Download SB401, a pollen-specific protein from Solanum berthaultii

The Plant Journal (2007) 51, 406–418
doi: 10.1111/j.1365-313X.2007.03153.x
SB401, a pollen-specific protein from Solanum berthaultii,
binds to and bundles microtubules and F-actin
Shuli Huang1,†, Lifeng Jin1,†, Jizhou Du1, Hua Li1, Qian Zhao2, Guangshuo Ou1, Guangming Ao2 and Ming Yuan1,*
1
State Key Laboratory of Plant Physiology and Biochemistry, Department of Plant Sciences, College of Biological Sciences,
China Agricultural University, Beijing 100094, and
2
State Key Laboratory of AgroBiotechnology, Department of Biochemistry and Molecular Biology, College of Biological
Sciences, China Agricultural University, Beijing 100094, China
Received 24 November 2006; revised 10 March 2007; accepted 23 March 2007.
*For correspondence (fax +8610 62733491; e-mail [email protected]).
†
These authors contributed equally to this study and are considered joint first authors.
Summary
We characterize a novel, pollen-specific, microtubule-associated protein, SB401, found in Solanum berthaultii.
This protein binds to and bundles taxol-stabilized microtubules and enhances tubulin polymerization in a
concentration-dependent manner, particularly at lower temperatures. Electron microscopy revealed that the
protein decorates the entire length of microtubules. Cross-linking and electrophoresis studies showed that
SB401 protein forms dimers, and suggest that dimerization could account for bundling. Double immunofluorescent staining of pollen tubes of S. berthaultii showed that SB401 protein co-localized with cortical
microtubule bundles. SB401 protein also binds to and bundles actin filaments, and could connect actin
filaments to microtubules. SB401 protein had a much higher affinity for microtubules than for actin filaments.
In the presence of both cytoskeletal elements, the protein preferentially bound microtubules to form bundles.
These results demonstrate that SB401 protein may have important roles in organizing the cytoskeleton in
pollen tubes.
Keywords: microtubule-associated protein, microtubules, actin, pollen tubes, Solanum berthaultii.
Introduction
Microtubule-associated proteins (MAPs) regulate the
dynamics and organization of microtubules (MTs). Recently,
an increasing number of MAPs or MT-related proteins have
been identified in plant cells. Some of these proteins types
have homologues in animal cells, whereas others are unique
to plants (Hussey et al., 2002; Lloyd and Chan, 2004; Lloyd
and Hussey, 2001; Sedbrook, 2004; Wasteneys, 2000). For
example, the plant MAP, MOR1 (microtubule organization 1)
is a homologue of Xenopus MAP215 and has an important
role in cortical MT organization (Whittington et al., 2001).
The cross-linking MAP65 has been identified in carrot,
tobacco and Arabidopsis (Chan et al., 1999; Jiang and
Sonobe, 1993; Smertenko et al., 2000, 2004), and is homologous to the spindle mid-zone proteins Ase1p, found in
yeast (Schuyler et al., 2003), and PRC1, found in human cells
(Mollinari et al., 2002). BY-2 cells contain a 190 kDa protein
that binds to both MTs and actin filaments, which suggests
that it might play a role in the interaction between these two
406
components of the cytoskeleton (Igarashi et al., 2000). A
katanin-like protein, which has the ability to sever MTs, has
been identified in Arabidopsis and alters the oriented
deposition of cellulose microfibrils (Burk and Ye, 2002). A
90 kDa phospholipase D from tobacco BY-2 binds to MTs
and the plasma membrane (Gardiner et al., 2001), and triggers the re-orientation of cortical MTs when activated
(Dhonukshe et al., 2003). In addition to these investigations
on the identity and function of MAPs, studies on the identity
and function of kinesin-related microtubule motor proteins
(KRPs) have also been performed in plant cells (reviewed by
Lee and Liu, 2004). Collectively, these studies on MAPs have
increased our understanding of the cellular functions of
MAPs and MT-related proteins in plants.
Pollen tube growth is essential for reproduction in higher
plants. Through tip growth, the pollen tube grows towards
the ovules and delivers the male germ unit to the embryo sac
for fertilization. Accordingly, pollen tube growth is a good
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd
SB401 binds to and bundles microtubules and F-actin 407
model system for investigating the control and regulation of
cell growth in plant. The cytoskeleton is crucial for the tip
growth of pollen tubes. A large body of evidence has
established that actin filaments are fundamental, not only
for delivering substances to the pollen tube tip by cytoplasmic streaming, but also because they are directly involved in
tip growth in response to environmental cues (reviewed by
Geitmann and Emons, 2000; Vidali and Hepler, 2001).
Although the precise functions of MTs in pollen tube growth
have yet to be elucidated, research has established that MTs
do participate in pollen tip growth. For example, treatment
with MT inhibitors partially blocks gymnosperm pollen
germination and growth of pollen tubes, which results in
an abnormal morphology and cytoplasmic architecture
(Anderhag et al., 2000). Treating pollen tubes of the tobacco
plant (Nicotiana sylvestris) with MT inhibitors results in
attenuated movement of the vegetative nucleus and the
generative cell from the pollen grain into the pollen tube,
and disruption of the cellular polarity that is normally
maintained by numerous cytoplasmic components (Åström
et al., 1995; Joos et al., 1994). In addition, MTs form unique
arrays and have special functions, such as mediating the
migration of the nucleus to the generative pole during pollen
development (Zonia et al., 1999). Although it is widely
recognized that the movement of organelles in pollen tube
cells involves actin, there is evidence that KRPs also
participate in this process (Romagnoli et al., 2003). Moreover, MTs may have an important role in the guidance of tip
growth, as seen in the tip growth of root hair cells of
Arabidopsis (Bibikova et al., 1999; Ketelaar et al., 2003).
Therefore, microtubules are probably involved in the tip
growth of pollen tubes, but little is known about the
dynamics and organization of MTs in this process. Furthermore, a pollen-specific MAP has not yet been identified.
The SB401 protein was first identified in 1997 by the
Thompson group from a cDNA library of in vitro-germinated
pollen of the diploid potato species, Solanum berthaultii (Liu
et al., 1997). SB401 belongs to the ‘late’ gene group of
pollen-expressed genes. It is exclusively expressed in
anthers: SB401 mRNA was not detected before the midbinucleate stage or in other vegetative tissues (Liu et al.,
1997). It is expressed in the late stage of pollen maturation,
throughout pollen germination and is enriched in extracts of
mature pollen grains and in vitro-germinated pollen. Most
interestingly, this protein contains six imperfectly repeated
motifs of the sequence V-V-E-K-K-N/E-E, which resembles a
repetitive domain responsible for MT binding of the microtubule-associated protein, MAP1B, found in murine cells
(Noble et al., 1989). However, preliminary experiments
showed that SB401 was not associated with MTs (Liu et al.,
1997), and so further investigation was necessary to establish whether SB401 is a MAP.
Here, we report our investigations into the properties of
SB401. These studies demonstrate that SB401 can indeed
bind to MTs and causes their bundling in vitro. Double
staining of MTs and SB401 showed that SB401 is associated
with cortical MTs in the cortex of pollen tubes. In addition,
SB401 also binds to and bundles actin filaments. Hence,
SB401 may play a role in the regulation of MT organization,
and function as a link between the actin and MT cytoskeleton.
Results
Purification of the recombinant SB401 protein
A construct of the cDNA responsible for encoding SB401
protein was created using pET-30a(+) vectors, and the
recombinant SB401 protein was purified (see Experimental
procedures). Figure 1 shows the mass of recombinant
SB401 protein determined using polyacrylamide gels with
various concentrations of SDS. The predicted molecular
mass of SB401 protein is 30.137 kDa. On 10% SDS–polyacrylamide gels, the molecular mass of SB401 is 50 kDa,
whereas it is 60 kDa on 8% SDS–polyacrylamide gels. Liu
et al. (1997) have described and discussed the mobility of
SB401 protein on SDS–polyacrylamide gels.
SB401 protein binds to MTs and enhances tubulin
polymerization
Co-sedimentation experiments were performed to determine whether the SB401 protein binds to MTs. After
incubation with taxol-stabilized MTs, SB401 protein was
co-sedimented with MTs by centrifugation. SB401 protein
1
2
3
4
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66
43
31
30
Figure 1. Coomassie blue-stained gel of expressed and purified recombinant
SB401 protein.
Lane 1, total extract from bacterial cells (10 lg); lane 2, total extract from
bacterial cells for SB401 protein without IPTG induction (10 lg); lane 3, total
extract from IPTG-induced bacterial cells for SB401 protein expression
(20 lg); lane 4, purified SB401 after Ni-NTA agarose purification (3 lg). The
purity of SB401 protein after purification was estimated to be about 95%,
according to gel scanning.
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Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 406–418
408 Shuli Huang et al.
(a)
(b)
Figure 2. Recombinant SB401 protein binds to taxol-stabilized MTs.
Pre-formed taxol-stabilized MTs polymerized from 2 lM tubulin were incubated with various concentrations of SB401, as shown.
(a) After centrifugation, the supernatants (S) and pellets (P) were analysed by
8% SDS–PAGE.
(b) Binding to MTs was saturated at a stoichiometry of 0.4 mol SB401 per mol
of tubulin dimer, estimated by gel scanning.
was not detected in the pellets in the absence of MTs (Figure 2a). The quantity of SB401 protein in the pellets
increased when a higher concentration of MT SB401 protein
was added. Saturation was reached when the concentration
of added SB401 reached 6 lM (Figure 2b). The binding ratio
between MTs and SB401 protein was 1:0.4 after measuring
the concentration of tubulin dimers and SB401 in the pellets
at saturation level (Figure 2b). This result indicates that
recombinant SB401 protein binds to taxol-stabilized MTs
in vitro.
To test whether SB401 protein has any effect on tubulin
polymerization, we performed turbidimetric assays. Figure 3
shows typical processes of tubulin polymerization in the
presence of various concentrations of SB401 protein at
various temperatures. At equilibrium, both the rate of
tubulin polymerization and the quantity of MTs were
increased significantly by adding SB401 protein. These
effects occurred in a concentration-dependent manner.
When tubulin polymerization was performed at 35, 30 and
20C, the enhancement effect on tubulin polymerization
after adding SB401 protein was more pronounced at low
temperatures (Figure 3). Tubulin usually did not polymerize
at 20C, but some polymerization did occur after adding
SB401 protein (Figure 3).
SB401 protein bundles MTs in vitro
To further investigate the effect of SB401 on MTs, we used
confocal microscopy to examine the effect of adding SB401
protein to rhodamine-conjugated MTs. In the presence of
Figure 3. SB401 protein enhances tubulin polymerization.
Tubulin (50 lM) was polymerized at 20, 30 or 35C in the presence of varying
concentrations of SB401 protein, and changes in the turbidity of the tubulin
suspension were monitored over 30 min. The polymerization rate and
quantity of MTs at equilibrium both increased significantly in the presence
of SB401 protein, especially at lower temperatures.
SB401 protein, MTs became organized into densely packed
bundles in a concentration-dependent manner (Figure 4).
When SB401 protein was absent, MTs were scattered individually throughout the solution (Figure 4a). After adding
1 lM SB401 protein, short and thin MT bundles appeared
(Figure 4b). When 2 lM SB401 protein were added, the MT
bundles became densely packed and formed disconnected
aggregates that ultimately meshed into a large meshwork
composed of long and wavy MT bundles (Figure 4c). The
addition of denatured SB401 protein, prepared by boiling
the native protein for 1 min, had no effect on MT bundling
(Figure 4d).
Although the formation of MT bundles proceeded rapidly
at high temperatures, the MT-bundling effect of SB401
protein was quite conspicuous even at low temperatures. At
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Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 406–418
SB401 binds to and bundles microtubules and F-actin 409
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
Figure 4. SB401 protein bundles MTs.
Samples containing 0.5 lM pre-formed taxol-stabilized rhodamine-conjugated MTs were incubated with varying concentrations of SB401 proteins at room
temperature for 5 min, and then observed under a confocal microscope.
(a) Single-filament MTs are scattered throughout the solution and no MT bundles are seen.
(b) Long, thick MT bundles appeared when 1 lM SB401 protein was added.
(c) After adding 2 lM SB401 protein, the MT bundles bunched further to form a wavy MT meshwork.
(d) No MT bundles were observed if the SB401 protein was denatured before adding it to the suspension. Loosening of MT bundles occurred when NaCl was added.
(e) MT bundles formed as the experiment in (c).
(f) The MT meshwork separated into small bunches of MT bundles when incubated with 50 mM NaCl.
(g) The bunches of MT bundles separated into thinner MT bundles when incubated with 100 mM NaCl.
(h) All MTs appeared as single MT filaments after adding 200 mM NaCl. Bar in (h) = 10 lM, and applies to (a–h).
(i and j) Electron micrographs of negatively stained samples taken from the experiment depicted in (c). (i) MTs in the absence of SB401 protein. (j) MTs in the
presence of 2 lM SB401 protein showing the tight bundles. Bar in (i) = 100 nm, and applies to (i) and (j).
(k) High-powered electron micrograph of negatively stained sections showing dot-like structures along the whole length of MTs in the bundles. Bar = 50 nm.
(l) Electron micrograph of a thin-section sample from the experiment depicted in (c), showing the distance between MTs in the bundles, estimated at approximately
6 nm. Bar = 50 nm.
0 or 4C, the taxol-stabilized MTs formed bundles about
10 min after the addition of SB401 protein. At 37C, bundles
formed almost immediately after addition of SB401 protein.
Once formed, the MT bundles persisted for as long as 24 h at
room temperature.
To investigate further the process of MT bundling, we
added increasing concentrations of NaCl to detach SB401
protein from the MTs (Figure 4). Before adding NaCl, MT
bundles were pre-formed using 2 lM SB401 protein (Figure 4e). Addition of 50 mM NaCl reduced the mass of MT
bundles. The large aggregated MT bundles lost their wavy
shape and were transformed into bundles in which the MTs
tended to be straight (Figure 4f). Further increases in the
NaCl concentration resulted in loosening of MT bundles.
Approximately half of the MTs remained in bundles after
addition of 100 mM NaCl (Figure 4g). When the NaCl
concentration was increased to 200 mM, the bundles broke
down, and apparently single, scattered MTs were observed
(Figure 4h).
Next, we examined the structure of SB401 protein–MT
bundles using electron microscopy. In the presence of
SB401 protein, the MTs were tightly bunched along their
whole length (Figure 4i,j). On the electron micrographs,
negatively stained dots were observed between the individual MT in the bundles (Figure 4k), and the distance between
them was about 6 nm (Figure 4l).
SB401 protein forms polymers
In order to answer the question of whether SB401 protein
formed dimers, we conducted cross-linking experiments and
gel analysis using native protein treated with EDC [1-ethyl-3(3-dimethylamino-propyl) carbodiimide]. After incubating
SB401 protein with EDC, the product was run on an SDS–
PAGE gel. Three bands appeared (Figure 5a, lane 2), whereas
only one band was present if the protein was not exposed to
EDC (Figure 5a, lane 1). To confirm this observation, we also
performed acrylamide gel analysis using native SB401
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 406–418
410 Shuli Huang et al.
(a)
SB401 protein co-localizes with MTs in pollen tubes
(b)
232
212
140
170
67
43
116
76
53
1
2
Figure 5. SB401 protein forms dimers.
SB401 protein dimers were analysed by EDC cross-linking experiments and
native acrylamide gels.
(a) SB401 protein was cross-linked by EDC and separated on a 10% SDS–PAGE
gel. Three major bands appeared on the gel (lane 2), while only one band was
present if the protein was not cross-linked by EDC (lane 1).
(b) Native SB401 protein was run on a native acrylamide gel and three major
bands were detected.
protein, and an identical result to that obtained using SDS–
PAGE was obtained. Three protein bands appeared on the
gel at 30, 60 and 120 kDa (Figure 5b). We concluded from
these experiments that SB401 protein can form 60 kDa
dimers in solution. The detection of a 120 kDa band suggests
that the protein may be also capable of forming tetramers.
(a)
To examine the association of SB401 protein with MTs, we
raised an SB401 protein antibody and observed the localization of SB401 protein in the pollen tubes of S. berthaultii
by double staining SB401 protein and MTs. Western blotting
established that the antibody was specific for SB401 protein
isolated from the protein extracts of S. berthaultii pollen
(Figure 6a). Under the confocal microscope, we observed
large bundles of cortical MTs all along the shank of the
pollen tube, but they were fragmented at the apical region
(Figure 6b). SB401 protein was distributed as dot-like structures throughout the cytoplasm and cortex (Figure 6c).
SB401 protein was found in the cell cortex and co-localized
mostly with large MT bundles (Figure 6d). Following treatment of the pollen tubes with 1.5 lM propyzamide and disassembly of the MTs, the SB401-labelled structures
dispersed (Figure 6e,f). Staining with pre-immune
serum showed no detectable signals in pollen tube cells of
S. berthaultii (Figure 6h–j), which confirmed the specificity
of the SB401 protein antibody.
SB401 protein binds to and bundles F-actin
The co-sedimentation experiments established that SB401
protein binds not only to MTs but also to actin filaments.
When increasing concentrations of SB401 protein were
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
Figure 6. SB401 co-localizes with MTs in Solanum berthaultii pollen tubes.
(a) Western analysis showed that the antibody specifically recognized SB401 from the protein extracts of S. berthaultii pollen tubes.
(b–g) Double staining of MTs and SB401 in S. berthaultii pollen tubes by confocal immunofluorescence microscopy was performed. (b) Cortical MTs formed large
bundles all along the shank of the pollen tube. (c) Dot-like structures, presumed to be SB401 protein, are visible in the cytoplasm and cortex. (d) Overlay image of (b)
and (c) showing that the SB401 protein (dots) at the cell cortex of the pollen tube shank were mostly co-localized with large MT bundles. (e) MTs after
depolymerization using propyzamide. (f) SB401 protein-labelled elements became more dispersed when MT aggregates were disassembled. (g) Overlay image of
(e) and (f).
(h–j) Staining with pre-immune serum showed no detectable signals in S. berthaultii pollen tubes. (h) Cortical MTs stained with tubulin antibody. (i) No signal was
detected with Pre-immune serum to stain SB401. (j) Overlay image of (h) and (i).
Bar in (j) = 5 lM, and applies to (b–j).
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 406–418
SB401 binds to and bundles microtubules and F-actin 411
(a)
(a)
(b)
(c)
(d)
(e)
(f)
(b)
Figure 7. Recombinant SB401 protein binds to F-actin.
F-actin was pre-formed in a 2 lM actin solution, and incubated with 0–8 lM
SB401.
(a) After centrifugation, the supernatants (S) and pellets (P) were analysed by
SDS–PAGE.
(b) Binding to F-actin was saturated at a stoichiometry of 0.22 mol SB401 per
mol of F-actin, estimated by gel scanning.
incubated with F-actin, the pellets following centrifugation
became enriched with SB401 protein in a concentrationdependent manner. The binding ratio of monomeric
actin:SB401 protein was approximately 1:0.22 at the saturation concentration (Figure 7). The presence and absence
of SB401 protein did not affect the extent of actin polymerization. When observed under a confocal microscope, the
actin filaments bundled together in a fashion similar to the
formation of MT bundles when SB401 protein was present
(Figure 8). Pre-formed single actin filaments remained
scattered throughout the suspension if SB401 protein was
not added (Figure 8a). However, actin bundles appeared
when 1 lM SB401 protein was added (Figure 8b). In the
presence of 3 lM SB401 protein, more actin bundles were
formed and these bundles aggregated (Figure 8c). It took
much longer for the actin filaments than the MTs to form
bundles (30 min versus 5 min). The addition of 200 mM NaCl
to detach SB401 protein from the actin filaments also
resulted in disassembly of the F-actin bundles (Figure 8d).
When observed using electron microscopy, actin bundles
were formed in the presence of SB401 protein (Figure 8e,f).
The distance between actin filaments in the bundles was
approximately 6 nm, the same distance as between MTs in
the SB401 protein–MT bundles.
SB401 protein preferentially bundles single-filament MT
compared to single-filament actin
In view of the results demonstrating that SB401 protein can
bind to both MTs and actin filaments, we considered
Figure 8. SB401 protein bundles F-actin.
Pre-formed F-actin (10 lM) was labelled with Alexa-488 phalloidin. Various
concentrations of SB401 to 0.5 lM actin were then added, and the mixture was
incubated for 60 min before observation. Confocal microscopic images of
F-actin (a) in the absence of SB401 protein, (b) after adding 1 lM SB401
protein, (c) after adding 2 lM SB401 protein and (d) after adding 200 mM NaCl
to the suspension containing 2 lM SB401 protein. The F-actin bundles were
dispersed into single actin filaments. Bar in (d) = 10 lM, and applies to (a–d).
(e and f) Electron micrographs of negatively stained samples of the
experiments depicted in (a) and (c). (e) Actin filaments in the absence of
SB401 protein. (f) Actin bundles with SB401. The distance between actin
filaments in the bundles is approximately 6 nm. Bar in (f) = 50 nm, and
applies to (e) and (f).
whether it could function as a connector between these two
cytoskeletal components. To address this issue, we performed several experiments involving 0.5 lM taxol-stabilized rhodamine-conjugated MTs and 0.5 lM F-actin
polymerized with 100 nM Alexa-488 phalloidin. In the absence of SB401 protein, F-actin remained as single filaments
(Figure 9a). When 0.25 lM SB401 protein was added to the
suspension, actin filaments were induced to form bundles
(Figure 9b). After the actin bundles formed, pre-formed
taxol–MTs were then added to the suspension. The MTs
became bundled within 5 min after their addition to the
suspension, with the bundles being mostly separate from
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Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 406–418
412 Shuli Huang et al.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
Figure 9. SB401 protein binds preferentially to MTs and connects MTs to actin filaments.
(a) Fluorescence image of 0.5 lM pre-formed F-actin (labelled with Alexa-488 phalloidin) in the absence of SB401 protein.
(b) The solution in (a) after adding 0.25 lM SB401 protein, showing the formation of actin bundles after 60 min.
(c) MTs formed bundles within 5 min of adding pre-formed 0.5 lM taxol MTs to the solution in (b).
(d) After 60 min incubation of the solution in (c), more MT bundles formed, and actin bundles were loosened into single actin filaments.
(e) Conversely, 0.5 lM taxol-stabilized rhodamine-conjugated MTs presented a single-filament pattern in the absence of SB401 protein.
(f) The solution in (e) after addition of 0.25 lM SB401 protein, showing bundling of MTs.
(g) MTs remained in bundles 5 min after adding pre-formed 0.5 lM F-actin to the solution in (f).
(h) After 60 min incubation of the solution in (g), MTs remained in bundles and no actin bundles formed.
(i) In the absence of SB401, actin filaments and taxol MTs (0.5 lM each) were scattered randomly throughout the suspension.
(j) MTs formed bundles 5 min after adding 1 lM SB401 protein to the solution in (i).
(k) As a result, actin bundles formed after 60 min and co-localized with MT bundles.
(l) MTs and actin filaments appeared as single filaments after adding SB401 protein antibody to the suspension in (k) and incubating for 30 min.
Bar in (l) = 10 lM, and applies to (a–l).
the actin bundles (Figure 9c). After 60 min, more MT bundles were formed, and the actin bundles separated into
single actin filaments (Figure 9d). When pre-formed singlefilament taxol–MTs (Figure 9e) were bundled by the addition
of 0.25 lM SB401 protein (Figure 9f), and then F-actin was
added to the suspension (Figure 9g), the MT bundles remained intact and actin bundles were not observed after
60 min (Figure 9h). This observation indicates that SB401
protein preferentially binds to MTs when it is not present in
sufficient quantities to cause bundling of single-filament
MTs and actin.
In a series of additional experiments, we first mixed
0.5 lM pre-formed MTs and 0.5 lM actin filaments in the
absence of SB401 protein. Both MTs and actin filaments
remained as distinct single filaments, scattered randomly
throughout the suspension. Co-localization of MTs and actin
filaments was not observed in the double-stained preparation (Figure 9i). After the addition of 1 lM SB401 protein, a
concentration four times greater than that used in the
previously described co-localization experiments involving
MT and actin, MT bundles were formed before the formation
of actin bundles (Figure 9j). This result confirms our previous finding that the time taken for actin bundles to form in
the presence of SB401 protein is longer than that needed for
MT bundles to form. With time, actin bundles began to colocalize with MT bundles (Figure 9k), which suggests that
actin bundles and MT bundles might be connected by SB401
protein. When SB401 antibody was added to the connected
MT and actin bundles in the presence of SB401 protein, the
bundles broke down (Figure 9l). For this breakdown to have
occurred, it is likely that a dynamic exchange took place
between the free SB401 protein in the suspension and the
bound SB401 protein on the MTs and actin filaments.
Accordingly, we suggest that SB401 protein plays an active
role in binding MTs to actin filaments.
Discussion
A previous report showed that the glutamic acid-rich protein, SB401, contains repeated motifs of the sequence V-V-EK-K-N/E-E (Liu et al., 1997), with the K-K-N/E-E core motif
resembling the MT-binding domain of murine MAP1B
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 406–418
SB401 binds to and bundles microtubules and F-actin 413
(Noble et al., 1989). This basic MT-binding region has no
structural relationship with the MT-binding domains of
other classes of MAP, such as kinesin, MAP2 or tau (Noble
et al., 1989).
A BLAST search showed that proteins containing this
imperfect repeated motif of sequence V-V-E-K-K-N/E-E are
present in a variety of plant species, including ST901 from
Solanum tuberosum cv. Desiree (accession number
AAS17876), a pollen-specific lysine-rich protein SBgLR
from S. tuberosum (accession number AAR29265, Lang
et al., 2004) and TSB from Lycopersicon esculentum
(accession number AAM53961, Zhao et al., 2004). A BLAST
search for sequences in the Arabidopsis genome identified
also a gene (At5g44610), located on chromosome 5, that
encodes a protein with unknown function and that contains
repeated V-E-E-K-K motifs. It would be of interest to
research the interaction of these proteins with MTs and
actin in the future. In addition, a recent report demonstrated that significant homology exists between the autophagic protein AtAtg8 and the microtubule binding, light
chain 3 of MAP1A and B (Ketelaar et al., 2004). Comparison
of these proteins with SB401 shows that ST901 from
S. tuberosum cv. Desiree has 73% homology, SBgLR
from S. tuberosum has 71% homology, and TSB from
L. esculentum has 66% homology. However, the two MTbinding proteins described in Arabidopsis do not exhibit
any homology with the proteins found in Solanum species.
Therefore, although the repeated motif is found in proteins
of several plant species, the SB401 protein appears to be a
genus-specific protein.
SB401 protein binds to both MTs and actin filaments
Preliminary experiments by Liu et al. (1997) indicated that
SB401 protein does not bind to MTs. Nevertheless, our
present study demonstrated that SB401 protein binds to
MTs. Several reasons may explain these different experimental results. Liu et al. (1997) reported that recombinant
SB401 protein formed inclusion bodies and therefore may
be inactive. In addition, the SB401 protein used in their
experiments was a truncated protein, starting at amino acid
23 and finishing at the C-terminal end. Whether this molecule possesses MT-binding activity has yet to be determined.
Our experiments using cross-linked proteins and native
gels suggest that SB401 protein forms dimers to bundle
MTs. The protein AtMAP65-1 from Arabidopsis can also
form dimers to link MTs, with the MT-binding site at its Cterminal end and bundling activity at its N-terminus
(Smertenko et al., 2004).
Various lengths for the inter-MT bridges induced by
MAPs have been reported. The proteins of the MAP65
family are capable of forming inter-MT cross bridges of 25–
30 nm in length (Chan et al., 1999; Smertenko et al., 2004).
This distance contrasts with the smaller cross-bridge
distance of 6 nm that we observed when SB401 protein
was used. The 65 kDa MAP isolated from tobacco BY-2
cells induces cross bridges of 10–12 nm length between
adjacent MTs (Jiang and Sonobe, 1993). The cross bridges
in the MT bundles induced by purified tobacco 65 kDa MAP
and in the cycled cortical MTs are 2–3 nm long, which is
much shorter than the cross bridges of 30–35 nm length
observed in isolated plasma membrane vesicles and isolated cortical MTs (Sonobe et al., 2001). The results of our
experiments also showed that the addition of SB401
protein induces the formation of wavy MTs. This formation
of wavy MT bundles by SB401 protein contrasts with the
action of other plant MAPs. For example, proteins of the
MAP65 family usually characteristically induce the formation of large and straight MT bundles (Chan et al., 1999;
Mao et al., 2005; Smertenko et al., 2004; Wicker-Planquart
et al., 2004). We conjecture that the variation of inter-MT
spacing in MT bundles might be related to the organization
of MTs.
Our studies also indicate that SB401 protein, similar to
MAP1B, binds to and bundles actin filaments, although the
protein binds preferentially to MTs. The KKEE motif, which
appears in both proteins, is also present in the F-actin binding
domain of villin (Friederich et al., 1992). It would be of interest
to establish whether these repeated motifs account for the
binding of SB401 protein to filaments of MTs and actin. If so,
this may well explain the nature of the competitive binding of
MTs and F-actin for SB401 protein. In the presence of MTs and
actin filaments, the SB401 protein binds preferentially to MTs
and binding to actin does not occur if free or excess SB401
protein is not available.
SB401 protein may play a role in mediating interaction
between microtubules and membrane organelles in pollen
tubes
We have shown here that the SB401 protein may be
associated with organelles in pollen tube cells. The decoration of large MT bundles in the shank, and shorter,
possibly fragmenting, MTs at the tip, are consistent with
the guidance or movement of materials to the tip. The
globular pattern of their decoration has also been observed with other plant MAPs. For example, the AtMAP656 isoform is associated with mitochondria and is seen as a
dot-like structure in a suspension of Arabidopsis cells (Mao
et al., 2005). Two proteins, with apparent Mr of 161 and
90 kDa and found in tobacco pollen tubes, bind to MTs and
are associated with the plasma membrane compartment
(Cai et al., 2005). In the present study, we observed dot-like
structures localized with cortical MTs. We propose that
these dots represent the punctate attachment of organelles
to the microtubular cytoskeleton. It is likely that SB401
protein targets specific organelles to MTs, and functions as
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 406–418
414 Shuli Huang et al.
a transport conduit or a platform for a subsequent reaction.
Does SB401 protein play a role in coordination of the
microtubule and actin cytoskeletons in pollen growth?
SB401 protein is specifically expressed in the late stage of
pollen maturation and throughout pollen germination (Liu
et al., 1997). Therefore, we wished to examine the protein’s
role in this process. The actin cytoskeleton is of fundamental
importance for pollen tube growth. Most of the actin filaments form bundles orientated longitudinally in cortical and
endoplasmic regions of the pollen tube cells, and are
organized into a reverse fountain pattern for cytoplasmic
streaming. In addition to the actin cytoskeleton, many MTs
are oriented longitudinally and sometimes adopt a slight
helical distribution in these cells (Geitmann and Emons,
2000; Hepler et al., 2001). Because treatment of pollen tubes
cells with an MT depolymerization agent has little effect on
germination or elongation of angiosperm pollen, MTs have
attracted relatively little attention regarding their role in
pollen tube growth. However, recent studies have shed new
light on their function in tip growth (Sieberer et al., 2005).
Apart from maintaining polarity in pollen tubes, movement
of the male germ unit and vesicle transport in pollen tubes,
MTs may also play a role in determining the direction of tip
growth. Depolymerization or stabilization of MTs resulted in
the formation of wavy root hairs (Bibikova et al., 1999).
Furthermore, the orientation of polarized growth of Arabidopsis root hairs depends on an intact MT cytoskeleton
(Ketelaar et al., 2003).
If MTs participate in directing tip growth and actin
filaments are involved cell elongation, how are these two
cytoskeletal activities coordinated? An analogous model for
studying how the pollen tube is directed in the style and
ovary is the study of cell movement in animals, especially
neuronal directing (Lord and Russell, 2002). MAP1B is
specifically expressed during neural development and has
a crucial role in axon formation and growth by stabilizing
MTs (Gordon-Weeks and Fischer, 2000; Mack et al., 2000).
Actin-based motility is utilized to produce persistent and
directed MT advance, steering the growth cone and guiding
axonal growth (Dickson, 2003).
In plant root hairs, increasing the instability of F-actin
results in broadening of the tip expansion area, whereas
depolymerization of MTs alters the orientation of polarized
tip growth (Ketelaar et al., 2003). This reinforces the idea that
both systems are required for tip growth. Moreover, this idea
is consistent with the emerging view that plant cells may use
MTs for polar-axis determination, and microfilaments for the
targeted delivery of components necessary for growth
(Mathur and Hülskamp, 2002; Sieberer et al., 2005). As a
protein expressed during tip growth, SB401 is likely to affect
both the actin and MT cytoskeletons. Therefore, we hypo-
thesize that it might serve to coordinate the function of the
two structures. There is increasing evidence that the actin
and MT cytoskeletons interact with each other in cells to fulfil
their functions. For example, the inter-digitating growth of
Arabidopsis pavement cells is controlled by two opposing
pathways that depend on the activity of Rho in plants (ROP)
GTPase. The ROP–RIC4 pathway promotes the assembly of
cortical microfilaments required for localized outgrowth, and
the ROP–RIC1 pathway promotes the organization of cortical
MTs locally to inhibit outgrowth of the cell (Fu et al., 2005).
Proteins interacting with both MTs and actin filaments are
also found in plants, such as the 190 kDa protein identified
from tobacco BY-2 cells (Igarashi et al., 2000), elongation
factor EF1-a (Durso and Cyr, 1994; Gungabissoon et al., 2001;
Yang et al., 1990) and plant-specific kinesin GhKCH1 (Preuss
et al., 2004). Our study demonstrated that SB401 protein also
binds to both MTs and actin filaments.
Although SB401 protein binds to actin filaments, our
results show that it binds preferentially to MTs rather than to
actin filaments when both of these cytoskeletal components
are present. Two putative casein kinase phosphorylation
sites within two units of the repeat motifs suggest that
phosphorylation may be crucial in the regulation of SB401
protein activity (Liu et al., 1997). Pedrotti and Islam (1996)
reported that dephosphorylated, but not phosphorylated,
MAP1B binds to microfilaments. Therefore, it is reasonable
to assume that phosphorylation could modulate the binding
of SB401 protein to MTs and actin filaments. In addition, a
recent study showed that the MT-bundling activity of a MAP,
NtMAP65-1, may be regulated by a mitogen-activated protein kinase in tobacco cells (Sasabe et al., 2006). The
interaction between AtMAP65-1 and MTs is also regulated
by phosphorylation and dephosphorylation of the protein
(Smertenko et al., 2006). Therefore, future experiments
could be aimed at exploring the role of phosphorylation of
SB401 protein in its MT-bundling activity.
Experimental procedures
Preparation of recombinant SB401 protein and its antibody
The cDNA sequence is available from EMBL Nucleotide Sequence
Database GenBank (accession number X95984). The GSB plasmid
containing the SB401 gene was kindly provided by Dr Junqi Liu at
China Agricultural University. The cDNA sequence encoding SB401
protein was amplified by PCR using two primers: forward 5¢GGAGGATCCATGGGTTGTGGGGAATC-3¢ (BamHI site underlined)
and reverse 5¢-GGGAAGCTTTAAAAGCAAGGATTTAA-3¢ (HindIII
site underlined). The full-length cDNA for the SB401 gene was
reconstructed into pET-30a(+) vector.
The recombinant protein with a six-His tag on the N-terminus was
expressed in Escherichia coli strain BL21 (DE3). Bacterial cultures
were grown in LB (Luria–Bertani) broth medium supplemented with
50 mg l)1 kanamycin at 37C until the culture reached an optical
density of 0.4–0.6 measured spectrophotometrically at 600 nm. The
induction of protein expression was performed by adding 1 mM
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 406–418
SB401 binds to and bundles microtubules and F-actin 415
ively, in PEMT buffer. After 30 min incubation at room temperature,
the samples were centrifuged at 25 000 g for 30 min at 25C. The
pellet was washed twice with PEMT, and resuspended with one
volume of SDS loading buffer (v/v). The samples were applied onto
8% SDS–PAGE gels. The gels were stained with Coomassie brilliant
blue R250. The amount of SB401 protein bound to MTs was determined by gel scanning with an Alpha 2200 (Alpha; http://
www.alphametals.com).
For the F-actin and SB401 protein co-sedimentation assay, rabbit
muscle actin and SB401 protein were centrifuged at 100 000 g for
60 min at 4C before use. Then, 2 lM F-actin and 0–8 lM SB401 were
incubated in a 100 ll volume of PEM buffer. After 30 min incubation
at room temperature, samples were centrifuged at 100 000 g for
60 min at room temperature. The protein concentration was measured three times using the method described above.
isopropylthio-b-galactoside, and allowed to progress for 4 h. The
bacteria were pelleted at 6000 g for 10 min at 4C, resuspended in a
protein extraction buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl,
20 mM imidazole) and sonicated. The bacterial lysates were centrifuged at 30 000 g for 30 min at 4C, and applied to a Ni-NTA agarose
resin column (Qiagen; http://www.qiagen.com/). The column was
washed sequentially with protein extraction buffers containing 20,
50 and 100 mM imidazole. The specifically bound proteins were
eluted with 250 mM imidazole, and proteins were dialysed overnight against PEM buffer (0.1 M PIPES, 1 mM EGTA, 1 mM MgSO4,
pH 6.9). The protein concentration was determined using a Bio-Rad
protein assay kit (http://www.bio-rad.com/), with BSA as a standard.
Protein samples were analysed by SDS–PAGE and visualized by
staining the gels with Coomassie brilliant blue R250 (Sigma-Aldrich;
http://www.sigmaaldrich.com/). Purified SB401 protein was stored
at )80C. The protein was centrifuged at 60 000 g (Beckman
Allegra 64R, http://www.beckmancoulter.com/) for 30 min at 4C
immediately before use.
Purified SB401 protein was used to elicit polyclonal antisera
production in rabbits. Blot affinity-purified antibodies were
prepared using the method described by Mao et al. (2005). To
further test whether the antibody was specific for SB401 protein,
protein extracts from S. berthaultii pollen tubes were prepared
according to the method described by Liu et al. (1997). The
protein sample was separated on 10% SDS–PAGE gels and
transferred to nitrocellulose membrane. The blots were probed
with purified SB401 protein antibody and pre-immune serum (as
control) diluted 1:500 with TBST (50 mM Tris, 150 mM NaCl,
0.05% Tween-20, pH 7.5).
To monitor the time course of tubulin polymerization, varying
concentrations (0, 0.2, 0.4 or 0.8 lM) of SB401 protein were added to
50 lM tubulin in PEM buffer containing 1 mM GTP. Microtubule
polymerization was monitored spectrophotometrically at OD350 in a
0.4 cm-wide quartz cuvette at 20, 30 and 35C for 30 min using a
DU-640 spectrophotometer (Beckman Coulter, http://www.
beckmancoulter.com) in a temperature-controlled compartment.
The polymerization experiments were repeated at least three times
at each temperature and with the various SB401 protein concentrations.
Preparation and polymerization of tubulin and actin
Microtubule and F-actin bundling assays
Porcine brain tubulin was purified according to the method
described by Castoldi and Popov (2003). NHS-rhodamine (5-(and 6-)
carboxytetramethyl-rhodamine succinmidylester) tubulin was prepared according to the method described by Keating et al. (1997).
Rabbit muscle actin was purified according to the method described
by Spudich and Watt (1971).
To polymerize the tubulin into MTs, NHS-rhodamine-labelled
tubulin was centrifuged at 30 000 g for 30 min at 4C before
polymerization. NHS-rhodamine-labelled tubulin was diluted with
PEM buffer (0.1 M PIPES, 1 mM EGTA, 1 mM MgSO4, pH 6.9)
containing 1 mM GTP (guanosine 5¢-triphosphate sodium salt
hydrate; Sigma) to a concentration of 40 lM, and incubated at
35C for 30 min. Two volumes (v/v) of PEMT (0.1 M Pipes, 1 mM
EGTA, 1 mM MgSO4, 20 mM taxol, pH 6.9) were added to the
solution, which was incubated at 35C for 15 min and centrifuged at
25 000 g at 25C for 10 min. The pellets were washed with PEMT
and resuspended gently with PEMT to give a final concentration of
10 lM tubulin.
Actin was centrifuged at 100 000 g for 30 min at 4C before
polymerization. Then, 10 lM actin in polymerization buffer (50 mM
KCl, 1 mM MgCl2, 1 mM EGTA, 0.2 mM ATP, 0.2 mM CaCl2, 0.5 mM
DTT, 3 mM NaN3, 10 mM imidazole, pH 7.0) was polymerized at 25C
for 30 min with 100 nM Alexa-488 phalloidin (Molecular Probes,
http://www.probes.com/).
Taxol-stabilized MTs (0.5 lM) were incubated with 0, 0.5, 1, 2, 3, 4 or
5 lM SB401 protein in PEM buffer at room temperature in a 100 ll
volume, and fixed with 1% glutaraldehyde after various incubation
periods (5, 30 or 60 min). Aliquots (1 ll) of the samples were placed
onto a slide and observed using a confocal microscope (Meta 510,
Zeiss; http://www.zeiss.com/) with a Zeiss 100· magnification oil
objective (NA 1.3).
F-actin (0.5 lM) was incubated with 0, 0.5, 1, 2, 3, 4 or 5 lM SB401
protein at room temperature for 5, 30 or 60 min, then fixed with 1%
glutaraldehyde. Aliquots (1 ll) of the samples were placed onto a
slide and observed using a confocal microscope. The results were
compared to those obtained using SB401 protein that had been
denatured by boiling for 1 min before adding to the incubating
solution.
Bundles of taxol-stabilized MTs were formed by adding 2 lM
SB401. NaCl was then added so that the final NaCl concentrations
were 50, 100, 150, 200 or 250 mM. The samples were incubated for
10 min and then observed under the confocal microscope.
For the electron microscopy studies, MTs or actin filaments were
observed either by negative staining or thin sectioning. Negative
staining was performed using saturated uranyl acetate. To prepare
the thin section, MTs were collected after centrifugation at 50 000 g
for 30 min at 25C. The pellets were fixed in 1% v/v glutaraldehyde in
PEM buffer followed by 1% w/v osmium tetroxide, and then
embedded in LR White acrylin resin. Samples were then sectioned,
and stained with uranyl acetate and lead citrate. The sections were
observed under a Hitachi 7500 electron microscope.
To observe the linkage of MTs and F-actin by SB401, 0.5 lM taxolstabilized MTs were mixed with 0.5 lM pre-formed actin filaments
polymerized with 100 nM Alexa-488 phalloidin, 1 lM SB401 protein
was added, and the reaction was fixed in 1% glutaraldehyde after 5
or 60 min incubation. Aliquots (1 ll) of the suspension were placed
Microtubule and F-actin co-sedimentation assays
Porcine brain tubulin and SB401 protein were centrifuged at
60 000 g at 4C for 30 min before use. The binding reaction was
performed in a 100 ll volume containing 2 lM pre-formed taxolstabilized MTs and 0, 0.5, 1, 2, 4, 6 or 8 lM SB401 protein, respect-
Microtubule polymerization assay
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 406–418
416 Shuli Huang et al.
on a slide and observed under the confocal microscope (Hitachi;
http://www.hitachi.com).
acquired using a Zeiss confocal microscope with a Zeiss 100· oil
objective (NA 1.4).
SB401 dimer assays
Acknowledgements
To assess the extent of dimerization of SB401 protein, cross-linking experiments were performed using EDC [1-ethyl-3-(3-dimethylamino-propyl) carbodiimide; Sigma] (Doi et al., 1987). EDC was
added to PEM buffer containing 16 lM SB401 protein to give a
final concentration of 4 mM. The sample was then incubated at
room temperature for 1 h. The cross-linking reaction was stopped
by adding SDS–PAGE loading buffer and then boiling for 5 min.
The proteins were separated on a 10% gel, running at 180 V for
120 min.
The acrylamide gel analysis was performed as described by
Daniel et al. (1996). Native SB401 protein and proteins of known
molecular weight (ovalbumin, 43 kDa; albumin, 67 kDa; lactate
dehydrogenase, 140 kDa; catalase, 232 kDa; ferritin, 440 kDa; thyroglobulin, 669 kDa) were electrophoresed on 6–12% native acrylamide gel. Protein mobility (Rf) was calculated by dividing the
distance of protein migration by the distance of migration of the dye
front. Plotting the log (100 · Rf) against the acrylamide concentration creates a line whose slope defines the molecular weight of the
protein. The slopes generated from the experiments show a linear
relationship with the molecular weight. The molecular mass of
SB401 protein and its oligomers were extrapolated from the
molecular weight standards.
The authors thank Professor Clive Lloyd at the John Innes Centre,
Norwich, UK, for critical reading, editing and comments on the
manuscript, and Dr Bo Liu at the University of California at Davis for
helpful discussions. Dr Junqi Liu at China Agricultural University
generously provided the plasmid containing the cDNA of SB401.
This research was supported by grants from the National Key Basic
Research Project of China (2006CB100101) and the National Natural
Science Foundation of China (30421002, 30370707 and 30570925) to
M.Y.
Localization of SB401 protein, microtubules and F-actin in
pollen tubes
Pollen of S. berthaultii was collected from the potato fields in
Zhangjiako, Hebei Province, China, and stored at )20C. The
pollen was germinated in germination medium (20-30 mg pollen
grains/ml) (H3BO3, 0.01% KNO3, 0.02% MgSO4Æ7H2O, 0.07%
Ca(NO3)2, 2% sucrose, 15% PEG-6000) at 25C for 1 h in the dark
(modified from a method described by Liu et al., 1997). The pollen tubes were fixed with 4% formaldehyde in 2% sucrose, 15%
PEG-6000, 50 mM PEM buffer, pH 6.9, for 45 min. The fixed
cells were treated with 1% w/v cellulase (Sigma, http://www.
sigmaaldrich.com/Local/SA-Splash.html) and 1% w/v pectolyase
(Fluka; http://www.sigmaaldrich.com/Brands/Fluka_Riedel_Home.
html) in PBS for 15 min. SB401 protein was probed with rabbit
SB401 antibody diluted 1:100 in PBS containing 3% w/v BSA
(Sigma) and 0.1% w/v Tween-20 at 4C overnight. The samples
were washed three times with PBS before adding mouse antia-tubulin (Sigma) diluted 1:500 in PBS containing 3% w/v BSA
and 0.1% w/v Tween-20, and incubated at 4C overnight. Secondary antibodies were Alexa-488-conjugated donkey antirabbit IgG
(Molecular Probes) and tetramethylrhodamine-5(and 6-)-isothiocyanate (TRITC)-conjugated goat antimouse IgG (Sigma), both
diluted 1:500 in PBS containing 3% w/v BSA and 0.1% w/v Tween20. The samples were incubated at 37C for 2 h, washed four
times with PBS, and mounted in 10 mM Tris, pH 9.2, 50% glycerol, 1 mg ml)1 o-phenylenediamine (Sigma) on slides. Staining
with pre-immune serum was used as a control.
To verify that the SB401 protein labelling was associated with
MTs, the treatment to disrupt MTs was performed before the
observation of SB401 protein. Pollen from S. berthaultii was
germinated in germination buffer containing 1.5 lM propyzamide
(Pestanal, 3,5-dichloro-N-(1,1-dimethyl-2-propynyl) benzamide;
Fluka) for 1.5 h. Pollen tubes were collected for the double immunofluorescence experiments as described above. The images were
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