Download Dissection of autophagy in tobacco BY-2 cells

RESEARCH PAPER
Plant Signaling & Behavior 10:11, e1082699; November 2015; © Published with license by Taylor & Francis Group, LLC
Dissection of autophagy in tobacco BY-2 cells
under sucrose starvation conditions using the
vacuolar HC-ATPase inhibitor concanamycin A
and the autophagy-related protein Atg8
Kanako Yano1, Takahiro Yanagisawa2, Kyosuke Mukae2, Yasuo Niwa1, Yuko Inoue2, and Yuji Moriyasu2,*
1
Graduate School of Food and Nutritional Sciences; University of Shizuoka; Shizuoka, Japan; 2Department of Regulatory Biology; Graduate School of Science and Engineering;
Saitama University; Saitama, Japan
Keywords: autophagy, autolysosome/autophagosome, concanamycin, E-64c, tobacco BY-2, vacuole
Abbreviations: ATG, autophagy-related; DMSO, dimethyl sulfoxide; GFP, green fluorescent protein; 3-MA, 3-methyladenine.
Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for
intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar HC-ATPase
inhibitors concanamycin A and bafilomycin A1, which caused the accumulation of autophagic bodies in the central
vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and
concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor
E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole,
but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with
concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the
pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker
protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence
microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP,
which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type
HC-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The
accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of
autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.
Introduction
Cells degrade their components in order to maintain and
remake themselves. Autophagy is one of the mechanisms responsible for the degradation of these constituents.
Autophagy is a process in which a cell transports a part of its
cytoplasm into lysosomes or vacuoles for degradation. It is classified as macroautophagy or microautophagy according to how the
part of the cytoplasm is sequestrated and transported into the
lysosomes or vacuoles.1,2 In macroautophagy, a special membrane sac called an isolation membrane first sequestrates a part of
the cytoplasm and makes a structure enclosed by a double membrane, an autophagosome. An autophagosome then fuses with a
preexisting endosome and/or lysosome to transform to an
autolysosome and degrades the enclosed cytoplasm. In microautophagy, a part of the cytoplasm is taken up into the lysosome by
invagination and fission of the lysosomal membrane, and is then
degraded.
The autophagy-related (Atg) protein Atg8 is a reliable marker
of macroautophagy in yeast cells. By the analysis of mutants
defective in macroautophagy, around 15 proteins responsible for
the formation of autophagosomes have been found.3-6 Of these
proteins, which are designated as autophagy-related proteins, a
ubiquitin-like protein Atg8 localizes to the structure called PAS
(preautophagosomal structure or phagophore assemble site) and
to autophagosomes.7 Atg8 protein is thought to work for the
elongation and fusion of the membrane of an autophagosome
precursor to complete the autophagosome structure.8-10 It can
© Kanako Yano, Takahiro Yanagisawa, Kyosuke Mukae, Yasuo Niwa, Yuko Inoue, and Yuji Moriyasu
*Correspondence to: Yuji Moriyasu; Email: [email protected]
Submitted: 08/07/2015; Accepted: 08/08/2015
http://dx.doi.org/10.1080/15592324.2015.1082699
This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The
moral rights of the named author(s) have been asserted.
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e1082699-1
therefore be used as a marker for the maturation process from a
PAS to an autophagosome in yeast cells. The Atg8 protein is conserved among most eukaryotic cells.11-15 The Arabidopsis
genome codes for 9 homologues of the yeast Atg8 protein, which
are designated AtAtg8a-i.16,17 By expressing a fusion protein of
one of these Atg8s or tobacco Atg8 and a fluorescent protein
such as green fluorescent protein (GFP) or other fluorescent proteins, the emergence and movement of putative autophagosomes
have been observed.18-23
Concanamycin A is a specific inhibitor of vacuolar HCATPase, and blocks the acidification of organelles depending
on this ATPase.24-26 Concanamaycin A inhibits vacuolar HCATPase activity and the acidification of organelles more
strongly than another inhibitor bafilomycin A1.27 Since acidic
organelles function in a variety of cellular processes, they
have various effects on cellular physiology.28-30 In Chara
cells, the pH of the central vacuoles is raised by treatment
with bafilomycin and concanamycin.31,32 In tobacco BY-2
cells, concanamycin inhibits the transport of vacuolar resident
proteins to the central vacuole, although it does not seem to
affect the pH of central vacuoles at the concentrations used
in this study.33 This suggests that the acidification of organelles other than the central vacuole by the HC-ATPase is
involved in the transport of vacuolar proteins. In the autophagic process of mammalian cells such as rat hepatocytes,
the inhibitors block autophagy at the step of transformation
from autophagosomes to autolysosomes.34
In plant cells, the pathway of macroautophagy has not been
elucidated clearly.35 When transgenic plants expressing Atg8
labeled with fluorescent protein were treated with concanamycin,
the structures derived from autophagosomes, which appear to correspond to autophagic bodies in yeast cells, are seen in the central
vacuole of Arabidopsis root and hypocotyl cells.18,20,36 These
results have been interpreted as showing that autophagosomes
directly fuse with the central vacuole and release their contents,
autophagic bodies, into the lumen of the central vacuoles.37 On
the other hand, electron microscopic observations have shown the
presence of autophagic vacuoles containing partially degraded
cytoplasm in Arabidopsis and tobacco cells cultured in sucrose-free
medium, suggesting that degradation of cytoplasm starts before
autophagosomes fuse with the vacuoles.38,39 Furthermore, in
tobacco BY-2 cells, 2 fates of fluorescent autophagosomes are
observed by fluorescence microscopy: one is direct fusion with the
central vacuole; the other is interaction with more small vesicles to
possibly become autolysosomes.21
Figure 1. Effects of concanamycin on the morphology of tobacco BY-2
cells. Four-day-old BY-2 cells were transferred to sucrose-free culture
medium, and the cellular morphology was observed by light microscopy.
(A) Immediately after transfer to the medium containing 0.1 mM concanamycin. (B) Cultured for 1 d in medium containing 0.1 mM concanamycin. (C) Cultured for 1 d in medium containing 1% (v/v) DMSO as a
solvent control. (D and E) Cells cultured in the presence of concanamycin for 2 d were observed under a light microscope with Nomarski (D) or
phase contrast (E) optics. Bar, 20 mm.
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Tobacco BY-2 cells cultured in sucrose-free medium perform macroautophagy. The addition of a protease inhibitor
such as E-64c or leupeptin into the medium blocks the process and causes the accumulation of numerous autolysosomes
containing undegraded cytoplasm in the perinuclear cytoplasm.40,41 The autolysosomes are acidic inside and contain
acid phosphatase and protease.39 It has been thought that
cysteine protease inhibitors retard the degradation of the
cytoplasm enclosed in the autolysosomes, and as a result,
autolysosomes containing undegraded cytoplasm accumulate
in the cells, probably because of physical interference by
accumulating cytoplasm.
In this study, we examined the pathway of autophagy in
tobacco BY-2 cells using the vacuolar HC-ATPase inhibitor
concanamycin and a fusion protein of GFP and AtAtg8.
We found that concanamycin has a different effect than the
cysteine protease inhibitor E-64c on cellular morphology.
We report that concanamycin distorts the pathway of macroautophagy in tobacco BY-2 cells, although it is still useful for
the detection of autophagy.
Results
Effect of vacuolar HC-ATPase inhibitor concanamycin A on
vacuolar morphology of tobacco BY-2 cells
The morphology of tobacco BY-2 cells substantially changes
under sucrose starvation (Fig. 1, see also Moriyasu and
Ohsumi40). In the logarithmic growth phase, the cells contained
many cytoplasmic strands (Fig. 1A), which were gradually lost
during starvation. Since vacuolar HC-ATPase is supposed
involved in various cellular processes including autophagy, we
first examined the effects of its inhibitor, concanamycin on the
morphological changes of BY-2 cells under sucrose starvation.
When 100 nM concanamycin was added to sucrose-free culture
medium, many particulate structures accumulated in the central
vacuoles (Fig. 1B vs. C). Because the structures were moving in
the central vacuole in the Brownian manner, and appeared to
be particles based on phase contrast microscopy (Fig. 1D, E),
we inferred that these intravacuolar structures were not strands
that go through the central vacuole but real particles that are
dispersed in the lumen of central vacuoles. Concanamycin did
not evoke such morphological change at 1 nM, whereas a small
amount of accumulation of vacuolar inclusions was observed at
10 nM. We confirmed that bafilomycin A1, another vacuolar
HC-ATPase inhibitor, evoked the same morphological change
at 5 mM, but not at 1 mM. This seems reasonable because concanamycin A inhibits vacuolar HC-ATPase more strongly than
bafilomycin A1.
Concanamycin at 100 nM did not cause this type of morphological change in medium containing sucrose (Fig. 2B vs. A),
showing that the accumulation of intravacuolar particles is
involved in cellular responses to sucrose starvation. However, at
1 mM, many vacuolar particles appeared even in nutrient-sufficient medium (Fig. 2C), and occasionally aggregated at corners
of the central vacuole (Fig. 2D).
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Figure 2. Effects of concanamycin on tobacco BY-2 cells cultured in the
presence or absence of sucrose. Four-day-old BY-2 cells were cultured in
medium without sucrose (A) or with sucrose (B, C and D) containing
0.1 mM (A and B) or 1 mM (C and D) concanamycin for 1 d. Bar in A and
B, 50 mm; bar in C and D, 20 mm.
Concanamycin A increases vacuolar pH
We examined whether concanamycin affects vacuolar acidification under the conditions used in this study. In order to roughly
estimate the effects of concanamycin on vacuolar pH, the cells
were prestained with the acidotrophic fluorescent dye quinacrine
and then kept in sucrose-free medium in the presence or absence
of 100 nM concanamycin (Fig. 3). Fluorescence microscopy
showed that less quinacrine fluorescence remained in vacuoles in
concanamycin-treated cells compared with untreated cells (Fig. 3,
E vs. F), suggesting that alkalization of the central vacuole
occurred in the presence of concanamycin. Such vacuolar alkalization occurred in less than 3 h at 1 mM concanamycin, whereas it
took more than 12 h at 100 nM (data not shown). Such
Figure 3. Effect of concanamycin on vacuolar pH. (A, B and C) Four-dayold tobacco BY-2 cells were kept in 0.1 M sorbitol, 5 mM HEPES-Na (pH
7.5) and 10 mM quinacrine for 5 min to stain their central vacuoles with
quinacrine. The cells were then cultured for 1 d in medium with sucrose
(A) or without sucrose (B and C) containing 0.1 mM concanamycin (A
and B) or 1% (v/v) DMSO as a solvent control (C). (D, E and F) Fluorescence images corresponding to Nomarski images A, B and C, respectively. Bar, 40 mm.
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Figure 4. Morphological changes of tobacco BY-2 cells after removal of
concanamycin. BY-2 cells that had been cultured in sucrose-free culture
medium containing 0.1 mM concanamycin for 1 d were cultured in the
presence (B and D) or absence (A and C) of sucrose for another 1 d (A
and B) or 2 d (C and D). Bar, 20 mm.
alkalization in the central vacuole also occurred in cells incubated
in sucrose-containing medium in the presence of 100 nM concanamycin (Fig. 3D), although no accumulation of vacuolar inclusions occurred (Fig. 3A). This result confirms that concanamycin
increases vacuolar pH both in the presence and absence of sucrose
in culture medium, thus showing that the difference in cellular
responses in the presence and absence of sucrose does not reflect a
difference in pH perturbation by concanamycin.
Effect of concanamycin A on the accumulation of vacuolar
particles is reversible
When cells accumulating many particles in the central
vacuoles, like the cells shown in Figure 1B, were transferred to
fresh sucrose-free culture medium in the absence of concanamycin, the particles disappeared within 2 d probably because they
were digested (Fig. 4A and C). When the cells were transferred
to MS medium containing sucrose, the vacuolar particles disappeared more rapidly (Fig. 4B and D), and concomitantly cells
resumed cell division and reconstructed many transvacuolar
strands. These results show that the effect of concanamycin on
the accumulation of vacuolar particles is reversible and thus suggest that it does not represent some adverse effect of concanamycin, leading cells to death. Taken together, the results show that
the central vacuole of a BY-2 cell is capable of digesting parts of
the cytoplasm taken up into the vacuole, as shown in an alga,
Chara corallina.42
Electron microscopy confirms that a vacuolar particle is a
membrane-enclosed vesicle containing part of the cytoplasm
Electron microscopy showed that the particles accumulating
in the central vacuoles in the presence of concanamycin are
parts of the cytoplasm (Fig. 5). Each of these cytoplasmic
drops seemed to be surrounded by membrane, and cellular
organelles such as mitochondria and ER appeared to be preserved in these drops (Fig. 5B, arrow). Figure 5C shows the
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presence of a membrane encircling a particle (arrowhead).
These structures resembled the autophagic bodies of yeast cells,
which are formed by the fusion of autophagosomes with the
preexisting vacuole, although the structures in BY-2 cells were
larger than those in yeast cells. Similar images have been
reported in cells of Arabidopsis roots and hypocotyls treated
with concanamycin, and in these reports, cytoplasmic drops
were thought to be formed in the same manner as in yeast
cells.18,20 Besides membrane-enclosed cytoplasmic drops,
membranous structures that seemed to have lost cytoplasm
were also seen in the vacuoles (Fig. 5B, arrowhead). Furthermore, in BY-2 cells treated with concanamycin, autophagosomes (Fig. 5D, arrow) and possibly their precursor structures
(Fig. 5D, arrowhead) were seen in the cytoplasm. These were
approximately 1 to 2 mm in diameter and seemed to be
enclosed by a double membrane. In addition to autophagosomes, structures that resembled autophagosomes, but were
different from typical autophagosomes in that they were larger
or contained more than 2 cytoplasmic drops inside (Fig. 5C,
E; arrow), were also seen in the cytoplasm. Here, these structures are called autophagosome-like structures. Autophagosome-like structures are likely to be formed through the
swelling of the outer membrane of a single autophagosome or
the fusion of 2 or more autophagosomes. Autophagosomes
and autophagosome-like structures were rarely seen in control
cells that were treated with only dimethyl sulfoxide (DMSO)
as a solvent control. These observations suggest that the number of autophagosomes and autophagosome-like structures
increase following treatment with concanamycin, and that
cytoplasmic drops in the central vacuole are structures released
into the central vacuole from autophagosomes and autophagosome-like structures.
In contrast to the effect of concanamycin, cysteine protease
inhibitors such as E-64c and leupeptin cause the accumulation of autolysosomes, which contain the degradation intermediates of parts of the cytoplasm.39,40 Parts of the
cytoplasm enclosed in autolysosomes are mostly degraded and
unidentified, although they are occasionally recognizable as
originating from mitochondria.39 Thus, the degradation of
cytoplasm enclosed in an autolysosome is at a more advanced
stage with respect to cytoplasmic degradation than cytoplasm
in cytoplasmic drops accumulating in the central vacuoles or
in autophagosomes and autophagosome-like structures. This
suggests that concanamycin works upstream from E-64c.
Taken together, it seems that concanamycin somehow blocks
the maturation of autophagosomes to autolysosomes in the process of autophagy in tobacco BY-2 cells induced under sucrose
starvation conditions. As a result of such inhibition, autophagosomes probably accumulate in the cytoplasm and the accumulation of a large number of autophagosomes may result in their
unusual fusion with the central vacuole to make cytoplasmic
drops in the central vacuole.
Concanamycin A inhibits net protein degradation
Treatment with the papain-type cysteine protease inhibitor
E-64c, as well as other inhibitors such as antipain and leupeptin,
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Concanamycin A suppresses
accumulation of autolysosomes
by E-64c treatment
When cells were treated
with E-64c and concanamycin
simultaneously, autolysosomes
did not accumulate and the cellular morphology was the same
as that of cells treated with only
concanamycin (Fig. S1). This
result strongly supports the
notion that the point of action
of concanamycin in the route of
autophagy is upstream from
that of E-64c.
The autophagy inhibitor 3methyladenine (3-MA)
suppresses the effect of
concanamycin A
3-MA blocks autophagy
induced by sucrose starvation in
tobacco BY-2 cells.43 Since the
accumulation of autolysosomes
is not seen in cells treated with
3-MA, the point of action of 3MA is thought to be upstream
from the formation of autolysosomes.43 If the cytoplasmic
drops that accumulate in the
central
vacuole
following
concanamycin treatment are
structures derived from autophagosomes, 3-MA should
Figure 5. Electron micrographs of tobacco BY-2 cells treated with concanamycin. Four-day-old BY-2 cells were
cultured for 16 h in sucrose-free culture medium containing 0.1 mM concanamycin. v, central vacuole. n,
abolish the accumulation of
nucleus. m, mitochondrion. p, plastid. (A) Many particles in the central vacuole. Bar, 5 mm. (B) Arrows, vacuolar
these structures. We used light
particles containing parts of the cytoplasm. Arrowheads, membranous structures in the central vacuole. Bar,
microscopy to examine whether
1 mm. (C) Arrows, autophagosome-like structures; arrowheads, membrane of a vacuolar particle enclosing a
3-MA blocks the accumulation
mitochondrion but having lost cytosol. Bar, 1 mm. (D) Arrow, autophagosome. Arrowheads point to a putative
of cytoplasmic drops in the cenprecursor of an autophagosome. Bar, 1 mm. (E) Arrows, autophagosome-like structures. Bar, 2 mm.
tral vacuole (Fig. S2). When
cells were treated with concanainhibits intracellular cysteine protease activity, and as a result mycin together with 3-MA, almost no accumulation of cytoblocks protein degradation through autophagy in tobacco BY-2 plasmic drops occurred in the central vacuole (Fig. S2). This
cells.40 In contrast, concanamycin treatment did not reduce pro- result supports the notion that vacuolar inclusions are derived
tease activity measured in vitro (Fig. 6A). However, like E-64c, from autophagosomes.
concanamycin blocked net protein degradation during sucrose
GFP-AtAtg8 protein is uniformly distributed in the
starvation, which is one of the signs of autophagy in BY-2 cells
(Fig. 6B). As shown in a previous study40, protein degradation cytoplasm and recruited to autophagosomes during autophagy
We transformed tobacco BY-2 cells to constitutively express
during sucrose starvation is mostly non-selective. We confirmed
this result and further found that almost all cellular polypeptides GFP with its C terminus fused to Atg8 protein from Arabidopsis.
seem to be rescued from degradation by concanamycin Four-day-old transgenic cells expressing the GFP-AtAtg8 fusion
(Fig. 6C), as is the case with E-64c. Thus the results confirm the protein appeared to have GFP fluorescence in much of the cytonotion that concanamycin works as a blocker of autophagy in sol (Fig. 7), suggesting that the expressed proteins are distributed
tobacco BY-2 cells. Although we found that some specific poly- almost uniformly in the cytosol. However, 6 to 24 h after the
peptides accumulated in the cells treated with concanamycin, we cells were starved for sucrose, many punctate structures with distinct GFP fluorescence showed up (Fig. 7B and C, arrows).
did not address our attention to these polypeptides in this study.
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These particles appeared to exist in the cytoplasm. At a higher
resolution, some of these structures seemed to be ring-shaped
(Fig. 7D, arrows). By 2 d after sucrose deprivation, these fluorescent particles mostly disappeared, and instead, somewhat bright
GFP fluorescence was observed in the lumen of the central vacuole (Fig. 7E). These results suggest that GFP-AtAtg8 in the cytosol was recruited to autophagosomes, which are recognized as
punctate or ring-shaped structures under fluorescence microscopy, and subsequently moved into the central vacuole during
the process of autophagy.
GFP fluorescence also appeared to be distributed in the cytoplasm of cells expressing native GFP protein (Fig. 7F). However,
no punctate structures having GFP fluorescence appeared even
when these cells were transferred to sucrose-deficient medium
(Fig. 7G). By 2 d after sucrose deprivation, significant GFP fluorescence was observed in the central vacuole of some cells
(Fig. 7H), suggesting that GFP in the cytosol is transported into
the central vacuole as a substrate for autophagy.
Autolysosomes accumulated by cysteine protease inhibitors
take over GFP fluorescence from autophagosomes
We examined whether the autolysosomes accumulated after
treatment with the cysteine protease inhibitor E-64c acquire
GFP fluorescence from autophagosomes labeled with GFP-Atg8.
The autolysosomes had distinct GFP fluorescence, confirming
that they are a successive organelle derived from the autophagosomes (Fig. S3).
The autophagy inhibitor 3-MA blocks the formation of
autophagosomes
When 3-MA was applied to culture medium, autophagosomes
did not appear (Fig. S4, A). In contrast, in control cells, where
water, the solvent for the 3-MA solution, was added to the medium,
fluorescent autophagosomes appeared (Fig. S4, B, arrows). Based
on counting of the autophagosomes, 3-MA substantially blocked
their formation (Fig. S4, E). The result is consistent with our previous result that 3-MA substantially blocked the formation of autolysosomes.43 After 2 d, autophagosomes were seldom seen, and a
significant portion of the GFP fluorescence moved into the central
vacuole in control cells (Fig. S4, D), whereas such movement did
not occur in 3-MA-treated cells (Fig. S4, C). This result suggests
that the final destination of autophagosomes is the central vacuole.
Figure 6. Effects of concanamycin on cellular protease activity and protein contents. Four-day-old tobacco BY-2 cells were cultured in sucrosefree culture medium containing no (DMSO and EtOH), 0.1, and 1 mM concanamycin (Con) for 1 d (A) or for 2 d (B and C). DMSO or ethanol (EtOH)
was added as a solvent control of concanamycin addition. tD0 means
immediately after the start of culture in sucrose-free medium. (A) Cells
were homogenized and cellular protease activities were measured at pH
5.5 using FTC-casein as a substrate. (B) Cellular protein contents were
measured. (C) Cellular proteins from the same volume of culture were
separated by SDS-PAGE and stained with silver. 0.1Con, 0.1 mM concanamycin for 2 d; 1Con, 1 mM concanamycin for 2 d. Molecular masses (kD)
of marker proteins are shown on the left.
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GFP-AtAtg8 is localized on cytoplasmic particles
accumulating in the central vacuole
To examine GFP-AtAtg8 localization, we cultured transformed cells expressing GFP-AtAtg8 in sucrose-free medium
containing concanamycin. Many particles were observed to be
moving in a Brownian manner in the central vacuole (Fig. 8A).
The lumen of the central vacuole also had GFP fluorescence, but
these particles had stronger GFP fluorescence. Not all particles in
the vacuole had GFP fluorescence, and in some cells, only a small
proportion of the particles had stronger GFP fluorescence
pervading the vacuolar lumen (Fig. 8B). It is likely that GFP
associated with the particles at first gradually became distributed
in the vacuolar lumen. In contrast, in control cells without
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concanamycin, autophagosomes were seen in the cytoplasm
(Fig. 8C). Autophagosomes were presumed to mingle with
cytoplasmic particles in the central vacuole in concanamycintreated cells (Fig. 8A); however, we could not strictly discriminate autophagosomes from many of the other vacuolar particles
by fluorescence microscopy. This observation supports the idea
that the particles accumulated by concanamycin treatment are
the structures formed by the fusion of autophagosomes with the
central vacuole, corresponding to autophagic bodies in yeast cells.
If a vacuolar particle is formed by the fusion of an autophagosome with the central vacuole, the membrane encircling the particle should become the inner membrane of the autophagosome
and be distinct from the vacuolar membrane. To try to prove
this, we used BY-2 cells that constitutively express a fusion protein of GFP and the vacuolar membrane protein VM23 from
radish.44 In the transgenic cells, the vacuolar membrane had
GFP fluorescence.45 When such cells were transferred to sucrosefree culture medium containing concanamycin, many particles
formed in the vacuole (Fig. 8D), but fewer had GFP fluorescence. Furthermore, the vacuolar lumen did fluoresce less than
GFP-Atg8-expressing cells (Fig. 8D vs. A and B), showing that
the membranes surrounding most vacuolar particles are distinct
from vacuolar membranes.
Taken together, the results from transgenic cells expressing
GFP-Atg8 and VM23-GFP support the notion that vacuolar particles are derived from autophagosomes. It should, however, be
noted that a significant portion of the vacuolar particles formed
by concanamycin seemed to have GFP-VM23 fluorescence, suggesting that invagination of portions of the vacuole membrane,
such as occurs during microautophagy, occurs under these experimental conditions.
GFP-AtAtg8 fusion protein is degraded into GFP in
response to sucrose starvation
To confirm that the fusion protein with the correct size is
expressed and to examine whether the expressed protein is altered
in response to sucrose starvation, we analyzed cellular proteins of
transformed cells that had been starved for sucrose (Fig. 9). The
pattern of polypeptides stained with silver did not change drastically during 1 d of sucrose starvation (Fig. 9A). Western blotting
with anti-GFP antibody detected the presence of only one band
of approximately 40 kD in 4-d-old cells, which are in the logarithmic growth phase (Fig. 9B, arrowhead). Antibody against an
N-terminal peptide of Atg8 protein also recognized this band
(Fig. 9C, arrowhead). Thus this band was regarded as the fusion
protein of 28 kD GFP and 12 kD Atg8. During sucrose starvation, the band corresponding to the fusion protein became faint,
and instead, a broad band, likely composed of several bands,
appeared around 28 kD. Antibody against Atg8 peptide did not
Figure 7. Morphological changes of transgenic cells expressing GFPAtAtg8 fusion protein in response to sucrose starvation. (A, B, C, D and
E) Four-day-old BY-2 cells expressing GFP-AtAtg8 (A) were cultured
under sucrose starvation conditions for 1 d (B, C and D) or 2 d (E), and
observed by a confocal laser microscope. (F, G and H) Four-day-old BY-2
cells expressing GFP (F) were cultured under sucrose starvation for 1 d
(G) or 2 d (H). For A, B, E, F, G and H, the Nomarski images are on the
left; the fluorescence images on the right. Bars represent 20 mm.
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Figure 8. Effects of concanamycin on changes in the morphology of
tobacco BY-2 cells expressing GFP-AtAtg8 fusion protein under sucrose
starvation conditions. BY-2 cells expressing GFP-AtAtg8 (A, B and C) or
VM23-GFP (D and E) were cultured under sucrose starvation condition
for 1 d in the presence of 0.1 mM concanamycin (A, B and D). DMSO
was used at 1% (v/v) as a solvent control of concanamycin addition
(C and E). The Nomarski images are on the left; the fluorescence images
on the right. Bars represent 20 mm.
recognize this band around 28 kD (Fig. 9C). The broad 28 kD
band was likely composed of GFP alone and GFP variants truncated at the N- and/or C-terminus. After 2 d of starvation, the
fusion protein nearly disappeared, whereas the intensity of bands
corresponding to GFP variants became stronger. Thus, the linkage between GFP and AtAtg8 is cleaved in response to sucrose
starvation. Under sucrose starvation, the GFP moiety appeared
to remain undegraded 2 d after sucrose deprivation, although
synthesis of the fusion protein seemed to stop.
Also in cells expressing GFP alone, a broad band of approximately 28 kD was detected, suggesting that the expressed GFP is
partially degraded. This band became faint in response to sucrose
deprivation, suggesting that net degradation of GFP occurs. Furthermore, a slight shift of the bands to a lower molecular mass was
observed, suggesting that GFP in the cytoplasm is transported to
lytic compartments by autophagy and further truncated.
In parallel with the effect of 3-MA on morphological changes,
3-MA inhibited the cleavage of GFP-AtAtg8 fusion protein to
GFP and Atg8 (Fig. S5). In the absence of 3-MA, the intensity
Figure 9. Cleavage of GFP-AtAtg8 fusion protein in tobacco BY-2 cells
under sucrose starvation conditions. BY-2 cells expressing GFP-AtAtg8
fusion protein or only GFP were transferred to sucrose-free culture
medium. Immediately, 1 d, and 2 d after transfer (0, 1, and 2, respectively), proteins were extracted from the cells. (A) Cellular proteins were
separated and stained with silver. The same amount of proteins was
loaded into each lane. Molecular masses (kD) of marker proteins are
shown on the left. (B) Western blotting using anti-GFP antibody. Arrowhead, GFP-AtAtg8 band.
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of GFP bands increased from day 1 to day 2. In addition, 3-MA
inhibited this increase. Unlike 3-MA, concanamycin did not
have any inhibitory effect on the cleavage of GFP-AtAtg8 fusion
protein. Rather, concanamycin promoted the accumulation of
bands corresponding to GFP in the cells. This is probably due to
concanamycin promoting the fusion of autophagosomes with the
central vacuoles. Alternatively, it is likely that concanamycin
raises the vacuolar pH and inhibits the degradation of GFP in
the vacuoles. E-64c neither inhibited cleavage of GFP-AtAtg8
nor enhanced the accumulation of GFP bands, suggesting that
the protease responsible for the splitting of the fusion protein is
not a papain-type cysteine protease.
Discussion
Our previous and present results obtained using 3 kinds of
inhibitors, the autophagy inhibitor 3-MA, the vacuolar
HC-ATPase inhibitors concanamycin and bafilomycin and the
cysteine protease inhibitor E-64c, clearly show a stepwise process
of autophagy in tobacco BY-2 cells. All three inhibitors block the
net protein degradation that occurs under sucrose starvation conditions, which is consistent with the idea that all 3 inhibitors
block autophagy, which occurs under starvation. 3-MA did not
cause the accumulation of any special structure under starvation
conditions, whereas the accumulation of autophagosome-related
structures and autolysosomes was respectively observed following
treatment with concanamycin and E-64c. When the cells are
treated with E-64c, autolysosomes, in which portions of the cytoplasm appear to already be partially degraded, accumulate. In
contrast, when the cells are treated with concanamycin, portions
of the cytoplasm with a boundary membrane, which resemble
autophagic bodies in yeast cells, are seen in the central vacuole as
well as autophagosomes and autophagosome-like structures in
the cytoplasm. Cells treated simultaneously with concanamycin
and E-64c exhibit the same morphology as those treated with
concanamycin alone, supporting the notion that the point of
action of concanamycin is upstream from that of E-64c. That 3MA suppresses both the effects of concanamycin and E-64c suggests that the point of action of 3-MA is upstream from those of
concanamycin and E-64c.
Membrane-enclosed parts of the cytoplasm, which are derived
from autophagosomes, accumulated in the central vacuole when
tobacco BY-2 cells were starved for sucrose in the presence of the
vacuolar HC-ATPase inhibitor concanamycin. Since vacuolar pH
increased following treatment with concanamycin, vacuolar
hydrolases that have their optimal pH in the acidic range should
be inhibited. Thus if parts of the cytoplasm were transported into
the central vacuole in the presence of concanamycin, they should
remain undegraded in the central vacuole. Indeed, our results
showed that high vacuolar pH is necessary for the retention of
parts of the cytoplasm taken up into the central vacuole, since they
disappeared after concanamycin had been washed out (Fig. 4).
However, we reason that the primary effect of concanamycin
on autophagy is distortion of the normal macroautophagic pathway in tobacco BY-2 cells. The accumulation of portions of the
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cytoplasm that occurs in the presence of concanamycin is likely
to be caused by a block in transformation of autophagosomes to
autolysosomes. The accumulation of numerous autophagosomes
in the cytoplasm probably leads to the fusion of the autophagosomes with the central vacuole and the accumulation of parts of
the cytoplasm in the central vacuole. Thus, we think that the
accumulation of many cytoplasmic drops in the central vacuole
does not mean that the central vacuole is the compartment where
degradation occurs during autophagy in tobacco BY-2 cells, but
rather reflects an artifact.
The following observations support the notion that almost no
autophagosomes directly fuse with the central vacuole in the normal autophagic pathway of BY-2 cells.
First, in mammalian and yeast cells, parts of the cytoplasm to
be degraded accumulate in the compartment for protein degradation during autophagy under condition inhibiting proteolysis.
When mammalian cells are treated with a cysteine protease inhibitor under nutrient-starvation conditions, parts of the cytoplasm
remain in the lytic compartments of mammalian cells, the lysosomes, and as a result, autolysosomes accumulate in the cells.46
In yeast cells undergoing nutrient starvation that are treated with
a serine protease inhibitor, phenylmethylsulfonyl fluoride
(PMSF), parts of the cytoplasm accumulate in the vacuole, which
is the lytic compartment of yeast cells.47 In addition to protease
inhibitor analyses, mutants that are defective in vacuolar protease
activities have been obtained in yeast cells. In these mutants, parts
of the cytoplasm, called autophagic bodies, accumulate in the
vacuole in the process of autophagy.3 We have shown that
tobacco BY-2 cells starved for sucrose in the presence of a protease inhibitor such as leupeptin or E-64c, cytoplasmic particles,
which represent partially-degraded parts of the cytoplasm, accumulate in autolysosomes, instead of in the central vacuole.40
Thus it is reasonable to suppose that the lytic compartments for
autophagy in tobacco BY-2 cells are autolysosomes.
Secondly, concanamycin evoked not only the accumulation of
autophagic bodies in the central vacuole, but also the accumulation of autophagosomes and autophagosome-like structures in
the cytoplasm. The transformation of autophagosomes to autolysosomes is blocked by bafilomycin A1 and concanamycin A in
mammalian cells.34 Thus it is reasonable to suppose that concanamycin blocks the maturation of an autophagosome to an autolysosome. Although the target HC-ATPase of these inhibitors is
unknown, it is likely that they inhibit membrane fusion by alkalizing the lumen of lysosomes and endosomes. Thus, autophagy
in BY-2 cells may be perturbed because concanamycin alkalizes
some acidic organelles related to autophagy in addition to the
central vacuole. Although we did not confirm the alkalization of
organelles other than the central vacuole, it is likely that other
acidic organelles are also alkalized by treatment with concanamycin. It is known that Golgi cisternae in BY-2 cells swell in the
same treatment, probably originating from their alkalization.48
In BY-2 cells, concanamycin inhibits the transport of proteins to
vacuoles although the vacuolar pH is normal.33
Taking this evidence together strengthens our notion that the
main lytic compartments of autophagy in tobacco BY-2 cells are
autolysosomes and not the central vacuole. Some autolysosomes
Plant Signaling & Behavior
e1082699-9
fuse with the central vacuole, and final degradation is in the central vacuole, but almost all autolysosomes complete the degradation of the cytoplasm enclosed in them. However, in some plant
species, autophagosomes appear to directly fuse with the central
vacuole in a normal autophagic process.49 Our observation of
barley and Arabidopsis root tips treated with the cysteine protease
inhibitor E-64d, an esterified form of E-64c, also demonstrated
that parts of the cytoplasm accumulate in the central vacuole as
well as in newly formed autolysosomes. Why the sites for degradation of cytoplasmic components in autophagy are different in
different cell types remains unknown. How the contribution of
these 2 organelles to autophagy is regulated in plant cells is an
issue for future research.
How transformation of autophagosomes to autolysosomes
occurs in tobacco BY-2 cells is not fully understood. In mammalian cells, it proceeds by the fusion of autophagosomes with preexisting lysosomes and/or endosomes. In the case of yeast, newly
formed autophagosomes fuse with the preexisting vacuole to
form a vacuole containing autophagic bodies. As noted above, in
some plant species, autophagosomes directly fuse with the central
vacuole, as in yeast cells. For tobacco autophagosomes to be
transformed to autolysosomes, there must be many lines of transport of membrane vesicles to autophagosomes or autolysosomes.
We previously found that the styryl dye FM4-64 on the plasma
membrane flows into autolysosomes probably through an endocytic pathway.45 Thus, it is probable that as in mammalian cells,
the fusion of autophagosomes with endosomes contributes to the
formation of autolysosomes in tobacco BY-2 cells. Our previous
results also showed that FM4-64 residing on the membrane of
central vacuoles and a protein existing in the lumen of central
vacuoles move to autolysosomes under sucrose starvation conditions, suggesting a flow of proteins and membrane lipids from
the central vacuole to autophagosomes/autolysosomes. Thus, it is
possible that hydrolytic enzymes supplied from the central vacuole to autophagosomes also contribute to the transformation of
autophagosomes to autolysosomes. It is therefore conceivable
that concanamycin blocks these processes by increasing the internal pH of endosomes and/or the central vacuole. In the absence
of E-64, protein degradation continues unabated. If digestion
occurs in autolysosomes before their fusion with the central vacuole, the number of empty lysosomes should increase under
sucrose starvation in the absence of E-64c. This notion is supported by the previous observation that the number of empty
vesicles, probably lysosomes, increases significantly in tobacco
cells starved for sucrose in the absence of E-64c.43
In this study, we succeeded in visualizing autophagosomes
using a light microscope. The recruitment of GFP-AtAtg8 protein to autophagosomes is a good marker of autophagy in living
plant cells as in yeast and mammalian cells. A large amount of
GFP-AtAtg8 is expressed in the cells. Comparing the results
from morphological analysis and immunoblotting, GFP-AtAtg8
is thought to be degraded into GFP and AtAtg8 moieties as
autophagy proceeds. The processing of GFP-AtAtg8 to GFP and
AtAtg8 can act as a biochemical marker of autophagy in tobacco
cells, and perhaps as a general biochemical marker of autophagy
in plant cells.
e1082699-10
Materials and Methods
Plant material
Cultured tobacco (Nicotiana tabacum) BY-2 cells were used.
The cells were cultured in Murashige and Skoog culture medium
containing 3% (w/v) sucrose and 0.2 mg/L 2,4-D as described
previously.40 The cells were starved for sucrose as described
previously.40
Chemicals
3-MA was purchased from Sigma. Concanamycin and bafilomycin were obtained from Wako Pure Chemicals Ind., Ltd
(Tokyo, Japan). and dissolved in DMSO or ethanol as 100x concentrated solutions. E-64c was from Peptide Institute Inc.
(Osaka, Japan). Fluorescein isothiocyanate-labeled casein was
prepared41 according to Twining.50
Light microscopy
Light microscopes (OptiPhoto, Nikon and BX-51, Olympus)
equipped with fluorescence and Nomarski differential interference contrast optics were used. Photographs were taken using a
photomicrographic camera (Microflex UFX-II, Nikon) and negative monochrome (Presto, ISO 400, Fuji) and color (Fujicolor,
ISO400, Fuji) film or a digital camera (DS-Fi1, Nikon). For
obtaining confocal images, confocal laser microscopes (LSM510,
Zeiss) were used. To stain cells with quinacrine, they were kept
in 0.1 M sorbitol, 5 mM HEPES-NaOH (pH 7.5) and 10 mM
quinacrine at room temperature for about 5 min.
Protease assays
Cells were collected on a glass filter (GF/A, 47 mm in diameter, Whatman) from 3 mL of culture, transferred to a Teflonhomogenizer and homogenized with 1 mL of 0.1 M acetateNaOH (pH 5.0) buffer containing 28 mM 2-mercaptoethanol.40
The homogenate was centrifuged at 15,000 rpm for 10 min. The
resultant supernatant (60 mL) and 40 mL 0.5% (w/v) fluorescein
isothiocyanate-labeled casein were mixed and incubated at 37 C
for 30 min. The protease reaction was stopped by adding
100 mL 10% (w/v) trichloroacetic acid. After centrifugation,
fluorescence at 525 nm was measured with an excitation wavelength at 490 nm.
Protein assay, SDS-PAGE and western blotting
Cells in 1 mL of culture medium were collected on a glass filter and transferred to a Teflon homogenizer. They were homogenized with 2 mL of 100 mM HEPES-NaOH (pH 7.5), 1 mM
EDTA, 100 mM leupeptin, 1 mM phenylmethylsulfonyl fluoride or 4-(2-aminoethyl)benzenesulfonyl fluoride, and 28 mL 2mercaptoethanol. The homogenate was centrifuged at
15,000 rpm for 10 min at 4 C. The protein content in the
supernatant was measured according to the method of Lowry
et al.51, modified by Bensadoun and Weinstein.52 BSA was used
as a standard. Proteins in the supernatant were also separated by
electrophoresis on SDS-polyacrylamide gradient gels (Ready Gel
10/20, Bio-Rad; or PAG Mini Daiichi 11/14 or 14/15, Daiichi
Pure Chemicals, Co., Ltd.) and visualized by silver staining.
Plant Signaling & Behavior
Volume 10 Issue 11
For protein gel blotting, proteins in a polyacrylamide gel
were electroblotted onto nitrocellulose membrane in a solution
consisting 25 mM Tris, 192 mM glycine and 20% methanol.
After transfer, the membrane was immersed in 10 mM TrisHCl (pH 8.0) containing 5% (w/v) skim milk, 150 mM NaCl
and 0.05% (w/v) Tween 20 at 4 C overnight. Anti-GFP antibody (GFP Polychlonal Antibody (IgG Fraction) #8363, Clontech Lab., Inc., CA, USA) was diluted 1:1000. The secondary
antibody used was anti-rabbit IgG antibody conjugated with
alkaline phosphatase (Anti-Rabbit IgG (Fc) AP Conjugate,
Promega). Binding of the primary and secondary antibodies to
membranes was done in 10 mM Tris-Cl (pH 8.0) containing
150 mM NaCl and 0.05% Tween 20. Nitro blue tetrazolium
and 5-bromo-4-chloro-3-indolyl phosphate were used as substrates for alkaline phosphatase.
Electron microscopy
Cells were fixed with 2% (w/v) glutaraldehyde and 1% (w/v)
formaldehyde in 100 mM sodium cacodylate-HCl (pH 6.9)
buffer at room temperature for 1 h and at 4 C overnight. After
the samples were treated with 1% (w/v) tannic acid for 1 h at
room temperature, they were fixed again with 1% (w/v) osmium
tetroxide for 2 h at room temperature. The samples were stained
en block with 2% (w/v) uranyl acetate for 2 h at room temperature. Thereafter they were dehydrated by an ethanol series and
propylene oxide, and embedded in Spurr’s resin. Thin sections
were made from these blocks and stained with uranyl acetate
and lead citrate. They were observed with an electron microscope
(H-7000, Hitachi).
Construction of plasmids
RNA was isolated from roots of Arabidopsis seedlings and single-stranded cDNA synthesized using reverse transcriptase. The
AtATG8d open reading frame was amplified by PCR using the
DNA as templates and the following 2 primers: the sense primer,
50 - tttgtacatggcgattagctccttcaa-30 , comprises the BsrG I site and
20 bases of oligoDNA from the 50 -end of the coding region on
the coding strand for AtATG8d DNA; the antisense primer, 50 ccgcggccgcttagaagaagatcccgaacg-30 , includes the Not I site and 20
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Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Funding
This work was supported by KAKENHI [grant number
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