other degradative mechanisms such as chaperone-mediated autophagy and microautophagy
also exist.13,15,16
Monodansylcadaverine (MDC) has been widely used as a specific marker for autophagic
vacuoles, since it was shown to accumulate in acidic compartments enriched in lipids.17

www.landesbioscience.com Autophagy 23
 The Dynamics of Autophagy Visualized in Live Cells

induced to undergo autophagy, for example after Table 1 Colocalization coefficient values for different autophagosomal
Indeed its staining intensity increases in cells

amino acid starvation18 or treatment with and endo/lysosomal markers

Marker A Marker B Cell type Mean (95% limits)

rapamycin.19 It has been proposed that the intensity
increase is due to its intercalation into the lipid
moiety of the double membraned structure of the EGFP-CD63 endo/lysosomes HeLa 74.0 (68.7–79.3)
autophagosome, an interaction that is retained in EGFP-CD63 other organelles HeLa 25.9 (18.1–30.9)
paraformaldehyde-fixed tissue.20 Based on the EGFP-LC3 endo/lysosomes HeLa, MCF-7 51.4 (46.0–56.1)
propensity of MDC to stack into adjacent mem- EGFP-LC3 other organelles HeLa 21.8 (12.0–31.6)
branes, a neutral derivative, monodansylpentane,
MDC EGFP-CD63 HeLa, MEFs (wt, atg5-/-) 88.0 (81.1–94.9)
was synthesized and this has been suggested to
locate more exclusively into double membraned MDC LTR HeLa, Cos7, MCF-7, NIH-3T3 79.5 (74.8–84.2)
21
structures. No quantitative studies have been MDC EGFP-LC3 HeLa, Cos7, MCF-7, NIH-3T3 37.0 (30.7–43.3)
conducted alongside cell markers of other organelles Mean colocalization coefficients and 95% confidence limits based on pooled data from different experiments with different cell types
to determine which fraction of the vesicles stained are shown. Endo/lysosomal markers refer to LTR (in live or post-fixed cells), and immunostaining with antibodies against LAMP-1,
by MDC and MDH are true nascent autophago- LAMP-2, CD63, cathepsin B, and LPBA. Other organelles include mitochondria (detected with dsRed-mito), and immunostained early
somes and not autophagosomes that have fused endosomes (anti-EEA1), ER (anti-KDEL), and Golgi (anti-GM130).

Munafo and Colombo18 have performed the most critical assessment MATERIALS AND METHODS
with endosomes, multivesicular bodies and lysosomes.

of MDC stained vacuoles, and have shown that a small proportion

of MDC-stained vacuoles are also stained with the autophagic marker Materials. LysoTracker Red (LTR) was obtained from Molecular Probes.
MAP-LC3, but the possibility that these are autolysosomes and not LAMP and CD63 antibodies were obtained from the Developmental
Studies Hybridoma Bank, University of Iowa (LAMP-1 clone H4A3,
autophagosomes was not excluded.
LAMP-2 clone H4B4, CD63 clone H5C6), anti-LBPA was a gift from
Recently, the molecular mechanisms by which autophagy is initi- J Gruenberg (Department of Biochemistry, University of Geneva,
ated and regulated have started to come to light. Studies using yeast Switzerland),35 anti-KDEL MAC256 was a gift from G Butcher (The
genetics have identified a range of mutants for the autophagic path- Babraham Institute, Babraham, UK)36 and anti-EEA1 and anti-Golgi
way, initially termed apg 22 or aut 23 genes, but recently redesignated GM130 were from BD Biosciences (Pharmingen). Secondary antibodies
as atg.24 MAP-LC3 is one of three mammalian homologues of yeast were from Jackson Immunoresearch Labs. Hanks balanced salt solution
Atg8, which is not only necessary for formation of autophagosomes, (HBSS), G418, MDC, NH4Cl, rapamycin, tamoxifen, and staurosporine
but also relocalises to and participates in the formation of the were obtained from Sigma. DMEM was obtained from Invitrogen.
autophagosomal membrane (reviewed in ref. 25). In yeast, this local- Boc-Asp(O-Me)fluoromethyl ketone (BAF) was obtained from Enzyme
Systems Products. Monodansylpentane (MDH) was synthesized as
ization requires both cleavage of Atg8 at it C-terminus and its described previously.21 pEGFP-LC3 was a kind gift of Tamotsu Yoshimori
modification by a phospholipid, processes controlled by two ubiqui- (National Institute for Basic Biology, Okazaki, Japan), pEGFP-CD63 was a
tination-like modifying systems.26,27 Consistent with this process, in kind gift of Paul Luzio (CIMR, University of Cambridge, UK), mRFP,
cells expressing EGFP-LC3, induction of autophagy by amino-acid kindly supplied by Roger Tsien (UCSD, Ca, USA)37 was inserted in place
starvation promotes relocalization of the protein to newly-formed of EGFP in the pEGFP-LC3 vector to create mRFP-LC3. The anti-LC3
vesicles14,28 and modification of endogenous LC3 to a more rapidly- antibody was a kind gift of Yasuo Uchiyama (Osaka University, Osaka,
migrating form can be observed on SDS-PAGE29 due in part to the Japan). The dsRed-mito plasmid was purchased from Clontech
C-terminal cleavage, but mainly to its lipid modification.30,31 LC3 (Invitrogen). EGFP-LC3 adenovirus was constructed using the AdEasy
system and used at an MOI of 75.
is either removed when autophagosomes fuse with lysosomes, and/or Cell culture. All cells (HeLa, Cos7, MCF-7 and SV40-immortalized
is degraded inside the lysosomes, the turnaround of individual atg5+/+ and atg5-/- MEFs) were routinely cultured in DMEM supplemented
vesicles being in the order of 10 minutes,28 as predicted by Pfeifer.32 with penicillin/streptomycin and 10% foetal calf serum (FCS). Autophagy
From these studies, the use of MAP-LC3 has been advocated as a was induced by several treatment regimes. For amino acid starvation, cells
mechanism to study autophagy in live cells. How consistent these were washed five times in Hanks Balanced Salt Solution (HBSS) and incu-
biochemical and cellular changes are remains to be thoroughly bated in HBSS for the indicated periods. For tamoxifen-induced autophagy,
documented. MCF-7 cells were maintained in DMEM without phenol red supplemented
In this paper we have examined the use of MDC and LC3 tagged with 10 mM HEPES and 10% charcoal stripped FCS. The day before
tamoxifen treatment, medium was changed to the same medium containing
to various fluorescent proteins as potential markers for the subcellular 3% charcoal stripped FCS and cells infected with EGFP-LC3 adenovirus.
compartments involved in autophagy in conjunction with the late Tamoxifen used at 1 µM was diluted from a 5 mM stock in 1:1 ethanol:
endosomal/lysosomal marker EGFP-CD63.33 We show that MDC DMSO; an equivalent volume of 1:1 ethanol:DMSO was added to control
is not a bona fide marker of nascent autophagosomes but it will stain cells.38 For rapamycin-induced autophagy, Cos7 cells were infected with
acidic compartments comprised of late endosomes and lysosomes EGFP-LC3 adenovirus the day before treatment and then incubated in full
that have recently fused with autophagosomes (known as amphi- medium in the presence of 200 nM rapamycin. Staurosporine and NH4Cl
+/+ and atg5-/-
somes34 and/or autolysosomes). In terms of MDC staining, therefore, treatments were administered in DMEM and 1% FCS. atg5
MEFs were prepared from fibroblasts from day 13.5 ATG5-/- and wild-type
these are not distinguishable from ordinary endo/lysosomes and
embryos,14 transformed with SV40 large T antigen (pEF321-T), and
hence cannot be used to indicate autophagosomal activity. On the immortalized cell lines were established.39
other hand, visualization of LC3 together with CD63 offers a Transfection and cell line selection. HeLa cells were transfected using
dynamic view of the entire process of autophagy in live cells. Lipofectamine Plus (Invitrogen) according to the manufacturer’s instructions.
Stable cell lines were selected using increasing concentrations of G418 over
three weeks and single clones picked to give clonal cell lines.

24 Autophagy 2005; Vol. 1 Issue 1

 The Dynamics of Autophagy Visualized in Live Cells

Figure 1. EGFP-CD63 is a reliable endo/lysosomal marker. EGFP-CD63 stably expressed in HeLa cells was examined for its fidelity as a lysosomal marker
in live cells (using 50 nM LTR, loaded for 30 min prior to observation) or in fixed cells after immunostaining with antibodies against LAMP-1, LAMP-2,
Cathepsin B or CD63 (to also obtain a measure of the fidelity of the correlation coefficient measurements), EEA1 (early endosome), LBPA (late endosome)
GM130 (Golgi), KDEL (endoplasmic reticulum) and dsRed mito (mitochondria). Confocal images were collected and are presented without modification.
Each correlation plot is derived from a field of view of 5–10 cells which contained the cell images shown. The mean correlation coefficient value (c) ± s.d.
of at least 5 images of 5–10 cells per image are shown on the plots. EGFP-CD63 is represented on the x-axis.

Dye loading. LTR was loaded at a concentration of 50 nM in serum free
DMEM for one hour. MDC was loaded at 50 µM for ten minutes or for the
MCF-7 experiments for one hour to match the conditions previously used.38
Immunocytochemistry. Cells grown on coverslips were fixed in 4%
paraformaldehyde for 10–15 minutes and post-fixed for five minutes in
100% methanol at -20˚C. For LBPA staining methanol was omitted and
0.05% saponin was used for permeabilization. All primary antibodies were
used at a concentration of 0.5–1 µg/ml in 10% goat serum in PBS. Staining
was carried out overnight at 4˚C. After 3 rinses in PBS, cells were incubat-
ed with 0.5 µg/ml anti-mouse or anti-rabbit Cy3 or Alexa 647 conjugated
antibodies as appropriate. Cells were counterstained with 2 µg/ml Hoechst
33342, rinsed a further three times and mounted in SlowFadeñ Light
(Molecular Probes).
Microscopy. Cells were observed with an Olympus IX70 microscope and
analyed using the UltraView confocal microscope system (PerkinElmer). All
images were confocal, except where using MDC, where fluorescence images
were taken. Note that for fluorescence images of LTR, acquisition had to be
immediate due to rapid photobleaching. Digital images were acquired and
colocalization analysis was performed using the UltraView software.
Colocalization is a quantitative measure of how many pixels detected by the
camera in each channel are coincident and is calculated by the following
equation, which gives a correlation coefficient, c:
where a = Channel1 - Channel1
and b = Channel2 - Channel2
The mean ± s.d. of c was calculated from at least 5 independent images
of cells (each image comprising 5–10 cells) and the 95% correlation coeffi-
cient was calculated after pooling data from different markers and/or cell
types (Table 1). Several methods were used to investigate the limits of values
of c that indicate high colocalization versus no colocalization (comparison of
repeated images of the same fluorescent structure in two different channels
in live cells, analysis of staining of the same organelle with two different
antibodies, staining with an antibody against the fused protein of an
EGFP-fusion product, or analysis of separate organelles). These analyses
demonstrate that c values above 0.7 indicate high colocalization while c
values below 0.4 indicate insignificant colocalization. Higher and lower values
respectively were usually obtained in fixed cells imaged in confocal mode. In
cells in which the pH gradient of the lysosomes was dissipated, MDC and
LTR stain filled the cytoplasm inevitably causing higher than background
colocalization with cytoplasmic organelles when viewed under the fluorescent
microscope. For clarity of viewing, MDC images are shown in greyscale, but
merged images using this marker assume it to have a blue colour (giving
cyan with green and magenta with red where colocalized). p values are based
on two-tailed Student’s t-test.
Western blotting. HeLa cells stably expressing EGFP-LC3 were treated
in DMEM containing 1% serum with either 200 nM rapamycin, or 50 mM
ammonium chloride, or 1 µM staurosporine and 100 µM BAF, or 20 µg/ml
cycloheximide, or left untreated. Proteins were extracted in SDS-PAGE
loading buffer as indicated and resolved on 10% SDS-PAGE gels. Proteins
were transferred to nitrocellulose, probed with anti-GFP antibody (1:500,
Clontech), and detected using the ECL detection system (Amersham).
Parallel cultures growing on coverslips were fixed at the same points for
microscopic observation. To confirm that EGFP-LC3 conversion matched

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 The Dynamics of Autophagy Visualized in Live Cells

Figure 2. EGFP-LC3 labels early autophagosomes and autophagosomes after their fusion with endo/lysosomes. (A) Unstimulated HeLa cells stably expressing
EGFP-LC3 and counterstained with an anti-CD63 antibody. (B) HeLa cells stably expressing EGFP-LC3, or EGFP-LC3 and dsRed-mito, were starved of amino
acids for two hours in HBSS. Live cells images were collected immediately in confocal mode as described in Figure 1, while other cultures were fixed and
immunostained with antibodies against EEA1, LBPA, GM130, KDEL, CD63, LAMP-1, LAMP-2 or Cathepsin B. Live cells were also imaged after loading with
LTR. Images, correlation plots and coefficients were calculated as described in Figure 1 and are shown alongside each pair of images. EGFP-LC3 is represented
on the x-axis. Note that EGFP-LC3 showed no significant colocalization with mitochondria, early endosomes, Golgi, or ER but ∼40% of the puncta colocalize
with endo/lysosomal markers in live cells (stained with LTR) or in fixed cells (stained with antibodies against endo/lysosomal markers).

that of endogenous LC3, extracts from control and NH4Cl-treated cells EA-Wax (EABiotech, Gentaur) as per manufacturer’s instruction. The
were separated on a 15% gel and the immunoblot was probed with autoradiographic film was developed after 12 days.

anti-LC3 antibody.
[3H]ethanolamine labelling. HeLa cells stably expressing EGFP-LC3
were labelled with 33.3 µCi/ml [3H]ethanolamine in growth medium for
14 hours and then one cohort was treated with 50 mM NH4Cl for 2 hours.
Cells were washed with PBS and then lysed in PBS containing 2% Triton
X-114 and protease inhibitors (CompleteTM, Roche). After tumbling end
over end at 4˚C for 1.5 hours, tubes were spun at 13,000 x g for 10 minutes
at 4˚C, a sample of the supernatant was removed (total lysate), and the tube
was heated to 37˚C for 10 minutes to obtain phase separation. Tubes were
spun 13,000 x g for 10 minutes and the upper aqueous phase was separated
from the detergent phase. After cooling to 4˚C, the aqueous phase was
reextracted with Triton X-114 and the entire incubation cycle was repeated.
In total the aqueous phase was reextracted 3 times. Proteins in the total
lysate, the first Triton X-114 fraction, and the thrice-extracted aqueous
phase were precipitated with acetone and pellets were dissolved in 50 mM
Tris-Cl pH 8.8 and 1% SDS. After normalising for protein, 20 or 50 µg of
each sample were separated by SDS-PAGE and blotted onto nitrocellulose.
The blot with 20 µg/lane was probed with anti-EGFP while the other blot
was exposed to X-ray film (Kodak, Biomax) after impregnation with
RESULTS
EGFP-CD63 marks late endosomes and lysosomes but not early endo-
somes, ER or Golgi. In order to label lysosomes in a pH-independent
manner and avoid induction of autophagic activity due to the transfection
process (discussed in ref. 40) we generated stable EGFP-CD63-expressing
HeLa cell lines. As shown in Figure 1, EGFP-CD63 showed high colocaliza-
tion with the late endosomal/lysosomal markers (defined forthwith as endo/
lysosomes) LAMP-1, LAMP- 2, cathepsin B and the lipid marker lysobispho-
sphatidic acid (LBPA) in fixed cells and also with LTR in live cells. The
correlation coefficient (c) value for EGFP-CD63 and LTR was 0.72, while
those for LAMP-1/2, LBPA, and cathepsin B antibodies were between
0.69–0.82 (combined mean ± s.d. 0.74 ± 0.06). In contrast, little colocaliza-
tion was found with markers of early endosomes (EEA1, c = 0.33), Golgi
(GM130, c = 0.32), endoplasmic reticulum (ER) (KDEL, c = 0.4) or mito-
chondria (dsRed-mito, c = 0.13) (combined mean ± s.d., 0.29 ± 0.12,
p<0.0003 compared to endo/lysosomal c value). In some cells a fraction of
EGFP-CD63 was also found on the plasma membrane, consistent with

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 The Dynamics of Autophagy Visualized in Live Cells

Figure 3. MDC does not label nascent autophagosomes prior to their fusion with the endo/lyososmal compartment. (A) HeLa cells stably expressing
EGFP-CD63 were labelled with 50 µM MDC for 10 minutes and imaged immediately or fixed with 4% PF for 15 minutes before imaging in fluorescent
mode. Correlation plots and coefficients were calculated as described in Figure 1 and are shown alongside each pair of images. MDC is represented on
the y axis. (B) HeLa cells stably expressing EGFP-LC3 were induced to undergo autophagy by amino acid starvation for two hours after which they were
stained with 50 µM MDC for ten minutes. Correlation plots show the left label of each pair on the x axis. Note that MDC is completely colocalized with
LTR, as in unstimulated cells (middle panel), but is only localized with ∼40% of the LC3- positive puncta (right panel), the same proportion of EGFP-LC3 that
colocalizes with the acidic endo/lysosomal marker LTR (left panel).

previous studies in platelets and other cells41 but there was little cytoplasmic
background. Moreover, EGFP-CD63 overexpression did not alter the
number or distribution of endo/lysosomal compartments when compared
with untransfected HeLa cells, both cells being stained for endogenous
CD63 (c 0.82 ± 0.003, data not shown). These data confirm that
EGFP-CD63 labels the acidic endo/lysosomal compartment with high
fidelity without altering their number or distribution.
All stages of autophagy before and after fusion with endo/lysosomes
can be followed with EGFP-LC3. HeLa cells were stably transfected with
EGFP-LC3 to investigate the formation of autophagosomes and their
segregation from lysosomes. In the absence of stimulation of autophagy the
majority of the EGFP-LC3 was diffusely localized, regardless of relative
expression levels, and showed little overt colocalization with any of the
organellar markers. Figure 2A, shows cells stained with anti-CD63 (c = 0.32,
see also Figs. 3 and 7). Stimulation of autophagy by amino acid starvation
for 2 hours (Fig. 2B) caused EGFP-LC3 to form into punctate structures as
reported previously.29 There was little colocalization of these puncta with
early endosomes (EEA1, c = 0.27), Golgi (GM130, c = 0.07), ER (KDEL,
c = 0.24) or mitochondria (dsRed-mito, c = 0.29) (combined mean ± s.d. 0.22
± 0.1). At this timepoint, about 60% of the EGFP-LC3-positive puncta

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 The Dynamics of Autophagy Visualized in Live Cells

Figure 4. The number of endo/lysosomes is significantly increased in tamoxifen-treated MCF-7 cells and rapamycin-treated Cos7 cells but MDC only colo-
calizes strongly with LTR-positive compartments. (A) MCF-7 cells infected with Ad-EGFP-LC3 were grown in phenol red-free medium containing 3%
charcoal-treated serum and treated with 1 µM tamoxifen over four days. Cells were loaded with 50 nM LTR and 50 µM MDC for one hour and fixed with
4% PF for 15 minutes before imaging in fluorescent mode. Cells were subsequently stained for LAMP-1. Note the remarkable increase in endo/lysosome
numbers reflected as increased coincidence of staining for MDC and LTR after three days exposure to tamoxifen. Correlation plots show MDC on the y axis.
(B) Ad-EGFP-LC3 infected Cos7 cells were treated with 200 nM rapamycin over two days. Cells were loaded with 50 nM LTR for one hour and 50 µM MDC
for 15 minutes and imaged live in fluorescent mode after which they were fixed and stained for LAMP-1. Areas of the coverslip that were not exposed to
UV fluorescence to visualise MDC are shown. Note that the increased intensity of MDC staining in rapamycin-treated cells matched by increased LTR and
LAMP-1 staining (average grain counts taken from 5 separate images, one of which is shown, increased from 29 to 86 grains per nucleus). Representative
correlation plots show LTR on the y axis and MDC on the x axis or MDC on the y axis and EGFP-LC3 on the x axis.

were also unfused with endo/lysosomes. However the remaining 40% was
equally colocalized with either of six endo/lysosomal markers (LTR, c = 0.54,
LAMP-1, c = 0.59, LAMP-2, c = 0.54, CD63, c = 0.48, cathepsin B, c = 0.44,
LBPA (c = 0.5) (combined mean ± s.d. 0.52 ± 0.05, p<0.002 compared to
value for other organelles). The overexpression of EGFP-LC3 did not alter
the number or distribution of endo/lysosomal compartments compared
with untransfected HeLa cells after both cell types were stained for LAMPs
(data not shown). These results indicate the rapid formation of amphisomes34
and autolysosomes from autophagosomes revealed previously by morpho-
metric studies32 or by visualization of EGFP-Atg5.14 Interestingly, when
LTR was colocalized with EGFP-LC3, we noted in live cells that the higher
the level of LTR staining the lower the level of EGFP-LC3 staining (data not
shown). These results fit with the previous data of Kabeya et al.29,30 who
showed that intralysosomal LC3 staining is gradually lost due to proteolytic
degradation while membrane bound LC3 is removed by Atg4B. The rela-
tively high proportion of fused autophagosomes with endo/lysosomes was
peculiar to cells in which there was a strong induction of autophagy around
a high density of perinuclear lysosomal clusters. In cells with more dispersed
lysosomes and fewer EGFP-LC3 puncta (some HeLa cells or NIH-3T3 cells
stably expressing EGFP-LC3) only about 5–16% of EGFP-LC3 puncta
colocalized with endo/lysosomal structure (see supplemental Fig. S1); this
lower level of colocalization most likely reflects the lower probability of the
two diffusely localized compartments encountering one another to fuse.
Thus we corroborate that EGFP-LC3 labels compartments that are distinct
from endo/lysosomes.
MDC only marks autophagic compartments after their fusion to acidic
endo/lysosomes. MDC has been widely advocated as a tool to label autophagic
vacuoles. To test to what extent MDC colocalizes with autophagosomes, we

28 Autophagy 2005; Vol. 1 Issue 1

 The Dynamics of Autophagy Visualized in Live Cells

Figure 5. MDC does not exclusively label nascent EGFP-LC3 positive autophagosomes accumulated under nocodazole treatment. HeLa cells stably expressing
EGFP-LC3 were treated with 2 µg/ml nocodazole during amino acid starvation in HBSS for two hours. Cells were then loaded with 50 nM LTR and 50 µM
MDC for 15 minutes and imaged live under fluorescent mode. Top row, a cluster of EGFP-LC3 positive vacuoles surrounded by noncoincident MDC and LTR
positive vacuoles, indicated by arrowheads; occasional colocalization of weak EGFP-LC3 with MDC and LTR was also observed, indicated by arrows.
Bottom row, an apparently giant LC3-positive vacuole (arrowhead) surrounded by noncoincident MDC and LTR positive vacuoles. Note the neighbouring
vacuole (arrow) which is surrounded by coincident MDC/LTR staining but which is EGFP-LC3 negative.

examined its colocalization with EGFP-LC3 under various autophagic con-
ditions. As a control we also examined to what extent it colocalized with
EGFP-CD63 and other endo/lysosomal markers. Figure 3A, shows that
MDC showed high colocalization with endo/lysosomal markers irrespective
of whether the cells were live or post-fixed after staining,17,18,20 (c values:
LTR, 0.76; EGFP-CD63, 0.91; EGFP-CD63/Fixed, 0.86; combined c
value ± s.d. 0.84 ± 0.08). In EGFP-LC3-expressing cells (Fig. 3B), however,
the proportion of EGFP-LC3-positive puncta colocalising with MDC was
dramatically reduced (c= 0.44 ± 0.08) and was indistinguishable from the
proportion of EGFP-LC3 that colocalized with LTR (c = 0.44 ± 0.05) while
LTR and MDC were highly colocalized (c = 0.86 ± 0.07). When HeLa cells
were treated with 50 mM NH4Cl, there was no loading of MDC or LTR
into punctate structures although EGFP-LC3 was highly punctate (see Fig.
8 below). These data suggest that at best, MDC will label acidified
endo/lysosomes that had fused with autophagosomes but not nascent
autophagosomes.
To further investigate whether MDC only labelled endo/lysosomes and
not newly formed autophagic vacuoles we studied MDC localization in
several models where an upregulation of MDC staining was observed after
treatments shown or postulated to enhance autophagy. Bursch et al.38
showed that tamoxifen induces the death of MCF-7 cells concomitantly
with induction of autophagy, detected by EM. This death was accompanied
by the accumulation of MDC-positive compartments suggested to be
autophagosomes. When we treated MCF-7 cells with 1 µM tamoxifen over
four days we observed a gradual increase in MDC labelling intensity and
number of puncta from an extremely low background, in agreement with
the original data (Fig. 4A). Moreover, this change was accompanied by
increased autophagy as indicated by the generation of EGFP-LC3 puncta
(Fig. 4A). However, there was little correlation between the increase in
MDC labelling and EGFP-LC3 puncta, as reflected by the wide standard
deviation around the low mean colocalization value (c = 0.43 ± 0.18). Thus,
some cells showed increased EGFP-LC3 puncta but no increase in MDC
loading (Fig. 4A, second row), some showed a proportion of EGFP-LC3
positive puncta costaining for MDC (Fig. 4A, third row), and some showed
increased MDC labelling without any apparent increase in EGFP-LC3
punctation (Fig. 4A, bottom row). In contrast, LTR showed near complete
coincidence with these MDC-positive compartments (c = 0.74 ± 0.06).
Moreover, LAMP-1 staining also showed a distinct increase over time from
a low background, as has previously been observed upon irradiation of
MCF-7 cells.42 These results indicate an increase in acidic endo/lysosomes,
rather than in autophagosomes per se, though in some cells in which
EGFP-LC3 compartments have fused with the endo/lysosomes the two are
congruent.
In Cos7 cells expressing polyglutamine repeat proteins that form inclu-
sions, an increase in MDC labelling was observed upon treatment with
rapamycin.19 Rapamycin blocks mTOR kinase activity and is widely used as
a rapid inducer of autophagy.43,44 When we added 200 nM rapamycin to
Cos7 cells, there was indeed an increase in the intensity of MDC staining
and apparent number of puncta after two days (Fig. 4B). This was accom-
panied by an increase in the punctation of EGFP-LC3, but as with the
tamoxifen-treated MCF-7 cells, the EGFP-LC3 puncta did not show a
strong colocalization with the MDC while all the MDC staining colocalized
with LTR (c = 0.8 ± 0.09). Cells that were post-fixed and stained for
LAMP-1 showed a 4-fold increase in LAMP-1 labelling consistent with the
increased intensity of MDC staining observed in our study and in the study
by Racikumar et al.19
In the third paradigm, MDC was added to EGFP-LC3-expressing HeLa
cells after amino acid starvation in the presence of the microtubule-disrupting
agent nocodazole (2 µg/ml). Disruption of microtubules by vinblastine
promotes the accumulation of giant vacuoles that were identified as
autophagosomes based on MDC staining.18,45 In keeping with these obser-
vations, aggregates of quite large EGFP-LC3-positive vacuoles or one giant
EGFP-LC3- positive vacuole were also induced by nocodazole (Fig. 5).
However, neither MDC nor LTR labelling was observed in many of these
structures; where it was seen the EGFP-LC3 was weak, perhaps indicative of
previous fusion and subsequent degradation of the internalized EGFP-LC3.
Moreover, a thin rim of MDC/LTR could be observed around the giant
LC3-positive vacuole, apparently consistent with MDC integrating into a
continuous membrane. However, the same pattern occurred around another
giant vacuole that was not stained with LC3. Whether this is one continuous
vacuole rim or an aggregate of tiny lysosomes require definition by electron
microscopy. Hence giant vesicles that are LC3-positive cannot be exclusively
detected with MDC.

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 The Dynamics of Autophagy Visualized in Live Cells

Figure 6. There is no difference in MDC localization in autophagy-proficient or deficient (Atg5 null) MEFs. SV40-immortalized Atg5+/+ and Atg5-/- MEFs
were transiently transfected with EGFP-CD63 and mRFP-LC3. Upon formation of mRFP-LC3 puncta (two days later; these were seen without stimulation, but
enhanced by amino acid starvation) cells were loaded with 50 µM MDC for 15 minutes and imaged live under fluorescent mode. Note that MDC shows
complete coincidence with the EFGP-CD63-positive endo/lysosomes, some of which are also mRFP-LC3- positive. However, all the mRFP-LC3-positive
puncta that are EFGP-CD63 negative are not stained with MDC. The same coincidence of MDC and EFGP-CD63 positive endo/lysosomes is retained in
Atg5-/- cells even though these lack mRFP-LC3 punctation.

To finally consolidate our findings, we also compared the localization of
MDC staining in SV40-immortalized Atg5+/+ and Atg5-/- MEFs, which are
incapable of forming autophagosomes.14 In Atg5+/+ MEFs expressing
EGFP-CD63 and mRFP-LC3, MDC showed virtual coincidence with the
EFGP-CD63 positive lysosomes but only partial colocalization with the
mRFP-LC3 compartment, as found in HeLa cells (Fig. 6). Moreover, the
same colocalized pattern of MDC and EFGP-CD63 was retained in Atg5-/-
MEFs, even though these cells were deficient in autophagy as indicated by
the lack of mRFP-LC3 punctation.
We also tested the staining pattern of monodansylpentane (MDH),
which lacks the amino group of MDC and was synthesized on the basis of
its being a solvent polarity probe, able to report on autophagic vacuoles
without the complication of requiring an acidic vacuole environment to
load.21 However, although MDH did form puncta, these failed to localise
with either EGFP-LC3 or EGFP-CD63 positive compartments, or to
change their pattern of staining upon stimulation of autophagy (see supple-
mental Fig. S2). The pattern of MDH puncta suggested that it is loading
into (or forming) very lipid rich environments.21 Indeed, when we examined
MDH staining in superior cervical ganglion neurons induced to accumulate
lipid droplets, MDH staining corresponded to the staining seen with the
lipophilic dye Oil-Red O (data not shown). Thus, MDH too is unlikely to
be useful as a marker of nascent autophagosome formation.
Together these data suggest that MDC is a good marker for endo/lyso-
somes, as it was originally defined, and it will also detect autophagosomes
that have fused with or acquired lysosomal properties at steady state. As the
number of lysosomes increases, so does MDC staining. It is however not a
reliable marker for autophagosome formation. Table 1 summarises all the
colocalization data for the various cell types and markers.
Formation of EGFP-LC3 puncta correlate with conversion to the
LC3-II form. Upon activation of autophagy, pro-LC3 is cleaved at its C-ter-
minus to form LC3 (LC3-I, apparent Mr 18 kDa). The free C-terminal
glycine is then modified, most likely by lipidation, to a faster migrating
form, LC3-II (apparent Mr 16 kDa).29 To examine whether EGFP-LC3
would form EGFP-LC3-II, and how reliably the relocalization of
EGFP-LC3 to punctate structures correlated with the appearance of the
faster migrating EGFP-LC3-II form, samples of cells were taken 1–24 hours
after application of various modulators of the autophagic process. To ensure
that inhibition of autophagy by cell division did not interfere with our
assay,46 especially as some of the treatments used inhibit cell division, we
performed these experiments in full medium containing 1% serum. As
shown in Figure 7, EGFP-LC3 migrated almost exclusively as a 45 kDa
band in unstimulated cells (top row), with no change in expression over 24
hours. Some background puncta were observed but most of the EGFP-LC3
was distributed diffusely throughout the cell, similar to profiles observed in

30 Autophagy 2005; Vol. 1 Issue 1

 The Dynamics of Autophagy Visualized in Live Cells

Figure 7. EGFP-LC3 puncta induced by autophagic stimuli correlates with its modification to a 43 kDa form. Stably transfected EGFP-LC3 HeLa cells cultured
in full medium containing 1% serum were treated with 200 nM rapamycin, 50 mM NH4Cl, 20 µg/ml CHX or 1 µg/ml staurosporine and 100 µM BAF.
Images were collected after 1, 2, 4, or 24 hours. Parallel cultures were collected at the same time and analysed for the presence of full length (45 kDa) or
the modified (43 kDa) forms of EGFP-LC3 by immunoblotting with an antibody against EGFP. The inset between rows 3 and 4 shows endogenous LC3 after
1 hour of treatment with NH4Cl. Note the tight correlation between the kinetics of gathering of diffuse EGFP-LC3 into fluorescent puncta and the presence
of the 43 kDa form of EGFP-LC3.

medium containing 10% serum, indicating that starvation did not occur. By
contrast, stimuli that produced punctation of EGFP-LC3 also produced a
notable product of 43 kDa. This product was most likely the lipidated form
of EGFP-LC3-II, as it—but not the 45 kDa form—segregated exclusively
with a Triton X-114 fraction, and was highly labelled with [3H]-
ethanolamine (see Fig. 8).
Rapamycin induced a slow change in the distribution of EGFP-LC3, no
observable changes in the cells being found within 4 hours (Fig. 7, second
row). However, both puncta and the 43 kDa form of EGFP-LC3 were
prominent after 24 hours with about a 3:7 ratio of 43/45 kDa. There was
also a general increase in the intensity of EGFP-LC3 staining and similarly,
an overall increase in the amount of total amount of EGFP-LC3 detected

Figure 8. NH4Cl-treated HeLa cells accumulate PtdEth-conjugated EGFP-LC3-II.

HeLa cells stably expressing EGFP-LC3 were labelled with [3H]ethanolamine
in growth medium for 14 hours and then one cohort was treated with 50 mM
NH4Cl for two hours. Cells were lysed in PBS containing 2% Triton X-114,
a sample of total lysate was removed and the remainder was treated through
cycles of phase separation and re-extraction as described in Materials and
Methods to obtain the aqueous and TritonX-114 extracts indicated. Proteins
(20 µg/lane for blot and 50 µg/lane for autorad) were separated by PAGE
and blotted onto nitrocellulose. Top, Immunoblotting for EGFP-LC3-I and -II
with the anti-EGFP antibody. Note that all the EGFP-LC3-II was extracted into
Triton X-114, leaving EGFP-LC3-I in the aqueous phase. Bottom, A parallel
blot probed for [3H]ethanolamine incorporation by autoradiography. Note
that the label is only contained in EGFP-LC3-II and not in EGFP-LC3-I. Bands
at 48 and 72 kDa represent the Golgi- and GPI-anchored forms of decay
accelerating factor (DAF).

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 The Dynamics of Autophagy Visualized in Live Cells

Figure 9. Mitochondrial fragments can be detected in autophagic and endo/lysosomal compartments. Live HeLa cells stably expressing dsRed-mito and
EGFP-LC3 or EGFP-CD63 were imaged under confocal mode. Note the occasional presence of mitochondrial fragments in EFGP-LC3 positive autophagosomes
and the frequent presence of mitochondrial fragments in EGFP-CD63-positive endo/lysosomes (arrows).

on the immunoblot. The increased amount of the 45 kDa form of
EGFP-LC3 compared to control cells may be due to perturbation of its
production and/or metabolism by rapamycin.
NH4Cl, which alkalinises lysosomes, is thought to prevent autophago-
some fusion with lysosomes and/or LC3 degradation, thereby causing
autophagosome accumulation and preservation.47 Application of 50 mM
NH4Cl caused rapid reorganization of EGFP-LC3 and its accumulation
into numerous small perinuclear puncta within one hour. Concomitantly
there was loss of the diffuse EGFP-LC3 fluorescence, especially noticeable
as decreased staining over the nucleus. (Fig. 7, third row). This change was
also concomitant with an almost complete conversion of EGFP-LC3 to the
43 kDa form. The effect of NH4Cl was not an artefact associated with
EGFP-LC3 as a similar conversion was observed in endogenous LC3 (panel
inset). Interestingly, as with rapamycin, a notable increase in EGFP-LC3
intensity was found in NH4Cl treated cells after 24 hours which correlated
with the appearance of about 50% more EGFP-LC3 on the immunoblot,
much of which was in the 45 kDa form.
As the effects of NH4Cl were so rapid and intense, we further investi-
gated the nature of the conversion to the LC3-II form. Extraction with 2%
Triton X-114 selectively isolates membrane proteins, including the modified
form of Atg8 in yeast.48 Similarly, extraction of lysates from NH4Cl treated
HeLa cells with Triton X-114 also selectively extracted EGFP-LC3- II, leaving
EGFP-LC3-I in the aqueous phase (Fig. 8). Moreover, extracted EGFP-
LC3-II showed high levels of incorporation of [3H]ethanolamine (Fig. 8),
suggesting that the protein is most likely modified with PtdEth, as is yeast
Atg8.48 EGFP-LC3-I in the aqueous phase was not labelled. The labelled 48
kDa protein found predominantly in the aqueous phase and the 72 kDa
protein found only in the Triton X-114 phase are most likely the Golgi- and
GPI-linked forms of decay accelerating factor (DAF), which has previously
been shown to be highly expressed in HeLa cells and to incorporate
[3H]ethanolamine;49 the 72 kDa species is known to possess a C-terminal
glycolipid structure, hence its separation with the Triton X-114 phase. These
data confirm that NH4Cl does lead to an accumulation of membrane
bound, PE modified EGFP-LC3-II.
The increase in intensity of EGFP-LC3 after prolonged treatments
prompted us to evaluate the natural turnover of the exogenous protein.
Cycloheximide (CHX) is both a protein synthesis inhibitor and an inhibitor
of autophagy.50,51 In keeping with both these functions, CHX added at 20
µg/ml, which suppresses protein synthesis by 99%, reduced the overall
intensity of the basal puncta within 1 hour and promoted slow loss of fluo-
rescence that was matched by a 50% decrease in the amount of the 45 kDa
band on the immunoblot (Fig. 7, fourth row). Thus, accumulation of 50%
more EGFP-LC3-I/II product compared to the starting levels of EGFP-LC3
(as found after 24 hours in the presence of NH4Cl or rapamycin) might be
explained by loss of the basal rate of degradation induced by these agents.
Finally, we examined whether an apoptotic stimulus would induce
autophagy that could be imaged using EGFP-LC3, as apoptotic stimuli have
been shown to induce autophagy, which under some circumstances may
contribute to mitochondrial elimination.52,53 In keeping with this observa-
tion, staurosporine (used in the presence of the caspase inhibitor Boc-
Asp(Omethyl) FMK to block the execution of apoptosis) caused a steady
increase in EGFP-LC3 punctation beginning at two hours (Fig. 7, bottom
row). These changes were accompanied by a time-dependent increase in the
conversion of the 45 kDa form to the 43 kDa form. Unlike rapamycin or
NH4Cl, however, treatment with staurosporine culminated in complete
conversion to the 43 kDa form by 24 hours, the arrangement of perinuclear
puncta and lack of nuclear EGFP-LC3 staining being very similar to that
induced by NH4Cl after one hour. There was thus no increase in total

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 The Dynamics of Autophagy Visualized in Live Cells

Figure 10. Imaging the fusion of autophagosomes and endo/lysosomes. Live HeLa cells stably expressing EGFP-CD63 and mRFP-LC3 were starved of amino
acids in HBSS for one hour and then imaged continuously for 15 minutes. An example taken at two minute intervals is shown. Note how the large
autophagosome (red) acquires a number of surrounding endo/lysosomes that eventually fuse to give a continuous green surround at the end of the record-
ing. See supplementary video for live cell recording.

EGFP-LC3 levels as found with rapamycin or NH4Cl, perhaps due to the
lack of sufficient energy to synthesise EGFP-LC3 due to loss of mitochondrial
function.
Together these data demonstrate that overall EGFP-LC3 punctation and
EGFP-LC3-II formation are correlated, as has been shown for the endoge-
nous murine LC3 in a transgenic mouse model overexpressing
EGFP-LC3.28 However, as the amounts of EGFP-LC3-II relative to the 45
kDa form were different for each type of autophagic stimulus at times when
the differences in the organization and percentage of the puncta were too
subtle to define by inspection of the cells, it appears that an insight into the
finer details of autophagic homeostasis are only provided by combination of
observation of the cells and by measurement of the amounts of
EGFP-LC3-I and -II.
Mitochondrial fragments can be detected in degradative compartments.
Any model of autophagy should be demonstrated to consume cytoplasmic
constituents. Mitochondria have been demonstrated to be degraded by
autophagy in liver of glucagon-treated rats in vivo.11,54 In cells doubly
labelled with dsRed-mito and EGFP-LC3 or dsRed-mito and EGFP-CD63,
we could clearly detect a number of vesicles containing fragments of
dsRed-positive mitochondria (Fig. 9). These were rare in EGFP-LC3 vesi-
cles, presumably reflecting the transient nature of this structure, but were
abundantly observed in the lumen of EGFP-CD63- positive compartments.
Taken with the previous data showing partial colocalization of punctuate
EGFP-LC3 with lysosomal markers, this finding suggests that we can follow
the autophagic degradation of a typical cytoplasmic organelle from the
autophagosome through to the autolysosome. Indeed, over a 15 minute
period of continuous recording of doubly labelled EGFP-CD63 and mRFP-
LC3 HeLa cells, we could detect numerous instances of fusion of red
autophagsomes and green lysosomal compartments in what was obviously a
highly dynamic process (Fig. 10; see supplementary video for real time images).
DISCUSSION
We have compared the efficacy of MDC and LC3 as reporters of
autophagy at the light microscopic level, opening up the opportunity
to study this process by live cell imaging, which is critical to our
understanding of this dynamic process. We have also followed the
process of autophagosome-endo/lysosome fusion using marker
proteins for each compartment. We demonstrate that the widely
used autophagic marker MDC cannot label nascent autophagosomes
at any stage prior to their acquisition of acidic properties. In all the
autophagy-proficient models tested (tamoxifen-treated MCF-7 cells,
rapamycin-treated Cos7 cells, nocodazole-treated HeLa cells, and
amino-acid starved MEFs), MDC shows complete localization with
the endo/lysosomal markers lysotracker red (LTR) or EGFP-CD63
but only shows localization with EGFP-LC3 vesicles when these are
also LTR positive. Moreover, the profile of MDC staining in amino
acid starved Atg5+/+ MEFs and in autophagy-deficient Atg5-/- MEFs
is identical. It has been suggested that autophagosomes acquire a
proton pump before their fusion with endo/lysosomes.55 If these
were acidic enough, such organelles would be likely to be stained by
LTR or MDC. However, we could not find any evidence for such
organelles as all the acidic vacuoles containing EGFP-LC3 had also
acquired endo/lysosomal markers whereas those that had not were
not labelled with LTR or MDC.
It has been previously suggested that MDC acts as a solvent
polarity probe and hence is a good autophagosomal marker especially
after fixation (when untethered MDC leeches out of the lysosomal
lumen and reduces background staining).20 In nocodazole-treated
HeLa cells there was evidence for MDC staining outlining two giant
vacuoles in what appeared to be a contiguous membrane. However,
this staining also coincided with LTR staining and was not specific
to EGFP-LC3-positive vacuoles. Whether the vacuoles are outlined
by a continuous membrane or are composed of tiny vacuoles
remains to be determined.
Although MDC may not stain nascent autophagosomes, there
was an increase in MDC staining intensity in the tamoxifen-treated
MCF-7 cells and rapamycin-treated Cos 7 cells, as previously report-
ed.19,38 This increase can be explained by a general increase in endo/
lysosome numbers as it was matched by an increase in LTR staining
and there was a notable increase in the number of LAMP-positive
compartments overall after both treatments. Indeed, initial observa-
tions on autophagy deficient Atg5-/- MEFs treated with rapamycin

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 The Dynamics of Autophagy Visualized in Live Cells
suggests that these cells also show an increased MDC and LAMP
staining after two days (unpublished observations). New endo/lyso-
somes are formed through vesicular trafficking from the Golgi and
retrieval of endosomes from the plasma membrane.56 Indeed,
increased endocytosis has been observed to occur during stimulation
of autophagy in several models of neurodegeneration.57-60 Thus,
enhanced endosomal trafficking could lead to an increase in
endo/lysosomal numbers. Such a mechanism also fits with evidence
for involvement of Skd1 AAA ATPase, Rab24 and Rab7, previously
implicated in endocytic processes, in autophagic vacuole turnover.
45,61,62,75 The impetus for increased lysosome numbers may also
arise due to activation of chaperone-mediated autophagy16 and
microautophagy15,63 alongside macroautophagy. Whilst only the
latter involves autophagosome formation, the other two types of
autophagy direct proteins directly to lysosomes and may necessitate
production of more lysosomes when activated.
Thus, MDC can indicate increased degradative activity, as a
faithful marker of acidic compartments, but it cannot be directly
linked to macroautophagy as has been previously implied. Problems
with its use may also arise from its ability to inhibit transglutaminase
activity, which can interfere with receptor-mediated endocytosis.64
Therefore use of MDC is not recommended for the specific study of
autophagy dynamics.
By contrast, the Atg8 homologue LC3 conjugated to fluorescent
proteins (EGFP or mRFP) appears to act as a good reporter of
autophagy as it: (a) is induced to a punctuate state under various
stimuli known to induce autophagy, (b) it reports on autophagy (or
perturbation of autophagy) under conditions where autophagy is
not necessarily predicted, for example after staurosporine treatment
in unstarved HeLa cells,53 (c) the number of nascent autophagosomes
and their fractional colocalization with auto/lysosomal compartments
can be visualized dynamically and quantified in live cells using, for
example, fluorescent CD63, a protein located in the membranes of
late endosomes and lysosomes,35,65-67 (d) used in conjunction with
other fluorescent labels it allows one to follow the autophagic degra-
dation of cytoplasmic constituents through autophagosome formation
and their fusion with endo/lysosomes (exemplified by mitochondria
labelled with dsRed-mito), and (e) the conversion of EGFP-LC3 45
kDa to the more rapidly-migrating 43 kDa form can be detected by
immunoblotting with anti-GFP, revealing underlying biochemical
dynamics of synthesis and degradation. Measurement of the ratio of
EGFP-tagged LC3-I to LC3-II circumvented the problem highlighted
by Kabeya et al.30 that endogenous LC3-II may be detected more
sensitively than LC3-I with an anti-LC3 antibody.
As the precise dynamics of LC3 punctation and LC3-I to -II
conversion have never been compared, we used several methods of
autophagic induction to examine how tightly the two are correlated.
NH4Cl, which alkalinises the lysosomes and inhibits enhanced
protein degradation associated with amino acid deprivation,47 led to
a rapid appearance of very small puncta within 1 hour. This change
was accompanied by a rapid conversion of all the LC3-I to LC3-II,
the most rapid and complete response to any treatment measured so
far, including amino acid starvation. As it appeared that most of the
NH4Cl-induced small puncta had not matured into lumenous
vacuoles after 1 hour, it may be that NH4Cl activated the formation
of isolation membranes independently of its ability to alkalinise
acidic compartments. Using phase separation into Triton X-114 and
[3H]ethanolamine labelling, we further confirmed that EGFP-
LC3-II is indeed membrane-bound and modified by PtdEth following
NH4Cl treatment. Interestingly, at 24 hours, we found an enrichment
34
of EGFP-LC3 intensity in the cells and a large increase in the
amount of EGFP-LC3-I on the Western blot. This may be due to
the return of some normal lysosomal function and reduction of stim-
ulus for autophagosome formation as after 24 hours we observed
renewed MDC and LTR loading into endo/lysosomes, albeit at
lower levels than in untreated cells (unpublished observations). Thus
the system may reset over long periods of treatment with NH4Cl,
perhaps due to active pumping of NH4+ from the cells,68,69 thereby
allowing LC3-I to accumulate once again. Our preliminary investi-
gations suggest that it is also possible that pro-LC3 may be converted
to LC3-I during this period, accumulating due to loss of stimulus
converting LC3-I to LC3-II.
In the case of rapamycin, the kinetics of puncta formation and
LC3-II accumulation were also correlated, both occurring rather
slowly. Considering that rapamycin inhibits S6 phosphorylation
(and presumably mTOR) within 30 minutes (Goemans C, unpub-
lished data) and in yeast Atg8 expression is increased within two
hours26,70 it is difficult to explain why autophagy induction is so
slow. Perhaps there is signalling compensation by amino acids
derived from other metabolic processes,71 the true impact of amino
acid signalling loss occurring only after these alternative pathways
have been used up. There was also a notable increase in overall
EGFP-LC3 levels indicating perhaps activation of EGFP-LC3
synthesis or conversion from pro-LC3 to LC3-I. These data imply
that rapamycin has additional effects aside from its inhibition of
mTOR, in keeping with recent evidence that the effects of treatment
with rapamycin and mTOR inhibition are not equivalent.72
We also investigated whether autophagic activity would be induced
by the pro-apoptotic drug staurosporine, as we have previously
shown that autophagic activity is induced by some apoptotic stimuli,
and that this activity is potentially responsible for the slow death of
cells when the apoptotic machinery is inhibited.52,53 Indeed, stau-
rosporine treatment (in the presence of BAF to inhibit the apoptotic
pathway) caused a marked punctation of EGFP-LC3 within two
hours, a rapid response that occurs well before any changes associated
with apoptosis in the cells. This change was associated with about
20% conversion from EGFP-LC3-I to LC3-II. As EGFP-LC3 puncta
intensity (compared to background fluorescence) increased over the
following 24 hours so there was a steady increase in conversion of
EGFP-LC-I to the LC3-II form. Interestingly, this was the only case
where there was a complete conversion to LC3-I to LC3-II after such
a long period of time. This change may be explained by loss of ATP
due to permeabilization of mitochondria, release of cytochrome c,
and loss of oxidative phosphorylation52 as even partial loss of ATP
will both prohibit de novo protein synthesis73 and ongoing
autophagy, including acidification of the lysosomes.74 As stau-
rosporine potentially inhibits numerous kinases, the linkage between
apoptosis and autophagy requires more careful assessment using
better-defined induction paradigms.
Evidently, LC3 punctation and LC3-II formation can have dif-
ferent dynamics depending on the individual pro- and anti-
autophagic compound used and hence the drugs in each class cannot
be considered equivalent. Identification of HsAtg4B as the enzyme
responsible for C-terminal cleavage of LC3-I and delipidation of
LC3-II30,31 and our ability to image lysosomes and autophagosomes
at the same time will help identify which steps are being regulated
under each condition.
Finally, to further evaluate the use of the markers of degradative
compartments, we also labelled mitochondria with dsRed2-mito and
followed the fate of mitochondria as an organelle that is typically
Autophagy 2005; Vol. 1 Issue 1
 The Dynamics of Autophagy Visualized in Live Cells
degraded by autophagy.54 Indeed, we detected small red fragments
of mitochondria in both EGFP-LC3 positive autophagosomes and
EGFP-CD63 positive endo/lysosomes although the former were
relatively rare compared to the latter. These latter compartments
may include amphisomes and autolysosomes as well as mature
lysosomes as in live imaging experiments we detected frequent
fusion of mRFP-LC3 positive autophagosomes with EGFP-CD63
positive endo/lysosomes. Two aspects of the fusion process were
striking: (1) there were always several endo/lysosomes ‘attacking’ an
autophagosome at any one time. (2) there appeared to be several
instances of ‘kiss and run’ before endo/lysosome(s) surrounded the
autophagosome. Thus, these markers may be used to follow the fate
of cytoplasmic material through the autophagic pathway though the
factors that control this fusion remain to be defined.
In summary, we have shown that MDC, a widely used marker of
autophagy, is not a bona fide indicator of autophagosomes, but
rather of general acidic compartments, and thus data using this dye
must be interpreted with care. In contrast, EGFP-LC3 reports on all
phases of autophagy from early autophagsomes through to fusion
with endo/lysosomes when colocalising with endo/lysosomal markers.
Moreover, conversion of EGFP-LC3-I to the -II form reveals bio-
chemical processes that may not be detected by simply examining
the state of cell punctation. Especially poignant for analysis of
autophagy is having both autophagosomes and endo/lysosomes
independently tagged, allowing the dynamics of this complex, regu-
lated system to be followed in real time.
Note
Supplementary Material can be found at:
www.landesbioscience.com/supplement/bampton1-1-sup.pdf
www.landesbioscience.com/supplement/bampton1-1-vid.mov
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