Báo cáo khoa học: GTP binding and hydrolysis kinetics of human septin 2 pot

GTP binding and hydrolysis kinetics of human septin 2
Yi-Wei Huang
1
, Mark C. Surka
1,3
, Denis Reynaud
2
, Cecil Pace-Asciak
2
and William S. Trimble
1,3
1 Program in Cell Biology, Hospital for Sick Children, Toronto, ON, Canada
2 Program in Integrative Biology, Hospital for Sick Children, Toronto, ON, Canada
3 Department of Biochemistry, University of Toronto, ON, Canada
Septins are a family of highly conserved guanine nuc-
leotide binding proteins that can assemble into fila-
ments and have been implicated in cytokinesis, cellular
morphogenesis, neuronal polarity, vesicle trafficking
and apoptosis in a wide range of organisms [1–7]. First
identified in Saccharomyces cerevisiae, septins Cdc3p,
Cdc10p, Cdc11p and Cdc12p localize to the mother–
bud junction where they play important roles in bud
emergence and cytokinesis [8,9]. At the bud neck their
appearance coincides with the presence of 10 nm fila-
ments that lie adjacent to the membrane in a concentric
pattern [8,9]. In mammals, at least 13 septin isoforms
are predicted from genomic analysis [10], named
SEPT1-SEPT13 in humans [11], but for most of them
their biological functions remain unclear. Septins can
be found in cytosolic fractions as heteromeric com-
plexes that appear to be comprised of most or all of the

septins expressed in a specific cell type [12]. In inter-
phase mammalian cells, most septins are organized into
filamentous structures that often colocalize with actin
bundles [13,14], or in some cases with microtubules
[15,16], implying that septin filaments may be an
important cytoskeleton component. Immuno-isolated
septin complexes from Drosophila [17] and yeast [18]
have been shown to polymerize into filaments under
favorable conditions. In addition, recombinant com-
plexes of SEPT2, SEPT6 and SEPT7 can be co-purified
in a 1 : 1 : 1 stoichiometry [12,19]. These complexes
can also polymerize into long filaments in vitro, indica-
ting the capacity of septins for self-directed
polymerization.
All septins possess a highly conserved central core
domain that has the potential to bind guanine nucleo-
tide and a variable length amino terminal domain.
Keywords
casein kinase II; GTP; GTPase kinetics;
phosphorylation; septins
Correspondence
W. Trimble, Program in Cell Biology,
Hospital for Sick Children, 555 University
Avenue, Toronto, Ontario M5G 1X8, Canada
Fax: +1 416 8135028
Tel: +1 416 8136889
E-mail:
(Received 24 February 2006, revised 8 May
2006, accepted 22 May 2006)
doi:10.1111/j.1742-4658.2006.05333.x

Septins are a family of conserved proteins that are essential for cytokinesis
in a wide range of organisms including fungi, Drosophila and mammals. In
budding yeast, where they were first discovered, they are thought to form a
filamentous ring at the bridge between the mother and bud cells. What
regulates the assembly and function of septins, however, has remained
obscure. All septins share a highly conserved domain related to those
found in small GTPases, and septins have been shown to bind and hydro-
lyze GTP, although the properties of this domain and the relationship
between polymerization and GTP binding ⁄ hydrolysis is unclear. Here we
show that human septin 2 is phosphorylated in vivo at Ser218 by casein
kinase II. In addition, we show that recombinant septin 2 binds guanine
nucleotides with a K
d
of 0.28 lm for GTPcS and 1.75 lm for GDP. It has
a slow exchange rate of 7 · 10
)5
s
)1
for GTPcS and 5 · 10
)4
s
)1
for GDP,
and an apparent k
cat
value of 2.7 · 10
)4
s
)1
, similar to those of the Ras

superfamily of GTPases. Interestingly, the nucleotide binding affinity
appears to be altered by phosphorylation at Ser218. Finally, we show that
a single septin protein can form homotypic filaments in vitro, whether
bound to GDP or GTP.
Abbreviations
GTPcS, guanosine 5¢-[c-thio]triphosphate; Sf21 cells, Spodoptera frugiperda cells; TBB, tetrabromobenzotriazole.
3248 FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS
Most also possess a carboxyl-terminal predicted coiled-
coil domain. The presence of characteristic motifs
within the GTPase region, including the P-loop (G-1
motif) as well as sequences resembling G-3 and G-4
motifs [20] has led to the classification of septins as a
novel group of GTPases [3]. Many GTPases possessing
these motifs, such as members of the Ras superfamily,
function as molecular switches regulating many essen-
tial cellular processes by cycling between a GTP-bound
active state and a GDP-bound inactive state [20,21].
Due to their low intrinsic GTPase activity and slow
GDP-GTP exchange rates, ras family GTPases require
additional factors to promote GTPase activity and
nucleotide exchange in order to inactivate and activate
them, respectively. Alternatively, for GTPases like
b-tubulin, the binding of GTP alters the conformation
of the protein to promote polymerization while hydro-
lysis of GTP serves as a timing mechanism to control
polymer turnover.
The high degree of conservation among the guanine
nucleotide binding domains of septins raises the possi-
bility that nucleotide binding and hydrolysis properties
may be important for septin function, but their precise

role remains unclear. Septin complexes isolated from
a variety of organisms and recombinant septin com-
plexes contain stoichiometric amounts of bound guan-
ine nucleotide with the majority being GDP [12,17,22].
Septins have been reported to bind guanine nucleotide
and hydrolyze GTP to GDP [13,17,23,24], but the sig-
nificance of this with respect to septin polymerization
has remained controversial. Mendoza et al. [23] have
reported that GTP binding induces Xenopus SEPT2
filament assembly in vitro, however, this polymeriza-
tion does not require GTP hydrolysis. Likewise, yeast
septins unable to hydrolyze GTP could form septin
neck rings in vivo while mutants unable to bind GTP
did not form neck rings and could not polymerize into
filaments in vitro [25]. Sheffield et al. [24] reported that
GTP binding and hydrolysis may be important for
mammalian septin heterodimer formation. In contrast,
Kinoshita et al. [12] demonstrated that septin com-
plexes can self-assemble into filaments while predomin-
antly bound to GDP and in the absence of guanine
nucleotide hydrolysis. Clearly, a thorough analysis of
the GTPase enzyme kinetics will be important in gain-
ing insights into the contribution of the GTPase
domain to septin function.
In this study, we examined the GTPase properties of
mammalian SEPT2. We show that recombinant
SEPT2 produced via baculovirus expression has meas-
urable GTP binding and hydrolysis kinetics compar-
able to that of Ras superfamily GTPases. The purified
protein has the capacity to polymerize into long fila-

ments when loaded with GTP or GDP. Moreover, we
show that the endogenous protein in HeLa cells, like
that produced in insect cells, is phosphorylated by
casein kinase II and that this phosphorylation alters
nucleotide binding.
Results
Septin 2 is phosphorylated by casein kinase II
Recombinant Septin 2 (SEPT2) was produced by bacu-
lovirus-mediated expression in Sf21 cells, and we previ-
ously examined its mass by laser desorption ⁄ ionization
quadrupole ⁄ time-of-flight tandem mass spectrometry
and noted that the recombinant protein was singly
phosphorylated. Direct peptide mapping and sequence
analyses on various enzymatic digests identified a novel
phosphorylated site at residue Ser218 in vivo. This
unique monophosphorylation at Ser218 was confirmed
by site-directed SEPT2 mutagenesis of Ser218 to Ala,
as similar analysis of the mutated protein showed no
evidence of phosphorylation [26]. Residue S218 is
detected by a variety of phosphorylation prediction
software to be a high probability casein kinase II site.
The detection of SEPT2 phosphorylation in Sf21
cells raised the question of whether endogenous septins
are phosphorylated in mammalian cells in vivo.To
begin to address this possibility HeLa cells were cul-
tured in the presence of [
32
P]-orthophosphate for 5 h
to radiolabel phosphoproteins in vivo. Cells were then
solubilized in a lysis buffer containing 0.5% Triton

X-100 and phosphatase inhibitors. The cleared lysate
was then subjected to immunoprecipitation using anti-
body specific to SEPT2. We have previously shown
[15] that immunoprecipitation of SEPT2 results in the
co-immunoprecipitation of septins 6, 7 and 9 in HeLa
cells. As shown in Fig. 1, several phosphoproteins were
immunoprecipitated with anti-SEPT2 antibodies and
the mobilities of these bands are consistent with each
of the septins that co-immunoprecipitate with SEPT2
from HeLa cells [15]. The position of SEPT2 was con-
firmed by western blotting (data not shown) and is
indicated by an asterisk.
As a means of identifying the possible kinases
responsible for SEPT2 phosphorylation we performed
an in-gel kinase assay. In this assay recombinant
SEPT2, expressed as a GST-fusion protein in bacteria,
is incorporated into the polyacrylamide matrix prior to
electrophoresis. Lysates of HeLa cells were electro-
phoresed through the gel, and then renatured to iden-
tify the presence of any kinases capable of using
SEPT2 as a substrate. In this assay, we found two
prominent bands between 40 and 50 kDa consistent
Y W. Huang et al. GTPase properties of human SEPT2
FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS 3249
with the mobilities of the a and a¢ subunits of casein
kinase II (Fig. 2A). To determine if these were casein
kinase 2, recombinant casein kinase II was electro-
phoresed beside the cell lysate and gave a similar
banding pattern. In addition, both the recombinant
casein kinase II and cell lysate bands were sensitive to

heparin, a casein kinase II inhibitor (Fig. 2B). The
identity of casein kinase II as the responsible kinase
was further supported by transfection experiments in
which a myc-tagged SEPT2 construct was expressed in
HeLa cells in the presence of [
32
P]-orthophosphate.
Cells were then incubated for 6 h with the casein kin-
ase inhibitor tetrabromobenzotriazole (TBB) at differ-
ing concentrations and then immunoprecipitated with
anti-myc IgG. As seen in Fig. 2C, myc-SEPT2 phos-
phorylation was inhibited in a dose-dependent manner
by TBB.
Prosite analysis of SEPT2 sequence revealed five
potential casein kinase II sites located at amino acids
84, 98, 120, 198 and 218. To determine if the site phos-
phorylated by Sf21 cells [26] was the same one phos-
phorylated in HeLa cells we utilized site-directed
mutagenesis to convert serine 218 to alanine. Cells
were then transfected with myc-SEPT2
WT
or myc-
SEPT2
S218A
, or mock transfected, labeled with
[
32
P]-orthophosphate, and immunoprecipitated with
anti-myc. As seen in Fig. 2D, only myc-SEPT2
WT

, but
not myc-SEPT2
S218A
, was phosphorylated in logarith-
mically growing HeLa cells.
Septin 2 binds and hydrolyses GTP
Like all septins characterized to date, SEPT2 can bind
and hydrolyze GTP in vitro, yet its kinetic properties
are not well known. To measure GTP binding and
hydrolysis kinetics, human SEPT2 was expressed in
Sf21 cells and purified from cell lysates with nickel-
chelated affinity chromatography to more than 95%
purity based on SDS⁄ PAGE (Fig. 3). Identity of the
protein was confirmed by immunoblot analysis using a
polyclonal antibody raised against SEPT2 [14]. We
found that purification in 20% glycerol was necessary
to stabilize this protein. As well, the addition of GDP
to the purification buffer system further stabilized the
protein, leading to full nucleotide binding activity (see
below).
We first sought to determine if baculovirus-expressed
SEPT2 co-purifies with nucleotide. To define the nuc-
leotide bound state of the purified proteins we used
reverse-phase HPLC and observed (Fig. 4A) that
1 mole of SEPT2 bound approximately 1.3 moles of
nucleotide, consistent with results reported for mam-
malian and Drosophila septin complexes [12,17]. The
GTP to GDP ratio was about 0.1, lower than that
observed for immuno-isolated Drosophila septins [17].
This is significantly different from Xenopus laevis Sept2,

which was found to be nucleotide free when purified
from bacteria [23]. We therefore examined the nucleo-
tide status of SEPT2 when purified from Escherichia
coli and found that 60% of the protein contained
guanine nucleotides in a GDP–GTP ratio of 1.4 : 1
(data not shown). Figure 4B shows that the nucleotides
are efficiently exchanged and can be removed from the
protein (about 10% of the nucleotides remain bound).
At high Mg
2+
concentrations GTPcS replaced GDP
efficiently, but could be readily stripped from the pro-
tein during wash steps when the protein is bound on
nickel beads in the low Mg
2+
concentrations due to its
rapid GTPcS off-rate under these conditions.
Using a filter-binding assay we measured both the
equilibrium constant (K
d
) and dissociation rate con-
stant (k
off
) of guanine nucleotides for SEPT2. To mon-
itor GTP binding, a nonhydrolysable GTP analog,
GTPcS, was used. Considering that Mg
2+
is known as
Fig. 1. Septin 2 co-immunoprecipitates with several phospho-
proteins. Immunoprecipitation with affinity purified anti-SEPT2

serum from cells labeled with [
32
P]-orthophosphate reveals that
SEPT2 and several co-precipitating proteins are phosphorylated in
vivo. No radioactive bands were detected when the preimmune
serum was used (left lane). The asterisk denotes the band detected
by affinity purified anti-SEPT2 serum on western blots (not shown).
GTPase properties of human SEPT2 Y W. Huang et al.
3250 FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS
an essential cofactor for many GTP-binding protein
functions [27], we examined whether septin nucleotide
binding and hydrolysis were affected by Mg
2+
concen-
tration. Figure 5A,B represents the binding results of a
set of independent experiments with different Mg
2+
concentrations for SEPT2. While binding of GTPcS
was saturable at all Mg
2+
concentrations, it was
clearly enhanced by Mg
2+
levels in the physiological
range (0.5–5 mm) (Table 1). The wild-type protein
showed a binding stoichiometry of 1, indicating that 1
molecule of protein bound 1 molecule of nucleotide
for both GTPcS and GDP in each of the Mg
2+
con-

centrations tested. The data revealed a hyperbolic
curve that was best fit to a single bimolecular binding
model. Scatchard analysis is shown in the inset and the
K
d
for GTPcS was measured to be 0.28 ± 0.06 lm in
5mm MgCl
2
and 3.37 ± 1.42 lm in 0.01 mm MgCl
2
.
Thus, the approximate 12-fold difference between low
and high Mg
2+
concentrations indicates the import-
ance of Mg
2+
in GTP-binding. Interestingly, this
Mg
2+
dependence was not observed for GDP binding
(Fig. 5B and Table 1). The K
d
values were very similar
in the low micromolar range at different Mg
2+
con-
centrations. Interestingly, when the nonphosphorylat-
able S218A form of SEPT2 was examined, it had a
much higher K

d
value of 2.5 lm for GTPcS and
4.4 lm for GDP (Table 1). This was very similar to
the values obtained for SEPT2 produced in E. coli
(1.7 lm for GTP cS and 6.4 lm for GDP) (data not
shown), suggesting that serine phosphorylation by
casein kinase II decreases the affinity of SEPT2 for
guanine nucleotides.
We next examined the effect of Mg
2+
on the dis-
sociation rate of GTPcS and GDP from SEPT2.
AB
C
D
Fig. 2. SEPT2 is phosphorylated at S218 by casein kinase II. (A) In-gel kinase assay reveals that Sept)2 is an efficient substrate for kinases
that have a mobility between 40 and 50 kDa. Gels were polymerized with GST (left lanes) or GST-SEPT2 (right lanes) and duplicate samples
of HeLa cell lysates were electrophoresed. The gels were renatured in the presence of [
32
P]-ATP as described in the methods and then
autoradiographed. (B) In-gel kinase assays show that commercial casein kinase II (left panel, right lane) gave bands comparable to the cell ly-
sate (left panel, left lane) and both bands were inhibited by heparin (right panel). (C) Increasing concentrations of TBB reduced SEPT2 phos-
phorylation. Cells were transiently transfected with myc-SEPT2, then immunoprecipitated, labeled with [
32
P]-orthophosphate, and then lysed
and lysates immunoprecipitated with anti-myc IgG. Increasing amounts of TBB caused a reduction in the labeling of myc-SEPT2. (D) Cells
were either mock transfected or transfected with myc-SEPT2, labeled with [
32
P]-orthophosphate, and then immunoprecipitated with anti-
myc as above. Autoradiography reveals that the wild-type protein is significantly labeled but SEPT2

S218A
is not (top panel). A western blot of
lysates was probed with anti-SEPT2 to reveal that both constructs were equivalently expressed (lower panel, upper band), and at levels not
greatly above endogenous SEPT2 levels (lower panel, lower band).
Y W. Huang et al. GTPase properties of human SEPT2
FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS 3251
Figure 6A,B represents a set of independent experi-
ments for the dissociation of GTPcS and GDP, respect-
ively, from SEPT2 with different Mg
2+
concentrations.
Fig. 4. Reverse-phase HPLC monitoring of guanine nucleotides
bound on SEPT2. Purified SEPT2 (240 pmol; A) was extracted as
described in the methods and bound nucleotides were fractionated
by HPLC on a Nova-PackÒ C18 column. (B) A three molar ratio of
GTPcS was added to the purified protein with 5 m
M MgCl
2
and
incubated at room temperature for 3 h to exchange GDP with
GTPcS. The protein was then reloaded onto a nickel column to
remove unbound nucleotides. Bound GTPcS quickly released and
washed away during wash steps with buffer A without Mg
2+
and
nucleotide
.
The protein was then eluted in 150 mM imidazole.
(B) shows only about 10% of the nucleotides still bound to the pro-
tein. (C) Control reveals the HPLC profile of a mixture of 240 pmol

of GDP, GTP and GTPcS. Absorbance at 254 nm is indicated in
arbitrary units.
Fig. 3. Purification results of His6-tagged SEPT2 wild-type over-
expressed in Sf21 cells. (A) Purification of SEPT2. A Commassie
brilliant blue stained 12% SDS ⁄ PAGE gel reveals the purification of
SEPT2. N-terminal His6-tagged SEPT2 proteins were overproduced
in Sf21 cells, accounting for about 10–15% of the total protein in
the insect cell lysate (L). Samples were sedimented at 110 000 g
and the majority of the SEPT2 protein remained in the supernatant
(S). After passage over Ni-NTA columns the flow-through (FT) was
depleted of SEPT2, which mainly remained bound to the column
during the washes (W1, W2) and eluted (E) as a single species in
150 m
M imidazole.
A
B
Fig. 5. Guanine nucleotide binding of SEPT2. (A) Equilibrium binding
curves of GTPcS with different concentrations of Mg
2+
. Data were
plotted by fitting to a bimolecular binding equation. Scatchard analy-
sis is shown in the inset with the ratio of the concentration of
bound GTPcS over the concentration of SEPT2 divided by the free
GTPcS ([b] ⁄ [SEPT2] ⁄ [f] – y axis) plotted against the ratio of bound
GTPcS over the SEPT2 concentration ([b] ⁄ [SEPT2] – x axis).
(B) Equilibrium binding curves of GDP with different concentration
of Mg
2+
. Data were plotted by fitting to a bimolecular binding equa-
tion. Scatchard analysis is shown in the inset with the ratio of the

concentration of bound GDP over the concentration of SEPT2 divi-
ded by the free GDP ([b] ⁄ [SEPT2] ⁄ [f] – y axis) plotted against the
ratio of bound GDP over the SEPT2 concentration ([b] ⁄ [SEPT2] –
x axis).
GTPase properties of human SEPT2 Y W. Huang et al.
3252 FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS
From these panels we can see the same phenomenon as
seen for the K
d
measurements, that at different Mg
2+
concentrations the dissociation of GTPcS is much more
greatly affected than that of GDP dissociation. Surpris-
ingly, the data best fit to either single or bi-exponential
decay models depending on the Mg
2+
concentration.
The summary of the k
off
rates of SEPT2 for GTPcS
and GDP fit to a single exponential decay model is pre-
sented in Table 2. The dissociation half-life for GTPcS
in low Mg
2+
is 1.24 min and in high Mg
2+
is 165 min.
The dissociation half-life for GDP in low Mg
2+
is

3.62 min and that in high Mg
2+
is 23.1 min. Again,
these results show Mg
2+
dependence for GTPcS disso-
ciation, which has > 130-fold difference between low
and high Mg
2+
concentrations while that for GDP has
only a six-fold difference. In similar experiments carried
out with the S218A mutant, we found that the k
off
for
GTPcS and GDP at high Mg
2+
are not significantly
different from those of SEPT2
WT
(Table 2) with the
dissociation half-life 182 min and 16.5 min for GTPcS
and GDP, respectively, at high Mg
2+
concentration.
Since the K
d
for GTPcS of the mutant is significantly
increased, it implies that the association rate constant
(k
on

) decreases.
In order to measure k
on
and hydrolysis rate con-
stants, it is first necessary to produce apoprotein
devoid of nucleotide, but all attempts to remove nuc-
leotide from the protein resulted in protein instability
and insolubility. Hence, it was not possible to measure
k
on
and hydrolysis rate constants by these means.
As we were unable to measure hydrolysis rate con-
stants directly, we used radioactive assays to measure
Table 1. Summary of the GTPcS and GDP dissociation constants and stoichiometrical binding site (n) for wild-type SEPT2 and S218A
mutant. The data for SEPT2 were best fit with a single binding model while. n represents the number of binding sites per protein molecule.
The values represent mean ± SD of at least three independent experiments. ND, not done.
MgCl
2
(mM)
GTPcS GDP
Wild-type S218A Wild-type S218A
K
d
(lM) nK
d
(lM) nK
d
(lM) nK
d
(lM) n

5 0.28 ± 0.06 0.98 ± 0.11 2.46 ± 0.6 0.73 ± 0.1 1.72 ± 0.15 0.94 ± 0.28 4.40 ± 1.8 0.50 ± 0.1
0.5 1.84 ± 0.1 0.93 ± 0.06 ND ND 1.19 ± 0.05 1.07 ± 0.1 ND ND
0.01 3.37 ± 1.42 0.70 ± 0.22 63.4 ± 10.8 0.73 ± 0.02 1.54 ± 0.37 1.02 ± 0.40 ND ND
A
B
Fig. 6. Guanine nucleotide dissociation from SEPT2. (A) GTPcS
dissociation curves with different concentration of Mg
2+
. (B) GDP
dissociation curves with different concentrations of Mg
2+
. Radioiso-
topes used for GTPcS and GDP binding were
35
S-GTPcSand
3
H-GDP, respectively. Data were plotted by fitting to single or bi-
exponential decay equations.
Table 2. Summary of the GTPcS and GDP dissociation rate con-
stants for SEPT2. The data were fit to a single exponential decay
model. The values represent mean ± SD of at least three independ-
ent experiments. ND, not done.
MgCl2 (m
M)
GTPcS k
off
( · 10
)3
s
)1

) GDP k
off
( · 10
)3
s
)1
)
WT S218A WT S218A
5 0.07 ± 0.02 0.06 ± 0.02 0.5 ± 0.1 0.7 ± 0.2
0.5 1.2 ± 0.1 ND 4.5 ± 1.6 ND
0.01 9.3 ± 2.5 ND 3.2 ± 0.8 ND
Y W. Huang et al. GTPase properties of human SEPT2
FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS 3253
SEPT2 steady-state kinetic constants, k
cat
and K
m
at
different Mg
2+
concentrations. In Fig. 7, we show a
representative experiment of SEPT2 at 5 mm Mg
2+
,
revealing a hyperbolic initial velocity curve that is a
function of GTP concentration, and fits well with the
Michaelis–Menten equation. A Lineweaver–Burke plot
of the data is shown in the inset. The apparent kinetic
constant for SEPT2 is summarized in Table 3. The k
cat

and K
m
values are not significantly different at 5 and
0.5 mm MgCl
2
; however, GTPase activity cannot be
clearly measured without Mg
2+
in the buffer system.
The k
cat
value for the SEPT2
S218A
mutant is similar to
that of the SEPT2
WT
. However, consistent with the K
d
value, K
m
is also four-fold larger than that of the wild-
type.
Previous studies had reported the ability of septins
to form filaments in vitro [12,17,18,24]. We therefore
set out to determine if baculovirus-expressed SEPT2
could form homo-oligomeric filaments in vitro. Consis-
tent with previously published results, we observed fila-
ments of a variety of lengths similar to those seen for
Xenopus SEPT2 [23] and for Drosophila and yeast
septin complexes [17,18]. SEPT2 filaments were detect-

able regardless of which nucleotide was present (GTP
or GDP) and typically these filaments were more than
5 lm in length and appeared to be bundles of fila-
ments of approximately 20–40 nm in diameter con-
taining several intertwined filaments in the bundles
(Fig. 8). However, we did note differences in the rate
of filament formation. Filaments formed within 30 min
when protein was loaded with GTPc S or GTP (not
shown), but took up to 6 h to achieve detectable fila-
ments when loaded with GDP (Fig. 8B).
Discussion
In this paper, we describe the first detailed kinetic
study of the GTP binding and hydrolysis properties of
a single septin protein and demonstrate that phos-
phorylation of serine 218 by casein kinase II alters
these properties. Previous studies have focused on sep-
tin complexes because single septin proteins expressed
in bacteria were found to be unstable and have
severely altered nucleotide binding [24,28] precluding
their analysis. Some information has been gained by
the study of septin complexes, but mammalian septin
complexes expressed in E. coli had extremely slow nuc-
leotide exchange rates [24]. This is similar to the poor
exchange rates seen for endogenous septin complexes
immunoisolated from Drosophila [17] and yeast [22].
Complexes comprised of different septin isoforms
would reflect the binding and hydrolysis properties of
each protein. Indeed, it has been reported that yeast
septins Cdc10 and Cdc12 contributed the majority of
Fig. 7. GTP hydrolysis kinetics of SEPT2. SEPT2 GTP hydrolysis

kinetic constants were measured in a mixture with 40 m
M Tris,
pH 7.5, 10% glycerol, 0.5 mgÆmL
)1
bovine serum albumin, 5 mM
MgCl
2
,1mM EDTA, 5 mM dithiothreitol, a fixed concentration of
purified protein and varying concentrations of
32
P-GTP at room tem-
perature. Data from a representative experiment show the hyperbo-
lic initial velocity curve as a function of GTP concentration and
were plotted by fitting with the Michaelis–Menten equation. The
Lineweaver–Burke plot of the data is shown in the inset. Reaction
conditions for all panels are specified in experimental procedures.
Figures are representative of the results of several independent
experiments.
Table 3. Kinetic constants of GTP hydrolysis of Sept2 wild-type and mutant S218A. The K
m
and k
cat
values represent mean ± SD of at least
three independent experiments. The k
cat
s values were calculated from the V
max
values with the molecular mass of 43.5 kDa. NM, not meas-
urable; ND, not done.
MgCl

2
(mM)
Wild-type S218A
k
cat
(x10
)4
s
)1
)
K
m
(lM)
k
cat
⁄ K
m
( · 10
)4
s
)1
ÆlM
)1
)
k
cat
(x10
)4
s
)1

)
K
m
(lM)
k
cat
⁄ K
m
( · 10
)4
s
)1
ÆlM
)1
)
5 2.7 ± 0.5 0.52 ± 0.05 5.2 4.6 ± 0.9 2.38 ± 0.08 1.9
0.5 2.5 ± 0.5 0.5 ± 0.04 5.0 ND ND ND
0NMNMNM NDNDND
GTPase properties of human SEPT2 Y W. Huang et al.
3254 FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS
the GTP binding to the yeast septin complex [25].
Also, complexes of mammalian septins comprised of
different septin isoforms also exchanged guanine nucle-
otides at different rates [24]. We therefore investigated
the possibility that insect cell expression may provide
protein more suitable for these types of analyses.
Moreover, we have recently shown that SEPT2
expressed in Sf21 cells is phosphorylated at a single
site [26], raising the possibility that the expression
system may affect its properties. We now show that

baculovirus expression of SEPT2 unable to be phos-
phorylated at this residue, as well as SEPT2 expressed
in bacteria and therefore not phosphorylated, had sig-
nificantly different kinetic properties. By examining a
single septin we have eliminated the complicating dif-
ferences in kinetics that likely exist between septin iso-
forms when one examines septin complexes.
Recombinant SEPT2 expressed in baculovirus was
stable for several months in )80 °C without significant
loss of binding activity (data not shown). However,
when the nucleotide was removed from SEPT2, the
protein quickly became unstable and lost nucleotide
binding and filament-forming properties. This phenom-
enon suggests that nucleotide binding is important in
maintaining proper protein conformation. This is also
consistent with what has been seen in many nucleotide
binding proteins. For example, removing the tightly
bound nucleotide from p21
H–ras
renders the protein
thermally unstable [29], although apoproteins can be
produced for Ras under specific conditions [30]. Con-
centrated nucleotide-free Ran, a Ras-related nuclear,
immediately precipitates on diluting to working con-
centration (1–2 lm) [31]. Similarly, addition of GDP
during the purification is critical in obtaining fully act-
ive Dictyostelium elongation factor 1A [32] while nuc-
leotide-free actin denatures rapidly at a rate of 0.2 s
)1
[33].

The GTPcS K
d
of SEPT2 is 0.28 lm and that of
GDP is 1.7 lm in the presence of physiological Mg
2+
concentrations. These values are about an order of
magnitude lower than those observed for complexes
of yeast septins expressed in bacteria. Complexes of
Cdc3p-Cdc12p, Cdc3p-Cdc11p-Cdc12p and Cdc3p-
Cdc10p-Cdc12p had K
d
values of 1.6, 6.2 and 5.9 for
GTPcS. However, they are much more reminiscent of
the K
d
values for GTPcS obtained for SEPT2 S218A
(2.5 lm) or for SEPT2 when expressed in E. coli
(1.7 lm). It would be of interest to determine if post-
translational modifications alter the kinetic properties
of yeast septins. These binding constants are signifi-
cantly different from those of the Ras family members
(from picomolar to nanomolar) but similar to those
of the Rho family of small GTPases [34,35]. Interest-
ingly, we observed a Mg
2+
-dependent phenomenon
Fig. 8. SEPT2 forms homotypic filaments in
GTP and GDP bound states. Negative stain
electron micrographs of SEPT2 filaments
found after loading the protein with GTP (A),

GDP (B) or GTPcS (C), followed by 6 h of
incubation. Scale bars on these figures rep-
resent 500 nm. Higher magnification of
GTPcS-bound filaments (D) reveals lateral
bundles of SEPT2 protein. Scale bar repre-
sents 100 nm.
Y W. Huang et al. GTPase properties of human SEPT2
FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS 3255
for GTPcS binding while GDP binding was Mg
2+
independent. Also, we made the surprising observation
that the decay fitting models best fit either single- or
bi-exponential curves depending on Mg
2+
levels. This
likely reflects different degrees of polymerization of the
protein that occur during the assay. Unfortunately, we
were unable to distinguish the properties of filamen-
tous septin complexes and monomeric septins since
SEPT2 spontaneously polymerized during the course
of the assays.
When comparing the off-rates, the value measured
for SEPT2 (t
½
¼ 165 min) appears to be quite similar
to that found for a SEPT2⁄ 6 ⁄ 7 complex expressed in
bacteria (t
½
¼ 150 min) [24]. This nucleotide binding
property is not fully consistent with either Ras or Rho

proteins. For example, p21
H–ras
shows Mg
2+
-depend-
ent guanine nucleotide binding behavior with a >500-
fold difference in GDP k
off
in the presence and absence
of Mg
2+
while Mg
2+
has no significant influence on
K
on
[30,36,37]. In the case of Rho family proteins,
RhoA, Cdc42 and Rac1 show similar K
d
values for
GTPcS and GDP binding in the presence or absence
of Mg
2+
although in the absence of Mg
2+
their off
rates significantly increased, indicating that their nuc-
leotide association rates increase in parallel and the
intrinsic catalytic activities are not significantly affected
by Mg

2+
[34,35]. Unfortunately, due to our inability
to produce SEPT2 in the nucleotide-free state, we were
unable to measure k
on
and hydrolysis rate con-
stants. The apparent k
cat
for SEPT2 as determined by
steady state kinetics is very low (2.7 · 10
)4
s
)1
or
0.016 min
)1
), remarkably similar to steady state values
measured from yeast Cdc3p⁄ Cdc12p binary complexes
0.019 min
)1
[28]. In this case, it is thought that, as
Cdc3p does not exchange GTP, this value entirely
derives from Cdc12p-mediated hydrolysis. These values
are more than an order of magnitude higher than the
value measured for singly expressed Cdc10p and
Cdc12p [25]. This K
cat
is similar to the intrinsic
GTPase rate of p21
ras

proteins (3.4–5 · 10
)4
s
)1
)
[20,38] and that of Rho family members RhoA, RhoB,
S. cerevisiae Cdc42 and Caenorhabditis elegans Cdc42
(3.4 · 10
)4
s
)1
) [39]. Like these proteins, SEPT2 does
not appear to have self-stimulatory GAP activity when
incubated at increasing concentrations (data not
shown). This is in contrast to several other Rho family
members such as RhoC, human Cdc42 and Rac2,
which contain a self-stimulatory GAP activity [39].
The k
cat
also depends on Mg
2+
since it cannot be
clearly measured following depletion of Mg
2+
, consis-
tent with the fact that Mg
2+
is an essential cofactor
for most GTPases. The crystal structure of RhoA
revealed that elimination of the Mg

2+
ion induced a
significant conformational change in the switch I
region that opens up the nucleotide-binding site and
suggested that a guanine nucleotide exchange factor
may utilize this feature of switch I to produce an open
conformation for GDP ⁄ GTP exchange [40]. The
Mg
2+
-dependent and -independent binding properties
of SEPT2 for GTPcS and GDP, respectively, may also
indicate different conformations in recognizing triphos-
phate and diphosphate guanine nucleotides. The simi-
larity of the K
d
, k
off
and k
cat
of septins and members
of the Rho family could be taken to imply that septins
require GTP exchange and GTPase activating proteins
to complete the GTP binding and hydrolysis cycle effi-
ciently. However, at present it is not known how rap-
idly these events would need to take place to support
septin function. Indeed, a recent report has suggested
that, as is the case for a-tubulin, the role of guanine
nucleotides in septins may be to ensure structural
integrity of the protein [22].
The ability of mammalian SEPT2 to polymerize

in vitro, similar to that seen for Xenopus SEPT2 [23],
indicates that the formation of septin filaments does
not require an ordered array of a set of septins from
distinct families, as has been recently postulated [41].
Whether other mammalian septins also have this capa-
city is not known, but it is interesting that immunopre-
cipitation of septins from cells routinely results in the
co-precipitation of other septins in near stoichiometric
ratios [12,15], indicating that the formation of homo-
polymers is not typical in vivo.
It was also noteworthy that the S218A substitution
near the C-terminus of the protein had a significant
effect on the GTP binding property of SEPT2. This
suggests that either the phosphorylation event results in
changes in intraprotein conformation that affect the
folding of the GTPase domain, or that inter-protein
interactions between the C-terminal domain of one
molecule of SEPT2 and the GTPase domain of another
influence its properties. We did not notice a difference
in the capacity of S218A mutant protein to polymerize
into long filaments, although it did appear to be less
efficient at forming thick bundles of filaments (data not
shown). It will be of interest to determine if septin poly-
merization is dynamically regulated in the cell, and if
so, to what extent phosphorylation status is involved.
Experimental procedures
Expression and purification of recombinant
SEPT2 and mutants
Human SEPT2 cDNA [14] (accession number T19030) was
subcloned into the baculovirus expression vector pFast-

GTPase properties of human SEPT2 Y W. Huang et al.
3256 FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS
BacHTb (Invitrogen, Burlington, ON, Canada) following
PCR amplification. The SEPT2
S218A
mutant was generated
using the QuikChange
TM
site-directed mutagenesis kit
(Stratagene, La Jolla, CA, USA). Subcloned and mutagen-
ized cDNA sequences were verified by dideoxynucleotide
sequencing. Note that in She et al. [26], the site of phos-
phorylation was indicated as Ser248 in the protein due to
the 30 amino acid tag added to its N-terminus. During the
course of these studies, we noted that a PCR error had
resulted in a R331H substitution that was found in all con-
structs after the completion of most of the studies described
herein. Given the conserved nature of this residue we there-
fore mutated the residue back to arginine and found that
none of the properties of the protein were altered by this
mutation (data not shown). This was not surprising, as a
large deletion including this region of the protein had previ-
ously been shown to have no effect on filament formation
or GTPase activity in X. laevis Sept2 [23]. The proteins
were overproduced in Sf21 insect cells as recommended by
the manufacturer. Briefly, cells were harvested 48 h postin-
fection by centrifugation at 1000 g for 10 min, washed once
with phosphate-buffered saline (NaCl ⁄ P
i
), pelleted again

and kept at )80 °C for later use. The purification steps
were carried out at 4 °C unless otherwise specified. Before
purification, cell pellets from about 6 · 10
8
cells were sus-
pended in 20–40 mL of buffer A (40 mm Tris, pH 8.0,
100 mm NaCl, 20% glycerol, 0.4 mm phenylmethylsulfonyl
fluoride, 1 lgmL
)1
of each leupeptin and pepstatin A,
40 lm GDP and 8 mm imidazole). Cells were disrupted by
sonication and centrifuged at 110 000 g for 60 min. The
supernatants were loaded onto Ni-NTA column (Qiagen,
Mississauga, ON, Canada) pre-equilibrated with the same
buffer, washed twice with buffer A without GDP in 10 mm
and 28 mm imidazole concentrations, respectively. Proteins
were eluted with buffer A without GDP in 150 mm imidaz-
ole. Proteins were routinely dialyzed in buffer D (40 mm
Tris, pH 7.5, 20 mm NaCl, 20% glycerol, 1 mm EDTA and
1mm dithiothreitol) although no difference was observed
when 200 mm NaCl was added. Protein concentration was
determined using the Bradford reagent (Bio-Rad, Mississ-
auga, ON, Canada). The dialyzed proteins were stored at a
concentration of 1mgÆmL
)1
at )80 °C and they were sta-
ble for several months.
In vivo [
32
P]-orthophosphate labeling and

immunoprecipitation
SEPT2
WT
or mutant SEPT2
S218A
were subcloned into a
modified pcDNA3.1 (Invitrogen) vector that contained myc
epitope at the N-terminus. HeLa cells were transiently
transfected with myc-tagged SEPT2
WT
or SEPT2
S218A
using
Lipofectamine (Invitrogen) according to the manufacturer’s
instructions. Eighteen hours post-transfection, cells were
washed twice with phosphate-free Dulbecco’s modified
Eagle’s medium. Cells were then grown in the presence of
0.75 mCi [
32
P]-orthophosphate for 5 h at 37 °C. Cells were
lysed with lysis buffer (40 mm Tris, pH 7.5, 20% glycerol,
0.2 m NaCl, 2 mm EDTA, 0.5% Triton X-100, 1 lm oka-
daic acid sodium salt, 50 mm NaF, 0.4 mm orthovanadate,
1mm phenylmethylsulfonyl fluoride, 1 lgÆmL
)1
of each of
the protease inhibitors leupeptin and pepstatin A. SEPT2
was immunoprecipitated with mouse antic-myc monoclonal
antibody IgG1 (9E10, Santa Cruz biotechnology, Santa
Cruz, CA, USA). The immunocomplexes were washed four

times with wash buffer (lysis buffer with 0.1% TritonX-100
and without proteases inhibitors). Proteins were eluted with
SDS ⁄ PAGE sample buffer, separated by SDS ⁄ PAGE and
blotted. Labeled SEPT2 was visualized by autoradiography
and quantitated using PhosphorImager.
For endogenous SEPT2 immunoprecipitations, HeLa
cells were grown in 10-cm dishes to 70% confluence
before proceeding to label and collect cells as described
above. Affinity purified anti-SEPT2 serum (3 l g) was added
to 40 lL of 50% protein-A agarose beads (Invitrogen) and
rotated for 1 h at 4 °C. The beads were washed three times
with HKA buffer, and the cell lysate was added along with
NaCl to a final concentration of 150 mm. The mixtures
were rotated at 4 °C for 1–2 h and washed five times with
ristocetin-induced platelet agglutination (RIPA) buffer (1%
Nonident P-40, 1% sodium deoxycholate, 0.1% SDS,
150 mm NaCl, 0.01 m sodium phosphate pH 7.2, 2 mm
EDTA) containing phosphatase inhibitors.
Inhibition of CK2 in vivo
HeLa cells transfected with myc-SEPT2
WT
for 18 h were
first washed twice with phosphate-free Dulbecco’s modified
Eagle’s medium and incubated with indicated concentra-
tions of TBB (Calbiochem, EMD Biosciences, San Diego,
CA, USA), a specific CK2 inhibitor [42], for 1 h. Cells were
then grown in the presence of 0.75 mCi [
32
P]-orthophos-
phate together with the same amounts of TBB for 5 h at

37 °C and treated as described above.
In-gel kinase assays
Purified GST or GST-Septin2 was copolymerized in the sep-
arating gel of a 10% SDS ⁄ PAGE at a concentration of
0.1 mgÆmL
)1
. Triton solubilized HeLa cell lysates (30 lg)
and ⁄ or 10 units of purified CK2 enzyme (Promega, Madi-
son, WI, USA) were loaded and electrophoresed. SDS was
removed from the gels by washing twice in buffer A (50 mm
Tris ⁄ HCl, pH 8.0 containing 20% 2-proponal) for 30 min,
followed by washing in buffer B (50 mm Tris ⁄ HCl, pH 8.0
containing 5 mm 2-mercaptoethanol). Proteins were dena-
tured by washing gels in two changes of buffer B containing
6 m guanidine chloride for 60 min. Renaturation was carried
at 4 °C for 30 min in buffer C (50 mm Tris ⁄ HCl, pH 8.0;
5mm b-mercaptoethanol; 0.04% Tween-20). The gels were
preincubated in kinase buffer (40 mm Hepes, pH 7.5,
Y W. Huang et al. GTPase properties of human SEPT2
FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS 3257
25 mm MgCl
2
,50mm dithiothreitol) for 30 min, and subse-
quently incubated with kinase buffer including 100 lm ATP
supplemented with 50 lCi of [c-
32
P]ATP (3000 Ci mmol
)1
)
(Amersham, GE Healthcare, Baie, d’Urfe, QC, Canada) for

1 h at room temperature. For those gels that were incubated
with heparin, 50 nm heparin (final concentration) was added
to the kinase buffer. All gels were washed five times with
5% trichloroacetic acid ⁄ 1% sodium pyrophosphate, dried
down, and subjected to phosphoimaging.
Nucleotide determination by reverse-phase HPLC
Analysis of bound nucleotides in the samples was carried
out on a Hewlett-Packard (1100 Series) HPLC to which was
attached a Waters programmable multiwavelength detector
(M-490). Nucleotide absorbance was measured at 254 nm.
Chromatographic separation of the compounds was carried
out on a 3.9 · 150 mm Nova-PackÒ C18 column (Waters,
Mississauga, ON, Canada) modified from the method of
Smith et al. [43]. Protein samples were diluted 1 : 1 with
HPLC buffer (100 mm KH
2
PO
4
⁄ K
2
HPO
4
, pH 6.4, 10 mm
tetrabutylamonium bromide), denatured in 1 : 10 volume
0.5% perchloric acid, neutralized by adding sodium acetate,
pH 5.4, to a final concentration 60 mm and centrifuged to
collect the supernatant prior to injection into the column
HPLC. The column was run in HPLC buffer at a flow rate
of 1 mLÆ min
)1

for 6 min, then the acetonitrile concentration
was increased in a linear gradient up to 10% over 9 min fol-
lowed by an additional 3 min at 10% acetonitrile.
Nucleotide binding assay, K
d
and K
d
measurement
For the GTPcS and GDP dissociation constant (K
d
) meas-
urement, the mixtures contained 40 mm Tris, pH 7.5, 20%
glycerol, 200 mm NaCl, 0.5 mg mL
)1
bovine serum albu-
min, 1 mm EDTA, 5 mm dithiothreitol and 0.01, 0.5 or
5mm MgCl
2
with 0.2–0.4 lm protein (calculated based on
the predicted SEPT2 monomer molecular weight 43.5 kDa).
35
S-GTPcS (specific radioactivity 7.2 mCiÆlmol
)1
)or
3
H-GDP (specific radioactivity 1.8 mCiÆlmol
)1
) (Perkin-
Elmer Life Sciences, Inc., Boston, MA, USA) in concentra-
tions that ranged from 0.04 to 60 lm were added. The

reactions were processed at room temperature for 3–6 h to
let the reaction reach equilibrium. The reactions were
stopped by diluting the samples into 2 mL of cold stop buf-
fer (20 mm Tris, pH 7.5 and 40 mm MgCl
2
) and filtered
through a 25-mm pre-wetted 0.2 lm nitrocellulose filter
membrane (Millipore, Cambridge, ON, Canada). The mem-
branes were washed with 9 mL of stop buffer and the
radionucleotides remaining bound to the protein were
quantified by scintillation counting. This procedure rou-
tinely yielded blank values of less than 0.3% of the total
radioactivity. To measure the nucleotide dissociation rate
constant, the reactions were processed in the same buffer as
in K
d
measurement. Protein (0.2 lm) was first mixed with
1 lm
35
S-GTPcSor
3
H-GDP for 0.5–1 h at room tempera-
ture, and 200 lm GDP or GTPcS were added to initiate
the dissociation. At different time intervals, aliquots were
taken out, diluted with 2 mL of cold stop buffer and were
treated as described above. Nucleotide exchange was con-
firmed by HPLC analysis of bound nucleotides.
GTP hydrolysis assay and steady-state kinetics
The initial velocity of GTP hydrolysis was measured by a
radioactive assay. It was started by the addition of 12 pmol

purified protein (0.2 lm)ina60lL reaction mixture (con-
taining 40 mm Tris, pH 7.5, 10% glycerol, 0.5 mgÆmL
)1
bovine serum albumin, 5 mm or 0.5 mm MgCl
2
,1mm
EDTA, 5 mm dithiothreitol, and 2 lm
3
H-GTP or
32
P-
GTP) and incubated at room temperature (23 °C±1°C).
At different time intervals (0, 10, 30 and 60 min), aliquots
were taken out and the reaction was stopped by adding
EDTA to a final concentration of 100 mm. The samples
were immediately applied onto PEI cellulose plate (Sigma-
Aldrich Canada Ltd, Oakville, ON, Canada) for TLC, sep-
arated by 0.75 m KH
2
PO
4
, pH 3.65 and quantified on a
Storm 860 Laser Scanner (Molecular Dynamic Co., Sunny-
vale, CA, USA) using ImageQuant software (ImageQuant,
GE Healthcare). The kinetic constants for SEPT2 were
determined in the above buffer system with a fixed protein
concentration and the
32
P-GTP concentrations varied from
0.2 to 15 lm (specific radioactivity 1.7 mCiÆlmol

)1
) (Amer-
sham, GE Healthcare). The initial velocity was measured
with less than 5–10% substrate conversion. At least three
independent experiments were carried out for each kinetic
constant. Kinetic constants were also measured without
Mg
2+
(the buffer as above but with 5 mm EDTA and with-
out MgCl
2
).
Data processing
Kinetic data were analyzed by nonlinear regression using
Origin 6.0 software (OriginLab, Northampton, MA, USA).
Equilibrium dissociation constants data were fitted to one-
site binding models and dissociation rate constants data
were fitted to single or bi-exponential decay models. GTP
hydrolysis kinetic data were fitted to the Michaelis–Menten
equation. The values of the catalytic constant, k
cat
, were
calculated from the V
max
values with the monomer molecu-
lar mass of 43.5 kDa. (k
cat
is the turnover number, i.e. the
number of moles of substrate transformed per second per
mole of enzyme).

SEPT2 polymerization
Polymerization was carried out in buffer D in the presence
of 15–20 lm SEPT2 and 5 mm MgCl
2
and 100 lm of each
GTPase properties of human SEPT2 Y W. Huang et al.
3258 FEBS Journal 273 (2006) 3248–3260 ª 2006 The Authors Journal compilation ª 2006 FEBS
GTP, GTPcS or GDP for 30 min to 6.5 h. The protein was
ultracentrifuged (280 000 g, 30 min at 4 °C) prior to the
experiments to remove formed filaments. For electron micro-
scopy (EM), samples were applied onto glow-discharged car-
bon-coated grids for 5 min. The grids were washed with one
drop of buffer D, stained with one drop of 1% uranyl acet-
ate in 30% ethanol and examined by electron microscopy
(Technai 20, Philips, FEI Systems, Toronto, ON, Canada).
Acknowledgements
The authors thank G. Boulianne for critical reading of
the manuscript, and B. Temkin and Y M. Heng for
EM data collection. WST is a CIHR Investigator.
YWH received a Postdoctoral Fellowship, in part,
through the Hospital for Sick Children Research
Training Centre. These studies were supported by
funds from the Canadian Cancer Society.
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