From the Divisions of Biochemistry and
¶ Virology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan,
§ Department of Pathology, Nagoya University School of
Medicine, Nagoya 466-8550, Japan, and the
Division of
Biological Sciences, Graduate School of Science, Nagoya
University, Nagoya 464-8602, Japan
Received for publication, May 28, 2002, and in revised form, January 31, 2003
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ABSTRACT |
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Septins are a family of conserved
proteins implicated in a variety of cellular functions such as
cytokinesis and vesicle trafficking, but their properties and
modes of action are largely unknown. Here we now report findings of
immunocytochemical and biochemical characterization of a mammalian
septin, MSF-A. Using an antibody specific for MSF subfamily proteins,
MSF-A was found to be expressed predominantly in mammary human mammary
epithelial cells (HMEC). MSF-A was associated with microtubules in
interphase HMEC cells as it localized with the mitotic spindle and the
bundle of microtubule at midzone during mitosis. Biochemical analysis
revealed direct binding of MSF-A with polymerized tubulin through its
central region containing guanine nucleotide-interactive motifs. GTPase activity, however, was not required for the association. Conditions that disrupt the microtubule network also disrupted the
MSF-A-containing filament structure, resulting in a punctate
cytoplasmic pattern. Depletion of MSF-A using small interfering RNAs
caused incomplete cell division and resulted in the accumulation of
binucleated cells. Unlike Nedd5, an MSF mutant deficient in GTPase
activity forms filament indistinguishable from that of the wild type in COS cells. These results strongly suggest that septin filaments may
interact not only with actin filaments but also with microtubule networks and that GTPase activity of MSF-A is not indispensable to
incorporation of MSF-A into septin filaments.
Septins, a family of heteropolymeric filament-forming proteins,
were originally discovered in yeast to be essential for budding, and
have since been identified in most eukaryotic organisms, with the
exception of plants (for review, see Refs. 1-6). Although septins have
25% or greater identity over their entire length, sequence similarity
is greatest in the central domain, which contains guanine nucleotide
interactive motifs homologous to those of ras-related small GTPases. In
addition to the conserved central domain, most septins have divergent
N- and C-terminal domains, some of which contain a predicted
coiled-coil region possibly involved in protein-protein interaction.
Although the ras-related small GTPases function as signal transducing
molecular switches through GTP/GDP-exchange and GTP-hydrolysis, little
is known of the physiological significance of mammalian septins.
Although septins were initially thought to play important roles in
controlling cytokinesis of budding yeast (7), it is now well
established that they are also required for localized chitin
deposition, bud site selection, cell cycle control, plasma membrane
compartmentalization, and regulation of some kinases (for review, see
Refs. 1-4). The accumulating biochemical and cell biological
observations on lower eukaryotic septins suggest that either they
comprise a novel cytoskeletal polymer or they function as scaffolds for
assembly of signaling complexes.
Numerous mammalian homologues of yeast septins have been identified
mainly based on random sequencing projects (reviewed in Ref. 6). Some
of the septins probably represent alternative splicing forms. The gene
mixed lineage leukemia septin-like fusion (MSF)1 has been identified as
a fusion partner gene of mixed lineage leukemia in a case of
therapy-related acute myeloid leukemia with a t(11,17)(q23;q25) (8, 9).
Thereafter, two alternative splicing variants, MSF-A and MSF-B, were
identified (10), and another report described the complicated
transcriptional pattern of MSF (11). MSF has been found to be deleted
in some cases of breast and ovarian cancers; hence, it was considered
to be a candidate for tumor suppressor gene (10, 12). These mutations may be associated with allelic loss of the 17q25 region (13-16). Although these findings provided insights into a possible role for MSF
in leukemogenesis and oncogenesis, not only the molecular mechanism(s)
regarding MSF function and tumorigenesis but also biochemical and
biological properties of MSF proteins remain to be elucidated. We did a
biochemical and biological characterization of MSF-A and found that
MSF-A interacts with microtubules in vitro and in
vivo. The biological significance of GTPase activity of MSF-A was
investigated and compared with that of well characterized septin, Nedd5.
Plasmid Construction--
Human MSF-A, MSF, MSF-B, Nedd5,
hSeptin2, and H5 were produced by PCR with Marathon-Ready cDNA
(human brain) (Clontech) then subcloned into
pGEX-4T3 and/or pRK5 vector containing Myc tag. The cDNA fragments
of MSF-A (aa 1-586), MSF-A-N (aa 1-283), MSF-A-Cent (aa 284-561),
and MSF-A-C (aa 562-586) were produced by PCR and subcloned into
pGEX-4T3 or pRK5 vector harboring Myc-tag. N312MSF-A, an MSF-A mutant
with a point mutation (Ser-312 to Asn) in the G1 box of the GTPase
region, was prepared using the QuikChange site-directed mutagenesis
kits (Stratagene). All constructs were verified by DNA sequencing.
Preparation and Characterization of Antibodies--
The
glutathione S-transferase-fused MSF fragment (aa
148-568) expressed in Escherichia coli served as the
antigen. A rabbit polyclonal antibody specific for MSF proteins was
produced and affinity-purified. Anti-Nedd5 antibody was kindly supplied
by Dr. M. Kinoshita (Harvard University, Cambridge, MA) (17). Western blot analysis was performed, and immunoreactive bands were visualized by making use of a horseradish peroxidase-conjugated anti-rabbit antibody and the enhanced chemiluminescence Western blotting detection system (Amersham Biosciences).
Cell Culture, Transfection, Immunofluorescence, and
Microinjection--
COS-7 and HeLa cells were grown in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum and
100 µg/ml penicillin in an air-5% CO2 atmosphere
with constant humidity. HMEC cells were maintained in RT-PCR--
Total RNA was purified from HeLa and HMEC cells by
Isogen kit (Nippon Gene Inc.). RT-PCR was performed with Ready-To-Go
RT-PCR beads (Amersham Biosciences) according to the manufacturer's
protocol. The following two primers were designed to amplify the
MSF-A-specific N-terminal cDNA segment (75 bp):
5'-ATGAAGAAGTCTTACTCAGGAGG-3' (sense) and 5'-TGGGCCACTGGAGTCACCAAGC-3' (antisense).
Expression and Purification of Recombinant Proteins--
MSF-A,
Nedd5, and their mutants were expressed in E. coli as
glutathione S-transferase fusion proteins and purified on
glutathione-Sepharose beads. The recombinant proteins were released
from the beads by cleavage with human thrombin. Protein concentration
was determined by the method of Bradford (36) and purity of the protein
preparations was confirmed on Coomassie Blue-stained SDS-polyacrylamide gels.
GTPase Activity Assay--
GTPase activity was determined as
described previously (18). Briefly, recombinant septin proteins (5 µg
of each) were incubated at 30 °C in 20 mM Tris/HCl, pH
7.5, containing 1 mM EDTA, 1 mM dithiothreitol,
0.1% lubrol, 25 mM MgCl2, 1 µM
[ Preparation of Tubulin and Microtubule-Binding
Analyses--
Pure tubulin was prepared from the bovine brain, as
described previously (19). The amount of MSF-A and mutants bound to microtubules was determined by cosedimentation assay, as described previously (20). Briefly, MSF-A or the mutants (0.25 µM)
and polymerized tubulin (5 µM) were incubated in 0.1 M PIPES buffer, pH 6.9, containing 0.5 mM
MgSO4 and 1 mM EGTA. After a 10-min incubation
at 37 °C, the samples were centrifuged at 100,000 × g for 30 min at 37 °C; then, aliquots of the supernatants
and pellets were examined using SDS-PAGE. Modifications made for
dose-response analyses; 2 µM tubulin and various amounts
of MSF-A were used.
RNA Interference (RNAi) with Small Interfering RNAs
(siRNAs)--
siRNA duplexes with the following sense and
antisense sequences were made: MSF-A, 5'-GAUCUUUUGAGGUCGAGGAGGdTdT-3'
(sense) and 5'-CCUCCUCGACCUCAAAAGAUCdTdT-3' (antisense). According to the recent report (21), siRNA duplexes with the following sense and
antisense sequences were also used as a positive control: MSFs,
5'-AUCAGCCGGAAGUCGGUGCdTdT-3' (sense) and 5'-GCACCGACUUCCGGCUGAUdTdT-3' (antisense). To control for the specificity of the knockdown, we used a
siRNA duplex, MSF-A-Mut, a single site-mutated version of the
inhibitory sequence: 5'-GAUCUUUCGAGGUCGAGGAGGdTdT-3' (sense) and
5'-CCUCCUCGACCUCGAAAGAUCdTdT-3' (antisense). siRNAs were supplied from Dharmacon Research Inc. (Lafayette, CO). Transfection was carried
out using Oligofectamine regent (Invitrogen). At 72 h after
transfection, immunofluorescence analysis with an Olympus BH2-RFCA
microscope or Western blot analysis was performed to analyze the
depletion of MSF-A.
Identification of MSF-A in HMEC Cells--
To characterize the MSF
subfamily of proteins, we first prepared a rabbit polyclonal antibody
against MSF then affinity-purified it on a column to which recombinant
MSF had been conjugated. Specificity of the antibody was confirmed with
various septin proteins overexpressed in COS-7 cells. As shown in Fig.
1A, the anti-MSF antibody
specifically recognized MSF-A, MSF, and MSF-B in Western blot analyses,
because the polypeptide used as an antigen contained a region common
among these three splicing variants. Because other septins tested were not recognized by anti-MSF, the anti-MSF antibody is specific for the
MSF subfamily of proteins. We next did a Western blot analyses to
detect endogenous MSF subfamily proteins in lysates of various
mammalian cell lines, including HeLa and HMEC cells (Fig.
1B). In the cell lysates tested, three proteins with
molecular masses of 76, 73, and 50 kDa, coincident with MSF-A, MSF, and MSF-B, respectively, were observed in different combinations. Preincubation of the antibody with recombinant MSF-A selectively inhibited the immunoreactivity (data not shown). RT-PCR analyses revealed the expression of MSF-A in HMEC cells as well as HeLa cells
(Fig. 1C). In addition, the expression level of the 76-kDa protein significantly decreased by MSF-A-RNAi (see Fig. 5A).
From these data, we concluded that the 76-, 73-, and 50-kDa proteins observed in HeLa cells are MSF-A, MSF, and MSF-B, respectively. MSF-A
was predominantly expressed in HMEC cell lysates (Fig.
1B).
MSF-A Colocalizes with Tubulin in HMEC Cells--
The mammalian
septins Nedd5 and H5 have been reported to localize along with actin
filaments (17, 22). We thus examined the subcellular localization of
MSF-A in HMEC cells, using a confocal microscope. HMEC cells were
double-stained with anti-MSF antibody and anti-tubulin antibody,
rhodamine-phalloidin, or anti-vimentin antibody. As shown in Fig.
2A, MSF-A was present in
filamentous structures. Double staining with an antibody to tubulin
revealed a significant overlap between filamentous MSF-A and
microtubules (Fig. 2, A and B). In addition,
colocalization of MSF-A with microtubule networks seemed to be
heterogenous, and the extent of overlapping differed from cell to cell,
which suggested dynamic features of interactions between MSF-A and
microtubules. In the mitotic cells, MSF-A was localized at the mitotic
spindle and the bundle of microtubules at the midzone (Fig.
2C). MSF-A was not evident at the spindle pole. We next
sought to determine whether MSF-A colocalizes with other major
cytoskeletons, actin filaments, and vimentin. Double staining showed no
apparent codistribution of MSF-A with actin (Fig. 2D) or
with vimentin filaments (Fig. 2E).
Direct Association of MSF-A with Microtubules in Vitro--
To
determine whether MSF-A directly interacts with microtubules, we
examined the in vitro association of MSF-A with
paclitaxel-stabilized microtubules, using a cosedimentation assay. We
also attempted to determine the essential domain of MSF-A for binding
with microtubules; for this, we used single point- and truncated MSF-A
mutants (Fig. 3A). In the
absence of microtubules, glutathione S-transferase-MSF-A was
not sedimented, but in the presence of microtubules, a substantial portion of glutathione S-transferase-MSF-A was precipitated
together with microtubules (Fig. 3B). We next determined
whether GTPase activity of MSF-A is important for association with
microtubules, because GTP hydrolysis is likely to play an important
role in in vivo Nedd5 filament assembly (17). We tested the
microtubule-binding activity of N312MSF-A, a GTPase activity-deficient
mutant in which Ser312 in the G1 box is changed to Asn (see Fig.
6A). Like wild-type MSF-A, N312MSF-A bound to microtubules,
thereby suggesting that the GTPase activity of MSF-A is not required
for the interaction between MSF-A and microtubules (Fig.
3C). Further analyses were done to identify the essential
region of MSF-A containing the microtubule-binding site. For this, the
binding between a series of truncated mutants of MSF-A and microtubules
was analyzed, using the cosedimentation assay (Fig. 3,
D-F). The central region containing guanine
nucleotide-interactive motifs of MSF-A was found to specifically interact with microtubules. MSF-A sedimenting with microtubules was
considered to be associated with the microtubule filaments via direct
binding. The direct interaction of MSF-A with microtubules was next
examined using a standard cosedimentation assay (Fig. 3G).
The binding of MSF-A to microtubules was saturable with an apparent
Kd of 0.1 µM.
Microtubule Network Is Essential for the Fibrous Distribution of
MSF-A in Interphase HMEC Cells--
The structural relationship
between microtubules and MSF-A-containing fibers was further examined
using reagents known to disrupt actin or microtubule bundles. When HMEC
cells were treated for 15 min with 5 µM cytochalasin B
(Sigma), a specific inhibitor of actin polymerization, actin filaments,
but not microtubules, were disrupted (Fig.
4, A-D and
F). Under these conditions, the fibrous distribution of
MSF-A was not affected (Fig. 4E). When HMEC cells were
treated for 100 min with 10 ng/ml demecolcine (Sigma), a specific
inhibitor of tubulin polymerization, microtubules were disrupted but
actin filaments were still observed (Fig. 4, G,
H, and J). Under these conditions, the fibrous
distribution of MSF-A and microtubules was disrupted (Fig. 4,
I and J). These results strongly suggest that
MSF-A interacts specifically with microtubules in HMEC cells and that
microtubule structure plays an important role in septin filament
structure containing MSF-A. On the other hand, overexpression of
N312MSF-A, a GTPase-deficient MSF-A mutant, MSF-A, or MSF-A-Cent (aa
284-561) had no effects on the microtubule structure (data not
shown).
Silencing Expression of MSF-A by RNA Interference Induces
Incomplete Cell Division in HMEC Cells--
In the next set of
experiments, we examined whether MSF-A-containing septin filaments
structure is essential for microtubule network organization. As shown
in Fig. 5A, protein level of
MSF-A drastically decreased by RNAi using MSF-A-duplex (lane
1), whereas the control siRNA duplex, MSF-A-Mut, had little effect
(lanes 2 and 6). Another duplex, tentatively
termed MSFs, the effects of which on silencing of expression of MSF
proteins have been reported in HeLa cells recently (21), was also used
as a positive control. Consequently, almost the same silencing effect
was observed (Fig. 5, lane 5). The level of GTPase Activity Is Not Required for Filament Formation of MSF-A in
COS Cells--
Because GTPase activity is noted to be essential for
the filamentous structure of Nedd5 (17), the functional relevance of GTPase activity of MSF-A was examined and compared with that of Nedd5.
As depicted in Fig 6A,
N312MSF-A and N51Nedd5, both of which were designed based on the well
characterized ras mutant with dominant-negative activity, showed a
highly reduced GTPase activity under conditions in which the wild type
of each septin has GTPase activity. The wild-type and mutant septins
were then individually overexpressed in COS-7 cells. As shown in Fig.
6B, N312MSF-A formed a filamentous structure
indistinguishable from that of the wild type, and the filament
structure was also observed when MSF-A and N312MSF-A were coexpressed
(Fig. 6C, a-c). In contrast, the introduction of
the GTPase-deficient mutation into Nedd5 significantly abolished
filament-forming activity (Fig. 6B). N51Nedd5, a
GTPase-deficient mutant, was not incorporated into the filament
composed of wild-type Nedd5, and the filament structure by wild-type
Nedd5 and aggregates composed of N51Nedd5 are independently present in
cells expressing them (Fig. 6C, d-f).
Taken together, these results suggest that guanine nucleotide-binding
and GTP-hydrolysis activities are not required for polymer formation of
MSF-A, whereas the activities are essential for polymerization of
Nedd5.
Although the GTPase activity of MSF-A is not required for its binding
with microtubules, it is possible that binding with microtubules may
affect the GTPase activity of MSF-A. We thus measured the GTPase
activity of MSF-A in the presence of various amounts of polymerized
tubulin, which has undetectable GTPase activity. Consequently, tubulin
had no effects on the GTPase activity (data not shown).
In the present study, we found that MSF-A, a member of the MSF
family and predominantly expressed in mammary HMEC cells, distributes along with microtubules in the cells. The septin filament structure containing MSF-A depends on the integrity of microtubules because microtubule disruption by demecolcine induced septin filament disruption in HMEC cells. When RNAi reduced expression of MSF-A in HMEC
cells, the cells displayed failed cell division more frequently than
control cells; consequently, 10-12% of the MSF-A-RNAi-treated cells
became binuclear. Additionally, ~14% of the cells with reduced level
of MSF-A showed lower levels of microtubules, although the physiological relevance of this phenotype remains to be elucidated. We
noted that MSF-A directly binds with polymerized tubulin in vitro through the center domain containing guanine
nucleotide-interactive motifs, although GTPase activity of MSF-A does
not seem to be required for binding to microtubules.
The function of septins in cytokinesis is likely to be conserved in
higher eukaryotic cells as well as in yeast (17, 23). Accumulating data
on mammalian septins indicate that some, such as CDCrel-1, ARTS, and
Nedd5, function in vesicle fusion processes (24, 25), apoptosis (26),
and neurodegeneration (27), although the precise molecular mechanisms
of these processes are largely unknown. Septins also seem to play
important roles in oncogenesis, because not only MSF (8, 9) but also
CDCrel-1 (28) has been identified as in-frame fusions with the mixed lineage leukemia/acute lymphocytic leukemia 1 protein. The presence of
a variety of septins in mammalian cells means that they are likely to
be involved in various as-yet-unidentified cellular processes.
Mammalian septins are thought to be related to actin filament
structures (17, 22). As for MSF-A, a recent study demonstrated that it
colocalizes with actin stress fibers as well as microtubules in HeLa
cells (21), suggesting interaction of MSF-A with actin filaments. On
the other hand, our data showed that the filamentous structure of MSF-A
in HMEC cells was conserved in cytochalasin B-treated cells. A possible
explanation is that efficient interaction of MSF-A with actin filaments
needs other molecule(s), such as other MSF subfamily of the proteins,
because HMEC cells predominantly express MSF-A, whereas HeLa cells
express MSF-A, MSF, and MSF-B equally.
It is notable that MSF-A-containing septin filaments in HMEC cells are
disrupted by demecolcine treatment and MSF-A interacts with
microtubules in vitro. The physiological significance of interactions between MSF-A and microtubules is an enigma; however, we
speculate that MSF-A plays a role in microtubule-dependent secretory pathways because 1) microtubules play important roles within
the secretory pathway in mediating the anterograde and retrograde
traffic of vesicular and tubular intermediates between ER and the Golgi
complex (for review, see Ref. 29), and 2) there is a large body of
evidence that microtubules are involved in post-Golgi protein
trafficking: microtubules are required for the efficient transport of
membrane proteins to the apical surface via a direct (30) or an
indirect transcytotic pathway (31, 32). It is also notable that MSF-A
localizes largely along the mitotic spindle in mitotic HMEC cells,
which suggests some function in mitotic processes. Although the
functional relevance of MSF-A in the mitotic process remains to be
elucidated, MSF-A may play a role in microtubule-dependent
mitotic processes and perturbation of the function might induce cell
cycle abnormalities resulting in oncogenesis.
Mammalian septins, such as Nedd5 and H5, were noted in
immunofluorescent studies to be localized along with actin stress
fibers (17, 22). Because H5 and Nedd5 are detectable in HMEC cells by
Western blot analyses,2
MSF-A, Nedd5, and H5 may function, in harmony with other septin molecules, to coordinate control of actin- and
tubulin-dependent cellular events. Another possibility is
that these septins function as filamentous scaffolds for organization
of proteins at a specific region inside the cell, because many proteins
depend on septins for localization (reviewed in Refs. 1, 3, 4). Budding yeast septins were found to function as a scaffold that allows for
assembly of multiprotein complexes required for activation of Gin4 and
Hsl kinases, which are activated and control bud growth during mitosis
(33, 34). In this context, we found that MSF interacts with mixed
lineage kinase 2 in vivo,2 which is distributed
together with activated c-Jun N-terminal kinase along microtubules
(35), meaning that septin scaffolds probably regulate mixed lineage
kinase 2-mediated c-Jun N-terminal kinase activation. The septin
complex may function as scaffolds in various cellular events that also
link actin and microtubule networks. Cytological investigations are
under way to clarify the physiological significance of the septin complex.
The results obtained by RNAi experiments are consistent with the recent
observation that MSF-A and/or the splicing variants play an important
role in cytokinesis in HeLa cells (21). In HMEC cells, MSF-A is
possibly involved in cytokinetic process cooperatively with other
septins such as Nedd5 (17) because it is a dominant MSF subtype in the
cells. In addition, a weak staining pattern of microtubules was
observed in ~14% of HMEC cells with reduced levels of MSF-A.
Although some cells with reduced levels of MSF-A became binucleated,
obvious correlation between the extent of reduction of MSF-A expression
and cytokinetic defect was not observed. Whether or not MSF-A-knockdown
affects the microtubule structure and how MSF-A plays a physiological
role in relation to microtubule functions and cytokinesis are
interesting areas for future examination. To test the role of MSF-A in
microtubule organization, anti-MSF antibody was microinjected into HMEC
cells. However, the antibody reacted with endogenous MSF-A and clearly colocalized with filamentous MSF-A; therefore, the antibody could not
induce morphological change of microtubules (data not shown). We assume
that the antibody recognized MSF-A but could not disrupt the
filamentous structure.
In the present study, we report the inter-relationship of a septin with
microtubules. Characterization of the MSF-A demonstrated here may
represent initiation of the unraveling of a septin function linking
actin and microtubule networks and various mammalian
septin-dependent key events.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimal
essential medium containing 12.5 mM HEPES, 35 µg/ml bovine pituitary extract, 12.5 ng/ml epidermis growth factor, 1 µg/ml insulin, 10 µg/ml transferrin, 10 nM
-estradiol, 3.5 µM hydrocortisone, 0.1 mM
phosphorylethanolamine, 0.1 mM ethanolamine, 1 ng/ml
cholera toxin, 2 mM L-glutamine, 50 µM ascorbic acid, 15 nM sodium selenite, and
1% calf serum. Transient transfection using COS-7 and HMEC cells was
carried out using the LipofectAMINE method (Invitrogen). For
immunofluorescence analyses, cells grown on 13-mm coverslips were fixed
in 3.7% formaldehyde in phosphate-buffered saline for 15 min, then
treated with 0.2% Triton X-100 for 5 min. To detect the MSF subfamily
of proteins, affinity-purified anti-MSF antibody was used as the
primary antibody, and Alexa 488 anti-rabbit IgG (Molecular Probes) was
used as the secondary antibody. To visualize Myc-tag, actin, vimentin,
or tubulin, cells were reacted with anti-Myc monoclonal (9E10),
rhodamine-conjugated phalloidin, anti-vimentin monoclonal antibody
(1B8), or anti-tubulin monoclonal antibody (Sigma), respectively.
Anti-Myc polyclonal antibody (Santa Cruz Biotechnology) was used where
indicated. Alexa 488-labeled anti-mouse antibody or FluoroLink
Cy3-linked anti-mouse antibody was used as a secondary antibody. When
analyzing the cells, we used an Olympus LSM-GB200 confocal microscope
(Figs. 2, 4 and 6) or an Olympus BH2-RFCA microscope (Fig. 5).
-32P]GTP (2500-3000 cpm/pmol). After incubation for
various times, the reaction was terminated by addition of ice-cold
2.5% (w/v) charcoal in 50 mM
NaH2PO4. The mixtures were incubated for 15 min
on ice and centrifuged for 15 min at 1000 × g at
4 °C. The amount of 32Pi released from
[
-32P]GTP was determined by counting the radioactivity.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of an affinity-purified
antibody for MSF proteins. A, lysates from COS-7 cells
transiently expressing Myc-tagged MSF-A, MSF, MSF-B, H5, Nedd5, CDC10,
FLJ10849, KIAA0202, and hSeptin2 were separated on a 10%
polyacrylamide gel and then subjected to Western blotting, using
anti-MSF antibody (top) or anti-Myc antibody 9E10
(bottom). B, lysates of HeLa (30 µg) and HMEC
(3 µg) cells were subjected to SDS-PAGE followed by Western blotting
using an anti-MSF antibody. C, analysis of RT-PCR products.
RT-PCR was performed in the absence ( ) or presence of the total RNA
(2 µg) from HeLa or HMEC cells. The products were separated on a 20%
acrylamide gel. The bands were visualized by UV light after ethidium
bromide staining.
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Fig. 2.
Colocalization of MSF-A and microtubules in
HMEC cells. A, HMEC cells were double-stained using an
anti-MSF antibody (green) and an anti-tubulin antibody
(red). The merged image was also shown. B,
enlarged images of the boxed area of A are shown.
C, HMEC cells during cytokinesis were double-stained
using an anti-MSF antibody and an anti-tubulin antibody as in
A. D, double-immunofluorescence analysis of MSF-A
immunoreactivity (left) and F-actin (center).
Right, merged image. E, double-immunofluorescence
analysis of MSF-A immunoreactivity (left) and vimentin
(center). Right, merged image. Scale bars, 20 µm.
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Fig. 3.
Cosedimentation of MSF-A and its mutants with
polymerized tubulin. A, schematic representation of
full-length MSF-A and its single-point and deletion mutants. Numbers
refer to amino acid positions. B, recombinant MSF-A was
incubated, with or without polymerized tubulin. Samples of supernatant
(sup) and pellet (ppt) fractions were then
separated on SDS-PAGE and stained with Coomassie Brilliant blue. The
cosedimentation analyses were also performed using N312MSF-A
(C), MSF-A-N (D), MSF-A-Cent (E), or
MSF-A-C (F) instead of wild-type MSF-A. G,
quantitative analysis of the binding between MSF-A and microtubules.
The figure represents three experiments that yielded similar
binding.
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Fig. 4.
Effects of treatment with demecolcine or
cytochalasin B on the MSF-A-containing filament structure. HMEC
cells were left untreated (A and B) or treated
with 5 µM cytochalasin B (Cyt.B) for 15 min
(C-F), or 10 ng/ml demecolcine (Demecol.) for
100 min at 37 °C (G-J), followed by confocal microscopic
analyses. Cytochalasin B and demecolcine effectively disrupted tubulin
and actin, respectively, under these conditions (A-D,
G, and H). Cells treated with
cytochalasin B were double stained for MSF-A (E) and actin
(F). Cells treated with demecolcine were
double stained for MSF-A (I) and tubulin
(J). Bar, 20 µm.
-tubulin was
little altered by the above siRNA treatments in Western blot analyses
(Fig. 5A, lanes 3, 4, 7,
and 8). However, when 500 cells were counted in which MSF-A
level was drastically lowered by immunofluorescent microscopy, 14% of
the cells showed reduced microtubule staining (Fig. 5B,
d-f), whereas the remaining cells with decreased
levels of MSF-A had almost intact microtubules (data not shown). In
control experiments with MSF-A-Mut, the filamentous structure of MSF-A and microtubules were little affected (Fig. 5B,
a-c). Under the conditions used, the actin filament
structure was not affected by the above RNAi treatments (data not
shown). It is notable that 10-12% of the cells with lowered levels of
MSF-A by MSF-A- or MSFs-RNAi failed to divide correctly, resulting in
binucleated cells (Fig. 5, B, d-f, and C).
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Fig. 5.
Effects of inhibition of MSF-A-expression on
cell division. A, lysates from HMEC cells transfected
with MSF-A-siRNA duplex (lanes 1 and 3),
MSFs-siRNAi duplex (lanes 5 and 7), or MSF-A-Mut
as a negative control (lanes 2, 4, 6,
and 8) were separated on a 10% polyacrylamide gel and then
subjected to Western blotting using anti-MSF antibody (lanes
1, 2, 5, and 6) or anti-tubulin
antibody (lanes 3, 4, 7, and
8). Molecular mass markers (in kilodaltons) are at
left. B, immunofluorescence analyses of HMEC
cells with MSF-A-Mut-siRNA (control; a-c) or
MSF-A-siRNA (RNAi; d-f). Immunofluorescence
analysis of MSF-A immunoreactivity (a and d),
tubulin (b and e), and DNA (c and
f) was carried out. Scale bar, 20 µm. C,
72 h after the transfection of MSF-A-Mut-, MSF-A-, or MSFs-siRNA
duplex, the cells were fixed and stained for MSF-A and DNA. The
percentage of cells with two nuclei was scored. Data are means ± S.E. of at least triplicate determinations. At least 200 cells per
sample were counted and at least three independent experiments were
performed.
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Fig. 6.
Effects of a GTPase-deficient mutation in G1
box on the filament-forming ability of MSF-A. A, time
course of GTPase activity of MSF-A, N312MSF-A, Nedd5, and N51Nedd5 was
examined. B, COS-7 cells were transfected with
pRK5-Myc-MSF-A, -N312MSF-A, -Nedd5, or -N51Nedd5. After 24 h of
transfection, these cells were stained using the 9E10 antibody.
C, COS-7 cells were double-transfected with pRK5-Myc-MSF-A
and pRK5-Flag-N312MSF-A (a-c) or pRK5-Myc-Nedd5 and
pRK5-Flag-N51Nedd5 (d-f). After 24 h of
transfection, these cells were double-stained using the anti-Myc
polyclonal antibody (green) and M2 antibody
(red). The merged image is also shown (c and
f). Scale bar, 20 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank M. Ohara for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid for scientific research and for cancer research from Ministry of Education, Science, Technology, Sports and Culture of Japan; by the Japan Society for Promotion of Science Research for the Future; by a grant-in-aid for the 2nd-Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare, Japan; and by the Princess Takamatsu Cancer Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed. Tel.: 81-52-762-6111 (ext. 7020); Fax: 81-52-763-5233; E-mail: minagaki@aichi-cc.jp.
Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M205246200
2 K. Nagata and M. Inagaki, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: MSF, mixed lineage leukemia septin-like fusion; aa, amino acids; HMEC, human mammary epithelial cells; RT, reverse transcription; PIPES, 1,4-piperazinediethanesulfonic acid; RNAi, RNA interference; siRNA, small interfering RNA.
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REFERENCES |
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