1 Boyer Center for Molecular Medicine, Department of Pathology, Yale University
School of Medicine, 295 Congress Avenue, New Haven, CT 06536, USA
2 NOVUS Biologicals, Inc. P.O. Box 802, Littleton, CO 80160, USA
3 Vita-Salute University School of Medicine, San Raffaele Scientific Institute,
Via Olgettina 58, Milano, 20132, Italy
Author for correspondence (e-mail: dario.altieri{at}yale.edu)
Accepted 23 October 2001
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Summary |
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Key words: Survivin, Microtubules, Mitotic spindle, Kinetochore, p34cdc2
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Introduction |
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Mammalian cell survivin (Ambrosini et
al., 1997) may share properties of IAPs involved in both apoptosis
inhibition and cell division control (Reed
and Bischoff, 2000
; Reed and
Reed, 1999
). Previous studies demonstrated that survivin is
expressed at G2/M in a cell cycle-dependent manner, and localizes to
components of the mitotic apparatus, including centrosomes, and mitotic
spindle microtubules (Li et al.,
1998
). In vitro, survivin bound polymerized microtubules with
µM affinity (Li et al.,
1998
), and a putative tubulin-binding domain was identified by
mutational analysis in the extended survivin C-terminal
-helix
(Verdecia et al., 2000
).
Moreover, forced expression of survivin counteracted cell death induced by
various apoptotic stimuli (Reed and
Bischoff, 2000
), whereas interference with survivin
expression/function by antisense or dominant negative mutants caused
spontaneous apoptosis and multiple cell division defects with supernumerary
centrosomes, multipolar mitotic spindles and multinucleation
(Chen et al., 2000
;
Li et al., 1999
;
Olie et al., 2000
). This
suggested that survivin may act both as a mitotic regulator and a
cytoprotective factor at cell division, a pathway potentially exploited in
cancer where the survivin gene is broadly upregulated
(Ambrosini et al., 1997
;
Velculescu et al., 1999
).
However, recent studies proposed a different subcellular localization of
endogenous (Uren et al.,
2000
), or transfected
(Skoufias et al., 2000
;
Wheatley et al., 2001
)
survivin, which was found associated with kinetochores of metaphase
chromosomes and the central spindle midzone at anaphase. This pattern was
reminiscent of `chromosomal passenger proteins', molecules that participate in
cleavage furrow formation (Giet and Glover,
2001
; Kaitna et al.,
2000
), and suggested that survivin and IAPs in yeast and C.
elegans (Speliotes et al.,
2000
; Uren et al.,
1999
) could participate in an evolutionary conserved pathway of
cytokinesis (Uren et al.,
2000
).
To gain further insights into the structure-function of the survivin
pathway, we have now revisited the topography and cell cycle regulation of
endogenous survivin with a novel panel of monoclonal and polyclonal
antibodies. Inconsistent with a proposed definition of `chromosomal passenger
protein' (Skoufias et al.,
2000; Uren et al.,
2000
; Wheatley et al.,
2001
), we found that only 20% of cellular survivin is actually
associated with kinetochores, whereas the majority of endogenous survivin is
bound to microtubules, complexed with p34cdc2-cyclin B1, and
required for the assembly of a normal bipolar mitotic spindle.
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Materials and Methods |
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Antibodies
Rabbit polyclonal antibodies (pAb) NOVUS (NOVUS Biologicals, Littleton, CO)
and BTD raised against full-length recombinant survivin were characterized in
previous studies (Grossman et al.,
2001; Grossman et al.,
1999
). An affinity-purified rabbit antibody recognizing survivin
phosphorylated on Thr34 by p34cdc2-cyclin B1
(
-survivinT34*) was generated and characterized in previous
studies (O'Connor et al.,
2000
). A mouse monoclonal antibody (mAb) 8E2 (IgG1, Neomarkers
Inc. Fremont, CA) raised against full-length recombinant survivin was
described previously (Li et al.,
1998
). A new mAb panel to survivin was generated in mice by
standard hybridoma technology using bacterially expressed, full-length
recombinant survivin as an immunogen. Antibody producing cells were fused to
NS1 myeloma cells, and hybridomas were cloned twice by limiting dilution.
Three new mAbs to survivin, 32.1, 60 and 58 (all of IgG1 subtype), were
established and confirmed for reactivity with recombinant survivin by ELISA,
and for recognition of
16.5 kDa survivin by western blotting of HeLa cell
extracts. A mouse mAb to ß-actin was from Sigma (clone AC-15, A5441). A
rabbit polyclonal antibody to FLAG was from Sigma (F7425). A mouse mAb 2E1
reacting with the p80 subunit of the Ku nuclear antigen was characterized
previously (Rothermel and Altieri,
1998
). A mouse mAb 1G12 to intercellular adhesion molecule-1
(ICAM-1) was characterized previously
(Duperray et al., 1997
).
Proteins, peptides and epitope mapping
Full-length human survivin in pGEX2T vector (Pharmacia) was expressed as a
GST fusion protein as described previously
(Li et al., 1998). The GST
frame was removed by overnight incubation with 10 U/ml thrombin (Sigma),
followed by incubation with 100 µl of benzamidine-coated beads (Sigma,
A8332) for 4 hours at 4°C. Two truncated survivin mutants,
Met1-Gly99 and Glu100-Asp142, were
also expressed as GST fusion proteins, released from the GST frame and
purified as described above. A series of partially overlapping synthetic
peptides was generated to duplicate the entire survivin sequence between
Met1 and Phe101, including
Met1-Pro12, Ala3-Ile19,
Pro12-Gly30, Leu28-Ala39,
Met38-Thr48, Pro47-Phe58,
Cys57-Trp67, Lys79-Lys90 and
Val89-Phe101. For epitope mapping, aliquots of the
various recombinant survivin proteins (1 µg/ml), or survivin synthetic
peptides (5 µg/ml) dissolved in 1% DMSO, were immobilized on plastic
microtiter wells (Immulon-2, Dynatech Laboratories, Chantilly, VA) in
bicarbonate buffer, pH 9.5 (100 µl/well) for 18 hours at 4°C. Wells
were blocked with 3% gelatin for 1 hour at 37°C, rinsed and incubated with
1:3 serial dilutions of culture supernatants of mAbs 32.1, 58, 60 or mAb 1G12.
In other experiments, serial dilutions of pAbs NOVUS or BTD, mAb 8E2 or
control rabbit serum were used as primary antibodies. After washes in TBS, pH
7.4, containing 0.1% bovine serum albumin and 0.5% Tween-20, binding of the
primary antibodies was revealed by addition of biotin-conjugated, rabbit
anti-mouse or goat anti-rabbit IgG for 1 hour at 37°C, followed by
streptavidine-alkaline phosphatase and determination of absorbance at
A405 using p-nitrophenyl phosphate (Sigma).
Subcellular fractionation
HeLa cells at 0.5-1x107 were lysed in two volumes of Hepes
buffer (25 mM Hepes, pH 7.5, 100 mM KCl, 2 mM EGTA, 1% Triton X-100, 1 mM
Na3 VO4, 1 mM PMSF) for 20 minutes at 4°C and
centrifuged at 900 g for 10 minutes at 4°C. The pellet-1
was collected as nuclear fraction and the supernatant-1 (post nuclear
sedimentation (PNS) fraction) was centrifuged at 2000 g for 10
minutes at 4°C. The supernatant-2 was centrifuged at 100,000
g in a Beckman TLA 100.4 rotor for 30 minutes at 4°C, and
the resulting supernatant (supernatant-3, cystosol) and the pellet-3
(cytoskeleton associated proteins) were collected. The nuclear and
cytoskeletal fractions were suspended in equal volumes of Hepes buffer and
analyzed for survivin expression by western blotting.
For sub-nuclear fractionation, HeLa cells at 2x107 were washed once in PBS, pH 7.4, and lysed in 1 ml of solution 1 (100 mM Pipes, pH 7.5, 1 mM EGTA, 1 mM MgCl2, 1% Triton X-100, 1 mM Na3 VO4, 1 mM PMSF). After centrifugation at 900 g for 5 minutes at 4°C, the supernatant was collected (PNS fraction) and nuclei (pellet-1) were further washed once with solution 1 and suspended in 1 ml of solution 2 (10 mM Pipes pH 7.5, 300 mM sucrose, 100 mM KCl, 3 mM MgCl2, 1 mM EGTA, 1 mM Na3 VO4, 1 mM PMSF) containing 200 µg/ml of DNAse. The resulting cell extract was incubated for 45 minutes at 33°C and then centrifuged at 1500 g for 10 minutes at 4°C. The supernatant-2 (DNAse released proteins, DRp fraction) was collected and the pellet-2 was resuspended in 1 ml of solution 2. 0.25 M ammonium sulfate (final concentration) was added dropwise and the extract was incubated for 5 minutes at room temperature. After centrifugation at 4000 g for 5 minutes at 4°C, the fraction containing nucleoplasmic proteins was collected (supernatant-3, NPp fraction) and the pellet-3 was suspended in 1 ml of solution 2. NaCl was added dropwise to a final concentration of 2 M, and the extract was further incubated for 10 minutes at 0°C and re-centrifuged at 8000 g for 5 minutes at 4°C. The supernatant-4 (remnant nucleoplasmatic and outer nuclear matrix proteins, ONMp fraction) and the pellet-4 suspended in 1 ml solution 2 (nuclear matrix, NM fraction) were collected and analyzed for survivin expression by western blotting.
Enriched centrosome fractions were prepared from HeLa cells as previously
described (Li et al., 1999).
Briefly, cells were solubilized in 50 ml of 1 mM Hepes, pH 7.2, 0.5% NP-40,
0.5 mM MgCl2, 0.1% ß-mercaptoethanol plus protease inhibitors.
The cell lysate was passed five times through a 10 ml serological pipette,
centrifuged at 2500 g for 10 minutes and incubated in 10 mM
Hepes and 1 µg/ml DNAse I for 30 minutes at 0°C. The mixture was
overlaid on a 5 ml 60% sucrose cushion containing 10 mM Hepes, pH 7.2, 0.1%
Triton X-100, and 0.1% ß-mercaptoethanol and centrifuged at 10,000
g for 30 minutes. The interface of the sucrose cushion
containing enriched centrosomal fractions was collected, separated on a 5-15%
SDS polyacrylamide gel and analyzed for survivin expression by western
blotting.
For microtubule co-sedimentation experiments, HeLa cell extracts (10 mg/ml) in MES buffer (0.1 M 2-[morpholino]ethane sulphonic acid, 1 mM EGTA, 1 mM MgSO4), pH 6.6, were subjected to three sequential rounds of temperature-dependent microtubule polymerization/depolymerization at 37°C or 0°C for 30 minutes each, respectively. After the last round of polymerization, microtubules were centrifuged through a sucrose cushion at 39,000 g for 30 minutes at 30°C and aliquots of pellet and supernatant were separated on a 5-15% SDS polyacrylamide gel and analyzed for survivin expression by western blotting. Protein extracts were quantified by Bradford protein assay.
Immunoprecipitation, immunoblotting and immunofluorescence
Asynchronously growing HeLa cells or HeLa cells arrested at the
metaphase-anaphase transition by treatment with 0.2 µM taxol (Sigma) for 16
hours at 37°C were solubilized in lysis buffer containing 0.03% CHAPS. 200
µg of precleared HeLa cell extracts were immunoprecipitated with mAbs 32.1
(10 µg/ml), or pAb NOVUS to survivin (4 µg/ml) for 16 hours at 4°C,
with precipitation of immune complexes by addition of 50 µl of a 50:50
protein A slurry, as described (O'Connor
et al., 2000). For immunoblotting, aliquots of the various
subcellular fractions or survivin immunoprecipitates were separated by
electrophoresis on a 5-15% SDS-PAGE, transferred to nylon membranes and
incubated with various primary antibodies (1-5 µg/ml), followed by
HRP-conjugated secondary antibodies (Amersham) and chemiluminescence
(Amersham).
Immunofluorescence and confocal microscopy were carried out as described
(Li et al., 1998). HeLa cells
grown on optical grade coverslips (12 mm diameter) were washed for 30 seconds
at 37°C in microtubule stabilizing buffer (MSB) containing 0.1 M Pipes, pH
6.9, 1 mM EGTA, 2.5 mM GTP, 4% polyethyl glycole 6000. Cells were incubated
for 3-8 minutes at 37°C in MSB containing 0.5% Triton X-100 or 0.1% NP-40,
washed twice in MSB for 30 seconds at 37°C, and fixed in MSB containing
3.7% EM grade formaldehyde for 20 minutes. Coverslips were incubated with mAbs
32.1 or 8E2 to survivin (10 µg/ml), 20C6 to tubulin, pAbs NOVUS or BTD to
survivin (5-10 µg/ml), or a human autoimmune CREST antibody (1:5000,
generously provided by Joseph Craft, Yale University School of Medicine). DNA
was stained with Hoechst 33342. Binding of the primary antibodies was revealed
by addition of fluorescein (FITC)- or Texas red (TR)-conjugated antibodies of
the appropriate specificity (Molecular Probes, Inc. Junction City, OR). After
washes, coverslips were mounted in Mowiol 4-88 (Hoechst, Frankfurt/Main,
Germany), and analyzed on a Zeiss Axiophot microscope or by confocal laser
scanning microscopy (CLSM Bio-Rad 1024). Files obtained from confocal
microscopy were assembled with Adobe PhotoShop 5.0.
In vitro kinase assay
p34cdc2 kinase activity was assessed in immunoprecipitates from
untreated or taxol-treated HeLa cells, as described previously
(O'Connor et al., 2000).
Briefly, cells were harvested under nondenaturing conditions and lysed using
sonication in 1x ice-cold lysis buffer (50 mM Tris, pH 7.5; 1% NP-40;
0.25% DOC; 150 mM NaCl; 0.2 mM EGTA; 1 mM EDTA, pH 8.0) containing 20 mM NaF,
1 mM Na3 VO4, plus PMSF. After centrifugation, the
supernatant was precleared by incubation with 10 µl MEP HyperCel
(Gibco/BRL) beads for 1 hour at 4°C, and incubated with a mAb to
p34cdc2 (Zymed) for 1 hour at 4°C followed by addition of 10
µl of MEP HyperCel beads overnight at 4°C. The immune complexes were
recovered and washed twice with lysis buffer and twice with reaction buffer
(20 mM Hepes, pH 7.4; 10 mM MgCl2; 0.5 mM DTT). The
p34cdc2 immunoprecipitates were incubated with 1 µg of histone
H1 (Boehringer Mannheim) and 5 µCi [
-32P]ATP (Amersham
Pharmacia) in 20 µl of reaction buffer, for 30 minutes at 30°C. The
reaction was terminated by addition of 2x SDS sample buffer, and samples
were separated on a 15% SDS-polyacrylamide gel followed by autoradiography or
western blotting with mAb 32.1 (1 µg/ml) or pAb NOVUS (1 µg/ml).
Lipid-based antibody loading of synchronized HeLa cells
HeLa cells were seeded at 0.5-1x105 in a 24-well plate and
synchronized at the G1/S boundary by a 16 hour culture in the presence of 2 mM
thymidine. 4 hours after thymidine release, cells were loaded with 2 µg of
mAb 8E2 or pAb NOVUS to survivin, or 2 µg control mouse or rabbit IgG in
250 µl of serum-free medium, in the presence of BioPORTERTM Protein
Transfection Reagent (Gene Therapy System, BP509604). After 4 hours of
incubation, the antibody uptake reaction was terminated by addition of 250
µl of complete growth medium containing 20% FCS. The efficiency of
intracellular antibody loading was determined using 1 µg of FITC-conjugated
goat IgG, followed by fluorescence microscopy analysis of transduced cultures.
Typically, this protocol resulted in expression of green fluorescence in
80% of treated cells. Cells were fixed 11 hours after thymidine release,
labeled with an antibody to tubulin and analyzed by immunofluorescence, as
described above. Images were collected using an IX70 Olympus inverted
microscope equipped with 40x (0.85 NA) and 60x (1.4 NA) objectives
and Inovision (Raleigh, NC) image analysis software.
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Results |
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Identification of immunochemically distinct subcellular pools of
survivin
In subcellular fractionation experiments, pAb NOVUS to full-length survivin
(O'Connor et al., 2000)
detected a
16.5 kDa survivin band both in cytosolic (Cy), cytoskeletal
(Csk) and nuclear (N) HeLa cell fractions, by western blotting
(Fig. 2A). In control
experiments, cytosolic and cytoskeletal fractions reacted with an antibody to
ß-actin, whereas nuclear fractions contained the p80 subunit of Ku
nuclear antigen, by immunoblotting (not shown). Surprisingly, the new mAb
panel exhibited a differential and mutually exclusive recognition of survivin
in isolated subcellular fractions. mAb 32.1 recognized a 16.5 kDa survivin
band in the nuclear, but not in the cytosolic fraction (PNS, post-nuclear
sedimentation comprising cytosolic and cytoskeletal extracts)
(Fig. 2B). Conversely, mAbs 58
or 60 bound to survivin exclusively in the cytosol, whereas no reactivity was
observed with nuclear extracts, by western blotting
(Fig. 2B). Similarly, in
extracts of HeLa cells over-expressing FLAG-tagged survivin, a nuclear pool of
survivin recognized by the antibody to FLAG or pAb NOVUS was entirely
unreactive with mAb 60 (Fig.
2C). By contrast, all three antibodies indistinguishably
recognized a more prominent (see below) cytosolic pool of FLAG-tagged,
over-expressed survivin by western blotting
(Fig. 2C).
|
The potential association of the various subcellular pools of survivin with
the mitotic apparatus was next investigated. As shown in
Fig. 3A, mAb 32.1 did not
recognize survivin bands in centrosome-enriched HeLa cell fractions,
consistent with its lack of reactivity with cytosolic survivin
(Fig. 2B). By contrast, mAbs 58
and 60 immunoblotted a 16.5 kDa centrosome-associated survivin band
(Fig. 3A), in agreement with
previous observations (Li et al.,
1999). In other experiments, none of the three mAbs 32.1, 58 or 60
recognized microtubule-associated survivin, co-sedimented after three rounds
of temperature-dependent microtubule polymerization/depolymerization
(Fig. 3B). However, a strong
survivin band was immunoblotted in association with polymerized microtubule by
pAb BTD to full-length survivin (Fig.
3B).
|
Distinct subcellular localization of endogenous survivin pools
In interphase HeLa cells, mAb 8E2 recognized survivin with a filamentous
pattern consistent with localization to cytoplasmic microtubules, by
immunofluorescence and confocal microscopy
(Fig. 4Aa). During mitosis, mAb
8E2 strongly labeled spindle poles and the entire length of mitotic spindle
microtubules at metaphase (Fig.
4Ab), and anaphase (Fig.
4Ac), and intensely stained midbodies at telophase
(Fig. 4Ad), in agreement with
previous observations (Li et al.,
1998; Wheatley et al.,
2001
). By dual immunofluorescence labeling, mAb 8E2 reactivity
remained restricted to mitotic spindle microtubules, and did not co-localize
with the punctate staining of a human anti-centromere CREST antibody with
kinetochores of metaphase chromosomes (Fig.
4Ae). Microtubule destruction by colchicine treatment did not
affect the reactivity of the CREST antibody with metaphase chromosomes,
whereas it abolished the labeling of mitotic spindle microtubules by mAb 8E2
(Fig. 4Af). In striking
contrast to the mAb 8E2 pattern, the novel mAb 32.1 did not react with mitotic
spindle microtubules, but exhibited a punctate labeling of metaphase
chromosomes that co-localized with the reactivity of the antiserum to CREST
with kinetochores (Fig. 4Ba).
At anaphase, mAb 32.1 labeling transferred to the microtubules of the central
spindle midzone, and concentrated in midbodies at telophase, whereas labeling
by the CREST antibody remained associated with separating sister chromatids
(Fig. 4Bb-d). Analysis of a
chromosomal spread revealed that mAb 32.1 reacted with kinetochores of
individual chromosomes (Fig.
4Cc, inset), and co-localized with the CREST labeling
(Fig. 4Ca-c). By contrast, mAb
8E2 did not react with individual chromosomes, but recognized a
colchicine-resistant structure that contains
-tubulin
(Fig. 4Cd,e).
|
Consistent with the observation that pAbs to full-length survivin recognized both nuclear and cytosolic survivin by western blotting (Fig. 2; Fig. 3), pAbs NOVUS (Fig. 5Aa-c) and BTD (Fig. 5Ad-f) simultaneously identified both pools of survivin within the same mitotic cell, with intense labeling of spindle poles and mitotic spindle microtubules (mAb 8E2 pattern), and a more limited reactivity with the central spindle midzone (mAb 32.1 pattern) (Fig. 5A). At telophase, both pAbs recognized survivin in midbodies (Fig. 5Ac,f), consistent with the individual reactivity of mAbs 8E2 and 32.1 (Fig. 4A,B). In peptide mapping experiments, pAbs NOVUS and BTD reacted with survivin epitopes Ala3-Ile19, Met38-Thr48, Pro47-Phe58 and Cys57-Trp67 (Fig. 5B). Although the mAb 8E2 epitope was localized to the peptide Cys57-Trp67 (Fig. 5B) and the inefficient protein recognition by western blotting (not shown), suggest that mAb 8E2 may react with a conformational epitope within Cys57-Trp67. In agreement with their lack of reactivity with nuclear survivin and microtubule-associated survivin, mAbs 58 and 60 did not provide clear patterns of survivin localization in mitotic or interphase HeLa cells, by immunofluorescence (not shown).
|
Differential cell cycle regulation of cytosolic and nuclear survivin
pools
In subcellular fractionation experiments, pAb NOVUS reacted with a 16.5 kDa
survivin band in cytosolic extracts and in a subnuclear fraction predominantly
containing nucleoplasmic proteins (Fig.
6A). By contrast, no survivin bands were detected in subnuclear
fractions containing DNAse-released proteins (DRp), outer nuclear matrix
proteins (ONMp), or nuclear matrix proteins (NMp)
(Fig. 6A). A ratio of 1:6
was derived for the survivin pool associated with nucleoplasmic proteins
versus cytosolic survivin, by quantitative western blotting with pAb NOVUS
(Fig. 6B). In synchronized
cytosolic extracts, a background level of survivin in interphase cells
detected by pAb NOVUS abruptly increased at mitosis
(Fig. 6C), 9 hours after
release from a mimosine block, as determined by DNA content analysis and flow
cytometry (Fig. 6E)
(Li et al., 1998
). This
coincided with mitotic phosphorylation of survivin on Thr34 by
p34cdc2-cyclin B1, as determined by western blotting with a
Thr34-phospho-specific antibody (
-survivinT34*)
(Fig. 6C), and in agreement
with previous observations (O'Connor et
al., 2000
). However, in synchronized nuclear extracts, survivin
detected by pAb NOVUS began to accumulate in S phase 6 hours after mimosine
release, thus potentially suggesting active nuclear import at this cell cycle
phase, and continued to increase throughout mitosis
(Fig. 6D) 9 and 12 hours after
G1 release (Fig. 6E). In
addition, no survivin bands phosphorylated on Thr34 were detected
in synchronized nuclear extracts at the various cell cycle phases, by western
blotting with
-survivinT34*
(Fig. 6D). In control
experiments, synchronized nuclear extracts, but not cytosolic extracts,
strongly reacted with mAb 2E1 to the p80 subunit of Ku nuclear antigen
(Rothermel and Altieri, 1998
),
thus corroborating the specificity of the subcellular fractionation protocol
(Fig. 6C,D).
|
Differential association of nuclear versus cytosolic survivin with
p34cdc2-cyclin B1
Survivin immunoprecipitated with pAb NOVUS from asynchronous or
taxol-arrested HeLa cells was found associated with p34cdc2, by
western blotting of the immune complexes with an antibody to
p34cdc2 (Fig. 7A,B),
and in agreement with previous observations
(O'Connor et al., 2000).
Consistent with its lack of reactivity for cytosolic survivin
(Fig. 2), mAb 32.1 did not
immunoprecipitate survivin from asynchronously growing HeLa cells
(Fig. 7A). By contrast, mAb
32.1 immunoprecipitated survivin from taxol-arrested HeLa cells after nuclear
envelope breakdown (Fig. 7B).
However, no association of p34cdc2 with survivin immunoprecipitated
by mAb 32.1 was demonstrated by Western blotting of the immune complexes
(Fig. 7B). Consistent with the
data presented above,
-survivinT34* strongly recognized a
Thr34-phosphorylated survivin band in pAb NOVUS immunoprecipitates
from taxol-synchronized cells, but not in mAb 32.1 immunoprecipitates under
the same experimental conditions (Fig.
7C). Analysis of kinase activity of p34cdc2
immunoprecipitates at various time intervals after taxol treatment revealed
the presence of a
32 kDa phosphorylated histone H1 band used as a
substrate, and a faster migrating band of 16.5 kDa consistent with the size of
survivin (Fig. 7D).
Immunoblotting with pAb NOVUS confirmed the identity of the phosphorylated
16.5 kDa band coimmunoprecipitated with p34cdc2 as survivin
(Fig. 7D). However, mAb 32.1
did not react with any bands in p34cdc2 immunoprecipitates under
the same experimental conditions (Fig.
7D). DNA content analysis by propidium iodide staining and flow
cytometry confirmed the sustained mitotic arrest induced by taxol treatment
(Fig. 7E).
|
Global antibody targeting of endogenous survivin causes defects of
spindle microtubules
Lipid-based intracellular loading of pAb NOVUS in synchronized HeLa cells
resulted in gross abnormalities of mitotic progression and a sixfold increase
in the number of cells with aberrant multipolar mitotic spindles, compared
with control cultures loaded with non-immune rabbit IgG
(Fig. 8A,B). A threefold
increase in aberrant multipolar mitoses was also observed after intracellular
loading of mAb 8E2, compared with non-immune mouse IgG (not shown). By
contrast, treatment with pAb NOVUS did not result in gross abnormalities of
cytokinesis, and formation of midbodies was unaffected
(Fig. 8A). Similar results were
obtained after microinjection in the nucleus of mAb 32.1 (data not shown). In
parallel experiments, loading of synchronized HeLa cells with pAb NOVUS
resulted in a reproducible increase in mitotic index, as compared with control
cultures (Fig. 8C).
|
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Discussion |
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The immunochemical differences between nuclear and cytosolic survivin may
explain, in part, the conflicting data of survivin localization reported in
the literature (Li et al.,
1998; Skoufias et al.,
2000
; Uren et al.,
2000
; Wheatley et al.,
2001
). Two regions in survivin that exhibited strikingly
differential antibody reactivity were identified here as
Cys57-Trp67, which is exposed in cytosolic and
centrosome-associated survivin, but masked in nuclear and microtubulebound
survivin, and Ala3-Ile19, which is accessible in
kinetochore-associated survivin, but not in cytosolic survivin. A plausible
interpretation of these data is that separate post-translational modifications
may differentially affect epitope accessibility of nuclear versus
cytosolic/microtubule-bound survivin in vivo. Consistent with this view, all
the new mAbs described here that exhibited differential reactivity with
survivin pools in vivo, indistinguishably reacted with bacterially expressed
recombinant survivin, in vitro. Together, these data may explain the
incomplete results of survivin localization to kinetochores reported by Uren
et al., which were obtained using only one antibody directed to the same
survivin sequence Ala3-Ile19
(Uren et al., 2000
). In
addition, simple over-expression experiments in the absence of corroborating
biochemical evidence are generally viewed as inadequate to determine protein
subcellular localization(s) (Skoufias et
al., 2000
; Wheatley et al.,
2001
), and the use of tagged molecules
(Skoufias et al., 2000
;
Wheatley et al., 2001
) can
notoriously generate incomplete or biased patterns of subcellular topography
(Ramanathan et al., 2001
).
This may be especially true for survivin, where addition of a sequence tag may
affect the N-terminus dimerization interface or the C-terminus
microtubule-binding domain in the extended
-helices
(Verdecia et al., 2000
), which
may explain the failure of Wheatley et al. to localize NH2-tagged
survivin to the anaphase central spindle
(Wheatley et al., 2001
).
Finally, although Wheatley et al. independently confirmed the spindle
microtubule localization of survivin using mAb 8E2
(Wheatley et al., 2001
), their
inability to observe metaphase spindle labeling with pAb NOVUS is inconsistent
with our findings, and may reflect inadequacies in experimental protocol, as
we have previously described a taxol-free microtubule-stabilizing procedure
for optimal detection of survivin on metaphase microtubules (see Materials and
Methods) (Li et al.,
1998
).
In addition to immunochemical differences, cytosolic and nuclear survivin
exhibited distinct kinetics of accumulation at mitosis, and only
microtubule-bound survivin physically associated with p34cdc2, and
was phosphorylated on Thr34 by p34cdc2-cyclin B1.
Previously, phosphorylation of survivin by p34cdc2-cyclin B1 was
identified as a requisite for apoptosis inhibition
(O'Connor et al., 2000), and
expression of a phosphorylation-defective survivin Thr34
Ala
dominant negative mutant caused apoptosis of cells traversing mitosis
(O'Connor et al., 2000
).
Intriguingly, the putative survivin-like IAPs in yeast and C. elegans
that have been solely implicated in cytokinesis
(Speliotes et al., 2000
;
Uren et al., 1999
), lack a
Thr34 consensus phosphorylation site, which is instead perfectly
conserved in genuine survivin homologs in Xenopus or avian genomes
(data not shown). This suggests that a more recent function of survivin in
cytoprotection may have evolved from a primordial role in cell division, and
coincided with the acquisition of a p34cdc2-cyclin B1
phosphorylation site on Thr34, a modification that remained
segregated with microtubule-associated survivin, but not with
kinetochore-bound survivin.
Whether the small subset of kinetochore-associated survivin participates in
an evolutionary conserved pathway of cleavage furrow formation
(Speliotes et al., 2000;
Uren et al., 1999
) is
currently unknown. Clearly, the proposed definition of survivin solely as a
chromosomal passenger protein (Skoufias et
al., 2000
; Uren et al.,
2000
; Wheatley et al.,
2001
) is inconsistent with the complexity of multiple survivin
localization, predominant association with microtubules and defects of mitotic
spindle assembly resulting from antibody (this study) or antisense
(Li et al., 1999
) targeting.
Rather, we propose a different model for the role of survivin at mitosis,
which involves regulation of microtubule function and formation of a normal
bipolar apparatus, a proposition fully consistent with the catastrophic defect
of microtubule assembly observed in survivin-knockout mice
(Uren et al., 2000
). Because
of the complexity and multiplicity of functions of the kinetochore
(Rieder and Salmon, 1998
), it
is also possible that kinetochore-associated survivin may not participate in
cytokinesis at all. Alternative functions of kinetochore-associated survivin
may involve regulation of sister chromatid separation at metaphase
(Dobles et al., 2000
) or, as
previously suggested (Li et al.,
1998
), the integrity of the mitotic spindle checkpoint
(Cahill et al., 1998
).
In summary, the data presented here identify a far more complex pattern of
survivin modifications and subcellular topography than recently claimed
(Skoufias et al., 2000;
Uren et al., 2000
;
Wheatley et al., 2001
) and
anticipate a requirement of the survivin pathway in controlling the assembly
of a normal bipolar mitotic apparatus. Future experiments will investigate the
dynamic relationship between nuclear and microtubule-bound survivin and
dissect their potential involvement in cytoprotection and mitotic control.
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