Purple acid phosphatase in the walls of tobacco cells

Article abstract of DOI:10.1016/j.phytochem.2008.07.008

Purple acid phosphatase isolated from the walls of tobacco cells appears to be a 220 kDa homotetramer composed of 60 kDa subunits, which is purple in color and which contains iron as its only metal ion. Although the phosphatase did not require dithiothreitol for activity and was not inhibited by phenylarsine oxide, the enzyme showed a higher catalytic efficiency (k_cat/K_m) for phosphotyrosine-containing peptides than for other substrates including p-nitrophenyl-phosphate and ATP. The phosphatase formed as a 120 kDa dimer in the cytoplasm and as a 220 kDa tetramer in the walls, where Brefeldin A blocked its secretion during wall regeneration. According to our double-immunofluorescence labeling results, the enzyme might be translocated through the Golgi apparatus to the walls at the interphase and to the cell plate during cytokinesis.

Full text of DOI:10.1016/j.phytochem.2008.07.008

Phytochemistry 69 (2008) 2546–2551
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Purple acid phosphatase in the walls of tobacco cells
a
Rumi Kaida a,b, Takahisa Hayashi b,c, , Takako S. Kaneko
*
a Department of Chemical and Biological Sciences, Japan Women’s University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan
b Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
c Institute of Sustainability Science, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 January 2008
Received in revised form 10 July 2008
Available online 31 August 2008
Purple acid phosphatase isolated from the walls of tobacco cells appears to be a 220 kDa homotetramer
composed of 60 kDa subunits, which is purple in color and which contains iron as its only metal ion.
Although the phosphatase did not require dithiothreitol for activity and was not inhibited by phenylar-
sine oxide, the enzyme showed a higher catalytic efficiency (kcat/Km) for phosphotyrosine-containing
peptides than for other substrates including p-nitrophenyl-phosphate and ATP. The phosphatase formed
as a 120 kDa dimer in the cytoplasm and as a 220 kDa tetramer in the walls, where Brefeldin A blocked its
secretion during wall regeneration. According to our double-immunofluorescence labeling results, the
enzyme might be translocated through the Golgi apparatus to the walls at the interphase and to the cell
plate during cytokinesis.
Keywords:
Nicotiana tabacum
Solanaceae
Tobacco
Purple acid phosphatase
Metallophosphatase
Wall
Ó 2008 Elsevier Ltd. All rights reserved.
Golgi apparatus
JIM 84
Brefeldin A
Translocation
1. Introduction
Despite the wealth of structural information about PAPs, there
have only been a few reports to date about their function because
Purple acid phosphatases (PAPs) are distinguished from other
acid phosphatases by their purple color, which is due to a tyrosine
to Fe(III) charge transfer transition (Antanaitis et al., 1983), and by
their insensitivity to tartrate inhibition (Doi et al., 1988). The first
plant PAP to be described was an enzyme from the red kidney
bean, a homodimeric glycoprotein that has a molecular mass of
approximately 110 kDa and contains a Fe(III)–Zn(II) metal center
in each subunit (Sträter et al., 1992; Suerbaum et al., 1993; Stahl
et al., 1994). The X-ray crystal structure of the enzyme showed
the Fe(III)–Zn(II) metal center coordinated by three histidines,
two aspartates, a tyrosine, and an asparagine (Sträter et al., 1995;
Klabunde et al., 1996). The structural analysis indicated a striking
similarity to both protein phosphatase 1 (Goldberg et al., 1995;
Egloff et al., 1995) and protein phosphatase 2B (calcineurin) (Grif-
fith et al., 1995; Kissinger et al., 1995) in the coordination environ-
ments of the active sites; the major difference is the replacement of
the tyrosinate ligand to a coordinated water molecule in the pro-
tein phosphatases (Funhoff et al., 2001).
their substrate has not yet been determined. Mammalian PAPs ex-
hibit a broad and non-specific activity towards phosphoproteins
(Ljusberg et al., 1999; Hayman et al., 1989). It has been proposed
that they are involved in iron transport (Nuttleman and Roberts,
1990), the generation of reactive oxygen species (Sibille et al.,
1987) and bone resorption (Ek-Rylander et al., 1994), among other
processes. We have shown that wall-bound PAP was involved in
wall regeneration in the protoplasts prepared from tobacco cells
(Kaida et al., 2003; Sano et al., 2003). Its main function, however,
is believed to be the release of inorganic phosphates as phosphorus
nutrients from phosphate esters in the soil as well as in the plant
(Cashikar et al., 1997; del Pozo et al., 1999; Miller et al., 2001; Boz-
zo et al., 2004; Zimmermann et al., 2004; Veljanovski et al., 2006;
Xiao et al., 2006).
We previously reported that purified PAP from tobacco cell
walls had a mass of 60 kDa with an additional smear band of
120 kDa (Kaneko et al., 1998); this measurement was made using
the reducing SDS–PAGE technique, in which the SDS sample buffer
contained b-mercaptoethanol. In this paper we report that tobacco
wall-bound PAP forms a tetramer of 220 kDa on SDS–PAGE in the
absence of a reducing agent. Furthermore, we explain the proper-
ties of PAP isolated from the walls of cultured tobacco cells, its sub-
strate specificity, and its translocation to the apoplastic spaces.
* Corresponding author. Address: Research Institute for Sustainable Humano-
sphere, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan. Tel./fax: +81 774 38
3618.
E-mail address: taka@rish.kyoto-u.ac.jp (T. Hayashi).
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.07.008

R. Kaida et al. / Phytochemistry 69 (2008) 2546–2551
2547
form a disulfide bridge is present at residue 379. One possibility
is that the four subunits of the phosphatase are non-covalently
bound to each other to form the tetrameric structure. It is known
that the acid phosphatase from yellow lupin forms a dimer of 50
and 44 kDa subunits without S–S linkage (Olczak et al., 1997).
The characteristic purple color of the purified enzyme could be
attributed to a charge transfer transition from a tyrosine, poten-
tially Tyr 201, bound to Fe(III) in the binuclear center (Antanaitis
et al., 1983). Our X-ray microanalysis system, fitted with a vari-
2. Results and discussion
2.1. Purification and characterization of purple acid phosphatase
Ionically bound cell wall PAP was extracted from a wall prepa-
ration of suspension-cultured tobacco cells by stirring with 0.7 M
NaCl, and then purified according to the purification procedure
we have described elsewhere (Kaneko et al., 1998), with modifica-
tions. The acid phosphatase activity was eluted from a hydroxyap-
atite column. First and second butyl-Toyopearl columns were then
subjected to gel filtration on Superdex 200. Table 1 summarizes the
purification of the wall-bound PAP including gel filtration.
The enzyme was purified 213-fold until it reached a final spe-
cific activity of 494 U mgÀ1. The ratio of 0.7 M NaCl-soluble wall-
bound proteins to the total number of proteins in the cells (includ-
ing wall proteins, cytoplasmic proteins, and proteins secreted to
the culture medium) was about 0.5%, and the level of purification
compared to the initial concentration was approximately 4000-
fold. The highly purified enzyme was purple, and we confirmed it
as NtPAP12 (Kaida et al., 2003) since its N-terminal sequence
(TVDMPLDSDVFRAPPGYNA) was identical to the N-terminal se-
quence of the deduced amino acid sequence in NtPAP12 by analy-
sis using DNAMAN (Lynnon Biosoft, Quebec, Canada).
able-pressure scanning electron microscope, detected Fe K
a emis-
sion as well as K emissions of C and O, constituent elements of
a
biological materials (Fig. 2). The enzyme might contain a mixed-
valent diiron unit in its catalytic active form, as is the case in mam-
malian phosphatases (Antanaitis et al., 1983) and sweet potato
(Waratrujiwong et al., 2006).
2.2. Enzymatic properties
As we show in Table 2, tobacco wall-bound PAP has a similar le-
vel of affinity with p-nitrophenyl-phosphate, ATP, phosphotyro-
sine, and phosphoserine, although phosphotyrosine had a higher
kcat/Km value for the phosphatase than did phosphoserine, by a fac-
tor of 2.3. The phosphatase had a higher affinity and catalytic effi-
ciency for two tyrosine phosphopeptides, ENDpYINASL (Daum
et al., 1993) and DADEpYLIPQQG (Zhang et al., 1993), than for
the serine/threonine phosphopeptide RRApTVA (Pinna and Ruz-
zene, 1996), unlike both ATP and p-nitrophenyl-phosphate. Gella-
tly et al. (1994) found a potato tuber acid phosphatase having a
significant phosphotyrosine phosphatase activity. In addition, a
few papers have reported findings indicating that PAPs could cata-
lyze the hydrolysis of phosphotyrosine in tomato (Bozzo et al.,
2002) and Arabidopsis (Veljanovski et al., 2006). Nevertheless, sev-
eral phosphoproteins occur in the walls in Arabidopsis (Kwon et al.,
2005).
In SDS–PAGE, the enzyme appeared as a single polypeptide
band with a molecular mass of 220 kDa under non-reducing condi-
tions (Fig. 1). Since only one band corresponding to a molecular
size of 60 kDa was detected in the presence of dithiothreitol
(DTT), the native subunits of the enzyme could exist as
a
220 kDa homotetramer composed of 60 kDa subunits. The phos-
phatase activity was not diminished in the presence of DTT. One
question that remains is how the polypeptide subunits (60 kDa)
form a tetramer (220 kDa), because, according to the deduced ami-
no acid sequence of NtPAP12, only one cysteine residue that can
Tobacco wall-bound PAP was insensitive to tartrate inhibition,
qualifying it as a tartrate-resistant acid phosphatase (Doi et al.,
1988) (Table 3). The activity of the phosphatase was significantly
Table 1
Purification of purple acid phosphatase from the walls of tobacco cells
Purification
step
Total
protein
(mg)
Total
activity
(U)
Specific
activity
(U mgÀ1
Purification Recovery
)
(fold)
(%)
13,000
cps
Crude extract
Hydroxyapatite
1st Toyopearl-
butyl
100
18.8
0.81
232.1
146.2
69.6
2.32
7.78
85.9
1
3.4
37.0
100
63
30
C
O
2nd Toyopearl-
butyl
Superdex 200
0.39
0.08
51.1
39.5
131
494
56.5
213
22
17
Fe
The activity was determined by using p-nitrophenyl phosphate as a substrate.
Mn
Zn
DTT
−
marker
keV
5
10
+
0
Fig. 2. X-ray spectrum of tobacco purple acid phosphatase in variable-pressure
scanning electron microscopy.
250 kDa
160 kDa
105 kDa
75 kDa
220 kDa
60 kDa
Table 2
Substrate specificity of tobacco purple acid phosphatase
Substrate
Km (mM)
kcat (sÀ1
)
kcat/Km (mMÀ1 sÀ1
)
50 kDa
35 kDa
p-Nitrophenyl phosphate
ATP
IDP
Phosphotyrosine
END(pY)INASL
DADE(pY)LIPQQG
Phosphoserine
RRA(pT)VA
0.33
0.40
1.45
0.41
0.03
0.02
0.40
0.22
125.0
133.2
60.4
62.4
32.8
26.7
26.8
2.0
379
333
42
152
1093
1335
67
30 kDa
Fig. 1. SDS–PAGE of tobacco purple acid phosphatase. The purified enzyme (1 lg)
9
was subjected to 10% SDS–PAGE in the absence (À) or presence (+) of 2 mM
The activity was determined as the amount of phosphate released.
dithiothreitol (DTT) and stained with silver.

2548
R. Kaida et al. / Phytochemistry 69 (2008) 2546–2551
Table 3
tide in the walls during wall regeneration (Fig. 3). This showed that
the tetramer could be formed from phosphatase subunits in the
walls. During the regeneration of cell walls in tobacco protoplasts,
PAP increased in the walls during cultivation while the level of pro-
tein in the cytoplasm remained constant. Upon the addition of Bre-
Effect of various substances on the activity of purple acid phosphatase
Additive
Concentration (mM)
Relative activitya (%)
None
100
63
24
3
21
7
73
99
47
98
98
93
94
98
97
85
98
103
93
Vanadate
Vanadate
Vanadate
Molybdate
Molybdate
NaF
Tartrate
ZnCl2
Okadaic acid
Microcystin LR
Phenylarsine oxide
CaCl2
MgCl2
FeCl2
FeCl3
MnCl2
EDTA
Dithiothreitol
0.01
0.1
1
0.01
0.1
2
2
2
0.01
0.01
2
5
5
5
5
5
2
2
feldin A (10 lg/ml), PAP in the walls was found to have decreased
after 30 min of cultivation. This observation showed that Brefeldin
A might block the secretion of PAP in protoplasts during wall
regeneration under conditions of high turnover for the phospha-
tase. This is in agreement with our previous observation showing
the inhibitory effect of Brefeldin A on the secretion of acid phos-
phatase activity in protoplasts regenerating their cell walls, in
which Brefeldin A caused the formation of stacks of hybrid ER-Gol-
gi membranes in tobacco protoplasts (Kaneko et al., 1996).
A double-immunofluorescence labeling experiment was per-
formed using rabbit anti-wall-bound purple acid phosphatase
antibody and JIM 84 for Golgi (Knox et al., 1989; Horsley et al.,
1993). The labeling pattern of the phosphatase was identical to
that of JIM 84, showing the localization of PAP in the Golgi appa-
ratus at the interphase of cells during the cell cycle (Fig. 4A). PAP
was also observed to localize in the Golgi at their metaphase be-
fore cell plate formation (Fig. 4B). These results indicate that pur-
ple acid phosphatase might be translocated through the Golgi
apparatus to the walls at the interphase and to the cell plate
during cytokinesis. We have concluded that wall-bound PAP oc-
curs as a 120 kDa dimer polypeptide in the Golgi, and that this di-
a
The activities represent the mean of three assays carried out for each sample.
inhibited by Zn2+, molybdate, and orthovanadate, but it was not
inhibited by phenylarsine oxide. Orthovanadate inhibits all protein
tyrosine phosphatases at concentrations between 10 and 100 lM,
while molybdate, phenylarsine oxide, and Zn2+ differentially inhi-
bit protein tyrosine phosphatases (Walton and Dixon, 1993). Oka-
daic acid and microcystin-LR, which inhibit protein phosphatases
1, 2A, and 2B (Takai et al., 1987; MacKintosh et al., 1990), had no
effect on the activity of wall-bound PAP; neither did Ca2+ or
Mg2+, which are required for the activity of protein phosphatase
2B and protein phosphatase 2 C, respectively (Cohen, 1989). The
activity of wall-bound PAP, which is sensitive to vanadate, insensi-
tive to okadaic acid, and does not require Ca2+ and Mg2+, seems to
be similar to that of protein tyrosine phosphatases (Luan, 2003).
Purple acid phosphatase is not classified among the protein
phosphatases (protein tyrosine phosphatase and protein serine/
threonine phosphatase), although the structures of the coordina-
tion environments of the active sites of PAPs are similar to those
of serine/threonine phosphatases. In our study, however, wall-
bound PAP showed a higher catalytic efficiency (kcat/Km) for phos-
photyrosine-containing peptides than for other substrates includ-
ing p-nitrophenyl-phosphate and ATP. Therefore, it is possible
that phosphotyrosine protein could be the substrate for wall-
bound PAP.
mer forms
translocation.
a 220 kDa tetramer in the walls during wall
3. Experimental methods
3.1. Culture of tobacco cells
The tobacco cell line XD-6 derived from Nicotiana tabacum L.
var. Xanthi was cultured in Murashige and Skoog medium
(40 ml) containing 3% sucrose and 2,4-dichlorophenoxyacetic acid
(1 mg LÀ1) in a 100-ml flask at 25 °C in the dark.
Five or 6-day-old cells in the logarithmic phase of growth were
used for all experiments.
3.2. Purification of wall-bound PAP from tobacco cells
Wall-bound PAP was purified according to the purification pro-
cedure described by Kaneko et al. (1998), with modifications. Sus-
pension-cultured tobacco cells (500 g) were homogenized in
50 mM HEPES-KOH (pH 7.0) buffer using a Teflon-glass homoge-
nizer at 1500 rpm for 20 min. The insoluble wall materials were
2.3. Translocation of PAP into the walls
collected on nylon-mesh (pore size, 50 lm) and washed, not only
5 times with 10 mM HEPES–KOH buffer (pH 7.0) by centrifugation
at 5000 Â g for 20 min, but also twice with 10 mM HEPES–KOH (pH
Western blot analysis showed that the phosphatase occurred as
a 120 kDa polypeptide in the cytoplasm and as a 220 kDa polypep-
Control
30 60 90 120
+Brefeldin A
30 60 90 120 min
0
0
220 kDa
wall
120 kDa
220 kDa
120 kDa
cytoplasm
Fig. 3. Immunoblot analyses of the wall and cytoplasmic fractions extracted from protoplasts regenerating walls. The protoplasts were cultured in the medium for wall
regeneration in the absence (control) or presence (+Brefeldin A) of Brefeldin A and harvested at the indicated times after the onset of culture. Five micrograms of total protein
were applied to SDS–PAGE under non-reducing conditions followed by immunoblot analyses.

R. Kaida et al. / Phytochemistry 69 (2008) 2546–2551
2549
Purple acid phosphatase
JIM 84
Merge
Fig. 4. Confocal immunofluorescence micrographs of purple acid phosphatase and Golgi apparatus in the interphase (A) and metaphase (B) of tobacco cells during the cell
cycle. Purple acid phosphatase and Golgi apparatus were stained with anti-wall-bound purple acid phosphatase antibody and JIM 84, respectively. Bar = 10 m.
l
7.0) containing 0.2 M NaCl. The wall-bound phosphatase was ex-
tracted with the same buffer containing 0.7 M NaCl. The extract
was concentrated by precipitation with (NH4)2SO4 at 65% satura-
tion. Following centrifugation at 14,000 Â g for 30 min, the pellet
was dissolved in 10 mM Tris–HCl (pH 7.0) and dialyzed against
strate, according to the method described by Van Veldhoven and
Mannaerts (1987). Each assay contained substrate and 50 mM so-
dium acetate buffer (pH 5.8) in a total volume of 0.1 ml. Reactions
were stopped at 3 min by the addition of Molybdate Dye/Additive
mixture (Promega, Madison, WI, USA). Controls were carried out
using the background levels of phosphate present at each substrate
concentration. One unit of activity is defined as the amount of en-
the same buffer. The sample was loaded onto
a column
(20 Â 250 mm) of hydroxyapatite (Seikagaku, Tokyo, Japan) pre-
equilibrated with 250 mM Tris–HCl (pH 7.0). The column was
washed with 200 ml of 250 mM Tris–HCl (pH 7.0), and then acid
phosphatase activity was eluted at a flow rate of 0.2 ml/min with
150 ml of 3 M Tris–HCl (pH 7.0). The fractions (30 ml) containing
activity were pooled, concentrated with (NH4)2SO4 at 65% satura-
tion, and dialyzed against 10 mM Tris–HCl (pH 7.0) containing
30% saturated (NH4)2SO4. The sample was applied on a column
(20 Â 220 mm) of Toyopearl-butyl (Tosoh, Tokyo, Japan) pre-equil-
ibrated with 10 mM Tris–HCl (pH 7.0) saturated 30% (NH4)2SO4.
Stepwise elution was performed with the buffer by decreasing
the concentration of ammonium sulfate (200 ml of 30%, 100 ml
of 20%, and 100 ml of 0%). Acid phosphatase activity was eluted
at a flow rate of 0.2 ml/min with 20% (NH4)2SO4. The fractions
(15 ml) containing activity were pooled, and precipitated with
65% (NH4)2SO4. This step was carried out twice, once with the first
Toyopearl-butyl and once with the second Toyopearl-butyl. The
sample was dissolved in 1 ml of 10 mM Tris–HCl (pH 7.0) contain-
ing 1 M NaCl, and applied onto a Superdex 200 HR 10/30 column
(10 Â 300 mm) (GE Healthcare, Chalfont St. Giles, Buckingham-
shire, UK) pre-equilibrated with 10 mM Tris–HCl (pH 7.0) contain-
ing 1 M NaCl. The activity was eluted at a flow rate of 2.0 ml/min
with 100 ml of 10 mM Tris–HCl (pH 7.0) containing 1 M NaCl.
The fractions containing phosphatase activity were precipitated
with 65% (NH4)2SO4 and the precipitate was stored at À80 °C.
zyme necessary to produce 1 lmol of phosphate per 1 min at 30 °C.
Protein content was quantified according to the method described
by Bradford (1976) using bovine serum albumin as the standard.
3.4. Electrophoresis
SDS–PAGE was performed with 10% acrylamide minigel accord-
ing to the manufacturer’s instructions (Atto, Tokyo Japan). For the
determination of molecular mass, a plot of log molecular mass ver-
sus retention factor (Rf) value was constructed using the standard
proteins of molecular weight marker (GE Healthcare, USA). Reduc-
ing and non-reducing conditions corresponded to the sample treat-
ment with SDS at 80 °C for 15 min in the presence and absence of
DTT.
3.5. Sequencing of the polypeptide
After SDS–PAGE was completed, the proteins were electrotrans-
ferred to PVDF membrane. The membrane was stained with Coo-
massie Blue and the stained polypeptide was subjected to
sequencing from the N-terminus with a protein sequencer (Applied
Biosystems, 477A, Foster City, CA, USA).
3.6. Determination of metal ion
3.3. Enzyme assays
The metal ion of tobacco wall-bound PAP was analyzed using an
EDX system (Horiba, EMAX-7000, Kyoto, Japan) fitted with a vari-
able-pressure scanning electron microscope (VP-SEM) (Hitachi, S-
3500N, Tokyo, Japan). The chamber pressure was 30 Pa. The accel-
erating voltage was 15 kV.
For the purification procedure, acid phosphatase activity was
determined by the hydrolysis of p-nitrophenyl phosphate. The
reaction mixture contained an enzyme preparation, 1 mM p-nitro-
phenyl phosphate and 50 mM sodium acetate buffer (pH 5.8) in a
total volume of 0.1 ml. After incubation at 30 °C for 3 min, the reac-
tion was stopped by addition of Na2CO3. One unit was defined as
3.7. Preparation and culture of protoplasts
1
l
mol of p-nitrophenyl phosphate hydrolyzed per min. For the
The preparation and culture of the protoplasts were performed
according to the procedure described in a previous report (Kaida
et al., 2003). Cells were incubated for 40 min at 30 °C with a
determination of kinetic parameters, acid phosphatase activity
was measured as the amount of phosphate released from sub-

2550
R. Kaida et al. / Phytochemistry 69 (2008) 2546–2551
mixture of 1.5% (W/V) cellulase (Cellulase Onozuka RS, Yakult, To-
kyo, Japan), 0.05% (W/V) pectinase (pectolyase Y-23, Seishin, To-
kyo, Japan), and 0.48 M mannitol at pH 5.2 to digest the cell
walls. The protoplasts were washed by centrifugation at 800 Â g
for 2 min in 0.48 M mannitol, and then incubated in the medium
described by Nagata and Takebe (1970) at a density of 105 protop-
lasts per ml at 26 °C. BFA (Wako, Osaka, Japan) was added to a final
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The localization of the Golgi apparatus and intracellular PAP
were observed using the method described by Yoneda and Hasez-
awa (2003), with modifications. The BY-2 cells were placed onto
coverslips coated with poly-L-lysine (M.W. 70,000–150,000, SIGMA,
Croydon, Australia). After 5 min, the cells were treated with 0.25%
(w/v) cellulase (Cellulase Onozuka RS, Yakult) and 0.05% (w/v) pec-
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3.7% (w/v) formaldehyde in the PMEG solution for 1 h and treated
with 0.5% (v/v) Triton X-100 in the PMEG solution for 20 min to
give them permeability; then they were washed with phosphate-
buffered saline (PBS, 20 mM Na-phosphate, pH 7.0 and 150 mM
NaCl). Prior to immunostaining, the cells were treated with 1%
(w/v) BSA, 0.1 M glycine, and 0.05% Triton X-100 in PBS for 20 min.
For double-immunofluorescence labeling, the cells were first
incubated with JIM 84 monoclonal antibody for 4 h, washed with
PBS, and then incubated with rabbit anti-wall-bound PAP antibody
for 1 h. After a second washing with PBS, the cells were incubated
with Alexa Fluor 488 conjugated anti-mouse IgM and Alexa Fluor
568 conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) for
1 h, then washed a third time with PBS. The cells were subse-
quently embedded in an antifading reagent (SlowFade Light; Invit-
rogen) and observed under a fluorescence microscope (Olympus
BX50, Tokyo, Japan) equipped with a confocal laser scanning head
system (Leica TCSNT, Solms, Germany).
Acknowledgments
We thank Paul Knox for providing JIM 84, and Seiichiro Hasez-
awa and Arata Yoneda for their helpful advice on immunofluores-
cence staining. This work was supported by the Program for the
Promotion of Basic Research Activities for Innovative Biosciences
(PROBRAIN). This paper is also a part of the outcome of the JSPS
Global COE Program (E-04): In Search of Sustainable Humano-
sphere in Asia and Africa.

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