The AP-1 m Adaptin is Required for KNOLLE Localization at the
Cell Plate to Mediate Cytokinesis in Arabidopsis
Rapid Paper
Ooi-Kock Teh1, Yuki Shimono1, Makoto Shirakawa1, Yoichiro Fukao2, Kentaro Tamura1,
Tomoo Shimada1 and Ikuko Hara-Nishimura1,*
1
Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502 Japan
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, 630-0101 Japan
*Corresponding author: E-mail, [email protected]; Fax, +81-75-753-4142.
(Received February 21, 2013; Accepted March 17, 2013)
2
Formation of clathrin-coated vesicles (CCVs) requires the
scaffolding adaptor protein (AP) complexes, which are
conserved across all eukaryotes. The Arabidopsis genome
encodes five AP complexes (AP-1 to AP-5), and each complex consists of four subunits. In this study, we characterized
the poorly defined AP-1 complex by using genetics, proteomics and live cell imaging. We showed that the AP-1 m
adaptin subunit (AP1M2) was localized to the trans-Golgi
network (TGN) and interacted physically with the AP-1 subunits in Arabidopsis. During treatment with brefeldin A
(BFA), the functional fluorophore-tagged AP1M2 relocated
to the BFA compartment. The AP1M2 loss-of-function
mutant ap1m2 displayed deleterious growth defects,
which were particularly evident in the compromised cytokinesis that was revealed by the presence of cell wall stubs in
multinucleate cells. Immunolocalization of the cytokinesisspecific syntaxin KNOLLE (KN) in ap1m2 showed that KN
was mislocalized and aggregated around the division plane,
while a secretory marker targeting to the cell plate remained
unaffected. Taken together, we propose that the AP-1 complex is required for cell plate-targeted trafficking of KN
in dividing plant cells, and that it has a common role in
mediating plant and yeast/animal cytokinesis systems
which are fundamentally different.
Keywords: Adaptin Adaptor protein complex Arabidopsis thaliana Cytokinesis KNOLLE Trans-Golgi
network.
Editor-in-Chief’s choice
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Abbreviations: amiRNA, artificial microRNA; AP, adaptor
protein; AP1M2, AP-1 m adaptin subunit; Ara, Arabidopsis
gene homologous to the mammalian ras gene; BFA, brefeldin
A; CCV, clathrin coated vesicle; CLSM, confocal laser scanning microscopy; GFP, green fluorescent protein; KN,
KNOLLE; mRFP, monomeric red fluorescent protein; PM,
plasma membrane; RT–PCR, reverse transcription–PCR;
secRFP, secreted red fluorescent protein; SEM, scanning electron microscopy; SNARE, soluble N-ethylmaleimide-sensitive
factor attachment protein receptor; SNX1, SORTING NEXIN1;
SYP, SYNTAXIN OF PLANTS; TEM, transmission electron
microscopy;
TGN,
trans-Golgi
network;
VHA-a1,
VACUOLAR H (+)-ATPase subunit a1.
Introduction
Delivery of proteins to their pre-defined destinations depends
on vesicle trafficking mechanisms that are mediated by vesicles
such as COPI, COPII and clathrin-coated vesicles (CCVs) in
eukaryotes. COPI and COPII are known to mediate the early
secretory pathways between the endoplasmic reticulum and
Golgi apparatus. CCVs are responsible for post-Golgi traffic,
which includes the late secretory pathway from the transGolgi network (TGN) to the plasma membrane (PM), the vacuolar trafficking pathway from the TGN to the vacuole, and
endocytosis from the PM to the TGN. In-depth analysis of
CCVs in yeast and mammalian systems reveals the detailed
composition of CCVs, which consist of the clathrin triskelion
(a three-legged protein made of three heavy chains and three
light chains) and the adaptor protein (AP) complex on the
vesicle surface.
The AP complexes play crucial roles in the formation of
vesicles and act as a major hub of interactions. Structurally,
the AP complexes mediate the scaffolding of the clathrin
triskelion at the budding site of CCVs on the membrane.
Functionally, the AP complexes recognize specific peptide
motifs in the cytoplasmic tails of cargo proteins (McMahon
and Boucrot 2011). Currently, five AP complexes (AP-1 to
AP-5) have been identified in eukaryotes: AP-1 and AP-2
were purified from enriched CCVs, whereas AP-3 and AP-4
were discovered by searching sequence databases for AP-1
and AP-2 homologs (Zaremba and Keen 1983, Robinson and
Bonifacino 2001). These five AP complexes are found in Homo
sapiens, Mus musculus and Arabidopsis thaliana. In contrast,
yeast, nematode and fruit fly possess only three AP complexes,
which correspond to the mammalian AP-1, AP-2 and AP-3
complexes (Boehm and Bonifacino 2002, Bassham et al.
Plant Cell Physiol. 54(6): 838–847 (2013) doi:10.1093/pcp/pct048, available online at www.pcp.oxfordjournals.org
! The Author 2013. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Plant Cell Physiol. 54(6): 838–847 (2013) doi:10.1093/pcp/pct048 ! The Author 2013.
Adaptor complex functions in cytokinesis
2008). The fifth AP complex, AP-5, was recently identified in the
human genome and its orthologs are widely conserved in the
genomes of other organisms (Hirst et al. 2011).
The structural composition of AP complexes is highly conserved across all eukaryotes. The complexes exist as heterotetrameric proteins that contain two large subunits (b1-5 and
one each of g/a/d/e/z), a medium subunit (m1-5) and a small
subunit (s1-5) (McMahon and Boucrot 2011). The AP complex
subunits are collectively referred to as adaptins. The m adaptin is
an essential subunit in AP complexes for the recognition of
YXX and dileucine-based ([DE]xxxL[LI]) sorting signals on
cargo proteins (Ohno et al. 1995, Bonifacino and Traub 2003).
Although adaptins play important roles in linking the cargo
proteins and membrane lipids to CCV formation, emerging
evidence identifies a large network of accessory proteins that
are able to replace the AP complexes functionally during CCV
assembly (Tebar et al. 1996, McMahon and Boucrot 2011).
In animal cells, current work indicates that all AP complexes
localize to the post-Golgi cellular compartments. AP-1 is found
on the TGN/endosomal membranes, while AP-3, AP-4 and AP5 localize primarily to endosomes, the TGN and late endosomal
compartments, respectively (Hirst et al. 2011). AP-2 localizes
exclusively to the PM and is involved in clathrin-mediated
endocytosis (Robinson 2004). Previously, independent genetic
screens in plants have isolated mutants of AP-3 b and m adaptin
subunits (Niihama et al. 2009, Feraru et al. 2010). Although the
subcellular localization of AP-3 remains elusive, analysis of the
AP-3 b adaptin mutant, protein affected trafficking2 (pat2),
shows that the mutant cells ectopically accumulate vacuolarand PM-localized proteins (Feraru et al. 2010). This suggests
that AP-3 may function in mediating vacuolar-targeted trafficking. Indeed, a recent report shows that the vacuolar localization
of SUCROSE TRANSPORTER4 is AP-3 dependent
(Wolfenstetter et al. 2012). At a physiological level, the AP-3
complex is required for mediating gravitropism in plants because the AP-3 m adaptin mutant zip4 exhibits a slightly
reduced gravitropic response (Niihama et al. 2009, Feraru
et al. 2010).
In this study, we characterize the poorly defined AP-1 complex in Arabidopsis. The A. thaliana genome encodes two
putative copies of each AP-1 adaptin: b (At4g11380 and
At4g23460), g (At1g60070 and At1g23900), m (At1g60780 and
At1g10730) and s (At2g17380 and At4g35410) (Bassham et al.
2008). These adaptins are specific to AP-1, except for the b
adaptins, which are presumably shared by AP-2 as revealed
by phylogenetic analysis (Dacks et al. 2008). Genetic analyses
of the AP-1 complex in yeast by disrupting the corresponding
s1, m1, b1 or g adaptins show no obvious deleterious effects on
protein sorting and growth (Phan et al. 1994, Stepp et al. 1995).
This result is surprising because AP-1 seems to be unimportant
for cell physiology in unicellular eukaryotic organisms. In contrast, knocking out the AP-1 m1 adaptins in Caenorhabditis
elegans and mice leads to embryonic lethality, indicating
an essential role for AP-1 in the development of multicellular
organisms (Meyer et al. 2000, Shim et al. 2000). Using the
combined experimental approaches of proteomics, genetics
and live cell imaging, we show that AP-1 is a TGN-localized
complex that is required to execute somatic cytokinesis properly in root and shoot cells in Arabidopsis.
Results
Identification of the AP-1 complex of Arabidopsis
To be comparable with the nomenclature for AP complexes of
the other organisms, the following nomenclature was used for
the Arabidopsis AP-1 subunit-encoding loci: AP1M1
(At1g10730, muB2); AP1M2 (At1g60780, muB1); AP1/2B1
(At4g11380); AP1/2B2 (At4g23460); AP1G1 (At1g60070);
AP1G2 (At1g23900); AP1S1 (At2g17380); and AP1S2
(At4g35410). The AP1M2 locus was formerly denoted as AtmB2-Ad (GenBank accession No. AC002292) (Happel et al.
2004). To better characterize the AP-1 complex, we first generated transgenic plants that stably express the green fluorescent
protein (GFP) fusion protein of AP1M2. The C-terminal GFP
AP1M2 fusion protein (AP1M2–GFP) was constructed as a
genomic fragment and expressed under the native promoter
of AP1M2. Anti-GFP immunoblot analysis using the total protein extracted from homozygous seedlings of the transgenic
plant detected a single band corresponding to 79 kDa, which
was in agreement with the predicted molecular weight of
AP1M2–GFP (Fig. 1A).
To determine the in vivo AP-1 adaptin subunit composition,
we performed immunoprecipitation using the AP1M2–GFP
transgenic plants. Total protein extracted from 6-day-old seedlings was subjected to anti-GFP immunoprecipitation. The
immunoprecipitates were separated by SDS–PAGE and stained
with fluorescent dye or analyzed on anti-GFP immunoblots.
The transgenic plant expressing free GFP was used as the negative control. Although free GFP appeared as a single 30 kDa
band, AP1M2–GFP pulled down an array of immunoprecipitated proteins of various sizes (Fig. 2A). To determine
the identities of proteins in the immunoprecipitates, mass
spectrometry analysis was performed with LTQ-Orbitrap on
peptides prepared from in-gel digestion. After cross-checking
with the protein pool from the free GFP control to eliminate
unspecific interactors, we showed that AP1M2 interacted with
five putative AP-1 adaptins encoded in the A. thaliana genome
(Fig. 2B, C).
AP1M2 is a TGN-localized adaptin and lies in a
BFA-sensitive trafficking pathway
Confocal microscopy analysis of the AP1M2–GFP transgenic
plants revealed that AP1M2 was expressed ubiquitously in all
tissues. It appeared as mobile punctate structures and diffused
fluorescence throughout the cytosol (Fig. 1B). To determine
the subcellular localization of AP1M2 in Arabidopsis, we
followed the co-localization, if any, between AP1M2–GFP and
the styryl dye FM4-64, which has been widely used as a marker
for endocytosis and endocytic vesicle trafficking in living
Plant Cell Physiol. 54(6): 838–847 (2013) doi:10.1093/pcp/pct048 ! The Author 2013.
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O.-K. Teh et al.
A
B
Cotyledon
kDa
Hypocotyl
Mature root
Root
elongaon zone
Root p
120
100
80
60
50
10 µ m
40
30
2 µm
Fig. 1 AP1M2 is expressed ubiquitously in Arabidopsis. (A) Anti-GFP immunoblot of a transgenic line stably expressing AP1M2–GFP. The
protein predominantly migrates as a 79 kDa band, in agreement with the predicted molecular mass of AP1M2–GFP. Protein from a stable
transgenic line expressing free GFP is used as control. (B) AP1M2–GFP is expressed ubiquitously in Arabidopsis. AP1M2–GFP labels mobile
punctate structures in the cotyledon, hypocotyl and root.
A
kDa
220
120
100
80
B
Ear
Ear
60
50
AP1M
AT1G60780
40
AP1S
30
AT2G17380
20
C
AGI code
AT4G11380
AT4G23460
AT1G60070
AT1G23900
AT2G17380
AT1G60780
Exp.1
1743
1667
1399
937
148
685
AP1/2B
AP1G
AT4G11380
AT4G23460
AT1G60070
AT1G23900
Score
Exp.2
2307
2157
1453
1364
358
340
Exp.3
613
650
237
71
29
964
Assigned name
AP1/2B1
AP1/2B2
AP1G1
AP1G2
AP1S1
AP1M2
Fig. 2 Compositional analysis of the Arabidopsis AP-1 complex
by mass spectrometry. (A) Fluorescent-stained SDS–polyaacrylamide
gel of AP1M2–GFP immunoprecipitates (right lane). Immunoprecipitates from free GFP-expressing transgenic lines (left lane) are
used as a control. (B) The composition of the AP-1 complex.
(C) Summary of the AP1M2-interacting proteins identified by mass
spectrometry analysis in three independent experiments. Values in the
score column represent MASCOT scores, which are the probabilitybased scoring of peptide sequence matching with the database.
840
eukaryotic cells (Bolte et al. 2004) because it sequentially
labels endomembrane compartments. Following intracellular
internalization, one of the earliest organelles labeled by FM464 is the TGN (Dettmer et al. 2006). AP1M2–GFP displayed
excellent co-localization with FM4-64-labeled punctate structures after 5 min of staining (Fig. 3A–C). This rapid co-localization suggests that AP1M2 may reside on the TGN. To show
that this was the case, we stained the AP1M2–GFP-expressing
seedlings with FM4-64 for 5 min followed by 1 h of 50 mM
brefeldin A (BFA) treatment. BFA is a fungal toxin that is
known to cause the agglomeration of the TGN and endosomal
membranes to form BFA compartments in Arabidopsis root
cells (Geldner et al. 2003). Therefore co-localization of
AP1M2–GFP with the BFA compartments would support the
notion that AP1M2 is localized to the TGN. As expected, BFA
treatment caused the relocation of AP1M2–GFP to the core
of BFA compartments that are labeled by FM4-64 (Fig. 3D–I).
This result was consistent with the rapid co-localization
between AP1M2–GFP and FM4-64 after just 5 min of incubation, and strongly indicated that AP1M2 resided on the TGN.
Notably, BFA treatment also diminished all AP1M2–GFPlabeled punctate structures and caused the cytosolic pool of
AP1M2–GFP to decrease (Fig. 3D, G). These results indicated
that AP1M2–GFP trafficking was in a BFA-sensitive pathway.
Next, we confirmed the subcellular localization of AP1M2–
GFP by performing a series of co-localization experiments using
well-established fluorescent markers. Transgenic lines stably
expressing each of a TGN marker [VACUOLAR H (+)-ATPase
subunit a1 (VHAa1)–monomeric red fluorescent protein
(mRFP) (Dettmer et al. 2006) and mRFP–SYP43 (SYNTAXIN
OF PLANTS43) (Uemura et al. 2012)], a Golgi marker
[mCherry–SYP32 (Geldner et al. 2009)] and pre-vacuolar
Plant Cell Physiol. 54(6): 838–847 (2013) doi:10.1093/pcp/pct048 ! The Author 2013.
Adaptor complex functions in cytokinesis
AP1M2-GFP
FM4-64
Merge
A
B
C
D
E
F
G
H
I
AP1M2 -GFP
compartment markers [mRFP–Ara7 (Ueda et al. 2004) and
SORTIN NEXIN1 (SNX1)–mRFP (Jaillais et al. 2006)] were
used for crossing with the AP1M2–GFP line. Confocal microscopy visualization showed that AP1M2–GFP co-localized exclusively with VHAa1–mRFP and mRFP–SYP43; no co-localization
was detected with mCherry–SYP32, mRFP–Ara7 or SNX1–
mRFP (Fig. 3J–X). Furthermore, AP1M2–GFP was sensitive to
the V-ATPase inhibitor concanamycin A (Fig. 4), which caused
the aggregation of the TGN (Dettmer et al. 2006). Taking these
results together, we concluded that AP1M2 is a TGN-localized
adaptin.
AP-1 is essential for the growth and development
of Arabidopsis plants
To investigate the cellular functions of AP1M2, we isolated the
T-DNA insertion mutant line FLAG_293C11 from the Versaille
collection to characterize the AP1M2 loss-of-function mutant.
Sequencing analysis showed that the T-DNA was inserted
into the sixth exon (Fig. 5A), and reverse transcription–PCR
(RT–PCR) analysis indicated that FLAG_293C11 was a null
allele (Fig. 5B). We subsequently renamed FLAG_293C11
as ap1m2-1. A homozygous ap1m2-1 was viable but showed
deleterious growth phenotypes (Fig. 5D, E). During germination, ap1m2-1 grew normally like the segregating wildtype or AP1M2+/ heterozygous seedlings. At 6 d after
germination, root elongation and leaf development in
M er ge
J
K
K
VHAa1-mRFP LL
M
N
mRFP-SYP43
O
Q
mRFP-Ara7
R
S
T
SNX1-mRFP
U
Merge
2 µM
Concanamycin A
P
VHAa1-mRFP
DMSO
AP1M2-GFP
5 µm
V
W
mCherry-SYP32
Fig. 4 Seedlings stably expressing AP1M2–GFP were treated with
2 mM concanamycin A or dimethylsulfoxide (DMSO) for 30 min
before proceeding to CLSM analysis. AP1M2–GFP (green) in the
root meristem showed distinct punctate structures that co-localized
well with VHAa1–mRFP (magenta) when treated with DMSO (upper
panel). Concanamycin A treatment caused VHAa1–mRFP to form
aggregations, as with the AP1M2–GFP.
X
5 μm
Fig. 3 AP1M2 is a TGN-localized adaptin. CLSM analysis of epidermal
cells in Arabidopsis root tip. (A–C) AP1M2–GFP (A) co-localizes with
the endocytic tracer FM4-64 (B) after 5 min incubation. White arrows
indicate perfect co-localization between AP1M2–GFP and FM4-64
(C). (D–I) AP1M2–GFP trafficking lies in a BFA-sensitive pathway.
After 1 h of BFA treatment, AP1M2–GFP relocalizes to the BFA compartment (G–I) compared with mock treatment with 0.1% (v/v)
Fig. 3 Continued
dimethylsulfoxide (DMSO; D–F), in which AP1M2–GFP remains in the
mobile punctate structures. (J–X) AP1M2–GFP co-localizes exclusively
with TGN markers VHAa1–mRFP (J–L) and mRFP–SYP43 (M–O), but
not with the pre-vacuolar compartment markers mRFP–Ara7 (P–R) and
SNX1–mRFP (S–U) or the Golgi marker mCherry–SYP32 (V–X).
Plant Cell Physiol. 54(6): 838–847 (2013) doi:10.1093/pcp/pct048 ! The Author 2013.
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O.-K. Teh et al.
A
FLAG_293C11
R
At1g60780
RT µ1AF0
LB4
LP2
amiRNA
B
At1g60780-R
Independent
transgenic lines
C
AP1M2+/+
ap1m2-1
AP1M2
#25
#3
#4
AP1M2+/+
AP1M2
ACTIN
T-DNA
D
E
AP1M2 +/+
AP1M2 +/-
ap1m2-1
ap1m2-1
#3
#4
#25
0.5 mm
1 cm
ap1m2-1
AP1M2+/+
F
G
50 µm
50 µm
Fig. 5 The AP1M2 loss-of-function mutant has severe growth phenotypes. Characterization of the Arabidopsis AP1M2 knock-out line.
(A) Schematic representation of primers (indicated as arrows) used
in genotyping and RT–PCR. For endogenous AP1M2 genotyping, primers R and LP2 are used. To genotype the T-DNA, primers R and LB4
are used. For semi-quantitative RT–PCR, primers RT m1AF0 and
At1g60780-R are used. The site targeted by the artificial microRNA
construct is indicated by the red line. (B) Semi-quantitative RT–PCR of
ap1m2-1 and wild-type AP1M2+/+. ACTIN is used as a loading control
and the cycle number is 28. The AP1M2 transcript is not detected in
ap1m2-1. (C) PCR genotyping of ap1m2-1 transgenic lines complemented with AP1M2–GFP. Numbers on top of the panel correspond
to the independent ap1m2-1 transgenic lines shown in (D). (D) The
AP1M2 loss-of-function mutant, ap1m2-1, shows severe growth arrest.
The growth defect is complemented by an exogenous copy of AP1M2
driven by its native promoter. Growth in the complemented ap1m2-1
(#3, #4, #25) is restored to resemble that of wild-type plants. Numbers
indicate the independent transgenic lines. (E–G) SEM analysis of the
ap1m2-1 aerial organs. Seedlings of the ap1m2-1 mutant show
distorted leaf development with abnormal curling (E) and unbranched trichomes (G) compared with wild-type AP1M2+/+ (F).
The stomata in ap1m2-1 develop normally and obey the one-cellspacing rule (D).
842
ap1m2-1 were arrested. The ap1m2-1 seedlings showed distorted apical growth with highly curled leaves and extremely
rough and bloated surfaces (Fig. 5E). While trichomes in
ap1m2-1 were unbranched compared with the wild type (Fig.
5F, G), the stomata developed normally with the one-cell-spacing rule strictly obeyed (Fig. 5G). The ap1m2-1 plants eventually entered the reproductive stage and generated
deformed flowers, from which a limited number of seeds
were produced.
To rule out the possibility that the arrested growth observed
in the ap1m2-1 mutant resulted from the indirect consequences of a second site mutation in unlinked T-DNA, we
generated transgenic plants in the Columbia (Col) background
that constitutively expressed an artificial microRNA (amiRNA)
that targeted AP1M2 (Fig. 5A, red line). These transgenic lines
phenocopied the arrested growth observed in ap1m2-1
(Supplementary Fig. S1). Next, we attempted to complement
the mutant phenotype in ap1m2-1 by using the AP1M2–GFP
construct described above. Because ap1m2-1 was sterile and
could not be used for transformation experiments, we transformed the heterozygous T-DNA plants (AP1M2+/) with
AP1M2–GFP and genotyped all transformants to identify
homozygous ap1m2-1. We successfully identified seven transformants that were all ap1m2-1 homozygous. All ap1m2-1
homozygotes that harbored AP1M2–GFP showed normal
plant growth with no obvious growth defects (Fig. 5C, D).
Therefore, complementation of the mutant phenotype in
ap1m2-1 with an exogenous wild-type copy of AP1M2 provided
unequivocal evidence that deletion of AP1M2 led to growth
arrest in Arabidopsis, and AP1M2 was required for normal plant
development from the seedling stage to the mature plant
stage. Furthermore, the complementation of the ap1m2-1
phenotype by AP1M2–GFP convincingly demonstrated the
functionality of AP1M2–GFP, therefore validating the TGN
localization of AP1M2.
The ap1m2-1 mutant has cytokinesis defects
Given the prominent roles of AP complexes in endosomal trafficking as revealed in mammalian systems, we first investigated
the dynamics of endocytosis in the ap1m2-1 mutant and in
wild-type plants. To this end, we followed the FM4-64 uptake
in a time-lapse manner. In wild-type AP1M2+/+ plants, FM4-64
reached its final destination at the vacuolar membrane after 1 h
of incubation at room temperature (Fig. 6, upper panel).
However, in ap1m2-1 cells, FM4-64 never reached the vacuolar
membrane within the same time frame. Instead, large membrane aggregates were formed after 35 min (Fig. 6, lower panel).
The uptake of FM4-64 in ap1m2-1 was not significantly different from that in the wild-type plants, although a slight delay
was observed (Fig. 6, lower panel). This result suggested that
AP1M2 was not be directly involved in endocytic uptake at the
PM. Detailed inspection of the FM4-64-labeled ap1m2-1 seedlings revealed peculiar structures that accumulated in the cotyledon epidermal cells. These structures were morphologically
distinct from the large membrane aggregates observed in the
Plant Cell Physiol. 54(6): 838–847 (2013) doi:10.1093/pcp/pct048 ! The Author 2013.
Adaptor complex functions in cytokinesis
15 min
35 min
60 min
ap1m2-1
AP1M2+/+
5 min
5 µm
Fig. 6 Endocytosis in ap1m2-1 is not impaired but is delayed. FM4-64 uptake is marginally delayed in ap1m2-1. Punctate structures labeled by
FM4-64 are readily detected in wild-type and ap1m2-1 root tips after 5 min of FM4-64 staining, although the number of punctate structures is
reduced in ap1m2-1. In wild-type cells, FM4-64 reaches the tonoplast after 1 h of incubation. In contrast, FM4-64 fails to stain the tonoplast in
ap1m2-1 within the same time frame; instead, FM4-64 stains aggregates that appear to increase in size with time.
root cells, and strongly resembled the BFA compartments that
represented endomembrane agglomeration in response to BFA
treatment.
Intriguingly, we observed a large number of cell wall stubs
in the cotyledon of ap1m2-1 compared with the wild type
(Fig. 7A, B), which suggested that there were defects in cytokinesis. Mutants that are defective in cytokinesis are usually
characterized by the presence of cell wall stubs and multinucleate dividing cells (Sollner et al. 2002). To determine if ap1m2-1
conformed to the criteria of a cytokinesis mutant, we examined
the ultrastructure of ap1m2-1 cells by transmission electron
microscopy (TEM). Cell wall stubs were readily detected in
the cotyledon (Fig. 7C, arrows) and root meristem (Fig. 7D,
E, arrows). Free (unfused) vesicles clustered around the cell wall
stubs (Fig. 7E, asterisks), which resembled those observed in
knolle and BFA-treated gnl1 seedlings (Reichardt et al. 2007). In
agreement with the cytokinesis defects, the curled leaves with
rough and bloated surfaces in ap1m2-1 mutant (Fig. 5E) are
likely consequences of the enlarged cell size due to incomplete
cytokinesis. The same phenotype had previously been
described in other cytokinesis mutants such as knolle, keule
and hinkel (Assaad et al. 1996, Lukowitz et al. 1996, Strompen
et al. 2002).
The cytokinesis-specific syntaxin KNOLLE
was mislocalized in ap1m2-1
dividing cells
The syntaxin KNOLLE (KN) is a cytokinesis-specific SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein that mediates vesicle fusion at the growing cell
plate (Lauber et al. 1997, Waizenegger et al. 2000). To facilitate
vesicle fusion, the Sec1/Munc18 protein KEULE stabilizes KN in
its open conformation at the cell division plane (Assaad et al.
2001, Park et al. 2012). The trafficking pathway of KN during
somatic cytokinesis has been mapped out previously (Reichardt
et al. 2007). Newly synthesized KN is delivered to the growing
cell plate via the secretory pathway; on mediating homotypic
fusion at the cell plate, the KN is targeted to the vacuole for
degradation by passing through the multivesicular body
(Reichardt et al. 2007, Van Damme et al. 2008). Because
AP1M2 is localized to the TGN, which is a sorting station
where secretory and endocytic pathways converge, it is possible
that the AP1M2-scaffolded vesicles may target KN to the cell
plate from the TGN or may remove KN from the cell plate
to the vacuole. Therefore, we examined KN localization in
ap1m2-1 dividing cells by using immunolocalization.
Plant Cell Physiol. 54(6): 838–847 (2013) doi:10.1093/pcp/pct048 ! The Author 2013.
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A
B
C
5 µm
5 µm
5 µm
D
E
*
*
5 µm
1 µm
F
G
H
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L
M
In wild-type root tip meristematic cells, KN appeared as
patches that were scattered in the cytoplasm during metaphase
(Fig. 7F) and anaphase (Fig. 7G). At the onset of telophase
(Fig. 7H) and throughout cytokinesis (Fig. 7I), KN-positive signals accumulated predominantly at the growing cell plate until
the completion of cell division (Fig. 7H, I). In ap1m2-1 dividing
cells, the localization pattern of KN was indistinguishable from
that in the wild-type control (Fig. 7J, K). However, during telophase, KN formed large aggregations around the cell plate,
although in some cases a portion of the KN-positive signals
was still localized correctly at the cell plate (Fig. 7L).
Aggregation of KN would eventually lead to the aberrant or
incomplete formation of the cell plate throughout cytokinesis
(Fig. 7M). The KN aggregation observed in the ap1m2-1 dividing cells was in sharp contrast to the largely unperturbed secretory marker, secRFP (Faso et al. 2009), targeting to the cell
plate. By stably expressing secRFP in the ap1m2-1 plants, RFP
signal was clearly observed at the cell wall stubs (Fig. 7N–W,
arrowheads), indicating that the secretory pathway to the cell
plate was normal before cell plate expansion was compromised.
Therefore, we concluded that the ap1m2-1 loss-of-function
mutation caused the mislocalization of the cytokinesis-specific
KN, which was responsible for the compromised cytokinesis
observed in ap1m2-1.
2 µm
N
O
P
Q
Discussion
R
10 µm
S
T
U
V
W
10 µm
Fig. 7 ap1m2-1 displays cytokinesis defects. (A and B) CLSM analysis
of ap1m2-1. The ap1m2-1 leaf epidermal cells (B) show irregular division patterns indicated by the cell wall stubs compared with the wildtype control (A) after 1 h of FM4-64 staining. (C–E) TEM analysis of
the ap1m2-1 cotyledon (C) and root tip meristem (D and E). Cell wall
stubs (arrows, C and D) are frequently observed in the ap1m2-1
cells, with unfused vesicles clustering around the cell wall stubs
(asterisks, E). (F–M) Immunolocalization of KN in the root tip.
Immunofluorescence of KN (green) in wild-type (F–I) and ap1m2-1
(J–M) cells is indistinguishable during metaphase (F and J) and anaphase (G and K). In wild-type control root meristem, KN localizes to
the growing cell plate from telophase (H) to late cytokinesis (I).
In contrast, KN fails to localize to the cell plate in the ap1m2-1 root
tip, but instead forms aggregates around the division plane during
telophase (L). In late cytokinesis (M), ap1m2-1 shows incomplete cell
plates and aberrant KN localization. Chromatin DNA is counterstained with 40 ,6-diamidino-2-phenylindole (DAPI; magenta). Cell
boundaries are indicated by dashed lines. (N–W) Serial images of
ap1m2-1 stably expressing the secretory marker secRFP. In two independent (N–R and S–W) T1 lines, cell wall stubs (arrowheads) are
clearly labeled by the secRFP (white) in leaf epidermal cells. Slice intervals are 1 mm.
844
Our results demonstrate that AP1M2 is a TGN-localized adaptin that has an essential function in Arabidopsis cytokinesis.
Cytokinesis in plants is fundamentally different from that in
animal cells. Plant cells utilize outgrowing, dividing cell plates
that eventually fuse with the parental PM, which is in sharp
contrast to the contractile ring in animal cells that constricts
the PM from the cell periphery (Jurgens 2005, Green et al. 2012).
This fundamental difference suggests a functional diversification between membrane trafficking mechanisms in plants and
animals during cytokinesis.
Membrane trafficking in plants during cytokinesis is poorly
understood. Expansion of the dividing cell plate is believed
to result from a series of homotypic fusions of vesicles that
originate primarily from the secretory pathway (Segui-Simarro
et al. 2004, Reichardt et al. 2007). Using genetic analysis, several
trafficking-related proteins have recently been identified
as key factors involved in cell plate expansion/elongation.
These include the interacting SNARE proteins KN, KEULE and
SNAP33, which mediate vesicle fusion at the cell plate (Assaad
et al. 2001, Heese et al. 2001, Park et al. 2012); the RabGTPases
Rab-A2 and Rab-A3, which localize to the growing margin of
the cell plate and could represent likely candidates for mediating KN-decorated SNARE complex docking at the cell plate
(Chow et al. 2008); and the adaptin-like TPLATE, which anchors
the cell plate with the parental PM at the cortical division
zone during late cytokinesis (Van Damme et al. 2011). Our
results show that the AP1M2 subunit is required for the
Plant Cell Physiol. 54(6): 838–847 (2013) doi:10.1093/pcp/pct048 ! The Author 2013.
Adaptor complex functions in cytokinesis
proper localization of KN at the cell plate, which further substantiates the significant involvement of endosomal trafficking
in the execution of cytokinesis.
Electron tomographic analysis of cytokinesis in Arabidopsis
shows that a large number of CCVs form around the cell plate
during later stages of cytokinesis (Segui-Simarro et al. 2004).
Given that the AP complex is the core component for CCV
formation, we hypothesize that the AP1M2-scaffolded CCVs
are involved in correctly mobilizing the cell plate-targeted proteins and/or removing the excess membrane materials from the
cell plate. Indirect evidence from specific localization of
AP1M2–GFP at the TGN but not on the cell plate appears to
support the former possibility (Supplementary Fig. S2). KN is
an obvious cargo candidate for the cell plate-targeted trafficking by AP-1. Interestingly the KN linker domain contains a putative tyrosine motif that could potentially serve as the sorting
signal for recognition by AP1M2 (Supplementary Fig. S3).
Indeed, our immunolocalization data show that in ap1m2-1,
KN localization at the cell plate is disrupted and a large
amount of KN aggregates around the cell plate. This KN aggregation is likely to be a consequence of disrupted TGN to cell
plate trafficking. Furthermore, we demonstrate that the secretory marker secRFP was able to traffic to the cell plates, suggesting that compromised KN localization at the cell plate was
a specific defect caused by the AP1M2 loss of function.
Surprisingly, we were unable to detect KN in the immunoprecipitates of AP1M2–GFP (Supplementary Table S1). Two
possibilities account for this. First, the interaction between
AP1M2 and KN may be too transient/weak for co-immunoprecipitation analysis. Secondly, KN is a cytokinesis-specific syntaxin that is only expressed in the dividing cells and the low
abundance of KN prevented KN from being detected in the
immunoprecipitates of AP1M2–GFP. Indeed anti-KN failed to
detect KN in protein extracts from the seedlings (Lauber et al.
1997).
Currently, there is no direct evidence to show that the
animal AP-1 complex is involved in mediating cytokinesis, as
the AP-1 knock-out mutants are embryo lethal, which prevents
characterization of the mutant phenotype (Zizioli et al. 1999,
Meyer et al. 2000, Shim et al. 2000). In contrast, knocking out
the AP-1 m and b adaptins in fission yeast (Schizosaccharomyces
pombe) and slime mold (Dictyostelium discoideum), respectively, causes cytokinesis defects (Kita et al. 2004, Sosa et al.
2012). Unlike plants, cytokinesis in fission yeast is similar to
that in animal cells, in which a contractile ring forms at the
cleavage furrow to mediate cell partitioning. It would be interesting to test if the Arabidopsis AP1M2 could functionally replace the fission yeast homolog, as this would provide clues
regarding the functional conservation of AP-1 between plants
and yeast. The current view holds that cytokinesis in plants and
yeast/animals is represented by two distinct models in which
the trafficking mechanisms are diversified for adaptive functions in the respective systems (Prekeris and Gould 2008).
Our results demonstrate that the highly conserved AP-1 is directly involved in the two fundamentally different cytokinesis
mechanisms. More importantly, the common role of AP-1 in
mediating plant and yeast/animal cytokinesis provides an evolutionary link between the systems.
Materials and Methods
Plant material
The T-DNA insertion line FLAG293_C11 was acquired from the
ABRC collection. Arabidopsis thaliana ecotypes Columbia and
Wassilewskija were used as the wild type in this study.
Immunoprecipitation and mass spectrometry
analysis
Immunoprecipitates of AP1M2–GFP were extracted from
6-day-old AP1M2–GFP-expressing seedlings using mMACS epitope tag protein isolation kits (Miltenyi Biotec). Mass spectrometry to identify the AP1M2–GFP immunoprecipitates
was performed as described previously (Tamura et al. 2010).
Immunolocalization
Five-day-old seedlings were fixed and immunolabeled as previously described (Chow et al. 2008). The primary antibody rabbit
anti-KN was used at 1 : 2,000; the secondary antibodies goat
anti-rabbit IgG Alexa Fluor 488 and goat anti-rabbit IgG Alexa
Fluor 405 were used at 1 : 400.
Chemical treatment
To visualize BFA compartments, the AP1M2–GFP-expressing
seedlings were stained with 5 mM FM4-64 for 5 min, followed
by 1 h incubation in 50 mM BFA at room temperature before
proceeding to confocal laser scanning microscopy (CLSM). For
concanamycin A treatment, seedlings were incubated in 2 mM
concanamycin A for 30 min. To measure endocytosis kinetics,
wild-type AP1M2-1+/+ and ap1m2-1 seedlings were stained for
2 min, and samples were immediately imaged with CLSM by
capturing images at 5 min intervals.
Confocal microscopy, SEM and TEM analysis
Confocal images were acquired using a Zeiss LSM780. For scanning electron microscopy (SEM) fixation, seedlings were incubated in fixation solution [6.8% (v/v) 37% formalin, 2.5% (v/v)
acetic acid, 45% (v/v) ethanol] overnight at room temperature.
Fixed seedlings were subjected to serial dehydration in
50 (twice), 60, 70, 80, 90, 99.5 and 100% ethanol for 20 min
for each step, followed by an overnight dehydration in 100%
ethanol. Finally, the seedlings were coated in gold using a JEOL
Fine Coater (model JFC-1200) and imaged with a table-top
microscope Miniscope TM-1000 (Hitachi). TEM analysis was
conducted by the Tokai Electron Microscopy service using
chemical fixation.
Molecular cloning
The AP1M2–GFP construct was synthesized as follows. A genomic fragment (including the promoter region) was amplified
Plant Cell Physiol. 54(6): 838–847 (2013) doi:10.1093/pcp/pct048 ! The Author 2013.
845
O.-K. Teh et al.
using primers At1g60780-F and At1g60780-R, sequenced, and
expressed as a GFP fusion protein from the binary vector
pGWB4. To construct mRFP–Ara7, the coding sequence of
Ara7 was amplified using primers Ara7-F and Ara7-R and
cloned into pENTR/D-TOPO, sequenced and subcloned into
pGWB655. For semi-quantitative RT–PCR of AP1M2, primers
RT m1AF0 and At106780-R were used. For genotyping of endogenous AP1M2 and T-DNA insertion, the primer pairs LP2+R
and LB4+R were used, respectively. See Supplementary Table
S2 for primer DNA sequences.
Supplementary data
Supplementary data are available at PCP online.
Funding
The Japan Society for the Promotion of Science (JSPS) [Specially
Promoted Research of Grant-in-Aid for Scientific Research to
I.H-.N. (No. 22000014)]; JSPS [O-K.T. is a postdoctoral fellow
under the JSPS Science Foreign Researcher Program (No. 2200088); JSPS [a research fellowship (No. 24005453) to M.S.].
Acknowledgments
We thank Gerd Jürgens (University of Tübingen, Germany) for
the kind donation of anti-KN, and Inhwan Hwang (Pohang
University, Korea) for discussion and data exchange.
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