Survivin exists in immunochemically distinct subcellular pools and is involved in spindle microtubule function
Paola Fortugno1,
Nathan R. Wall1,
Alessandra Giodini1,
Daniel S. O'Connor1,
Janet Plescia1,
Karen M. Padgett2,
Simona Tognin3,
Pier Carlo Marchisio3 and
Dario C. Altieri1
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
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Fig. 1. Characterization of new mAbs to survivin. (A) Reactivity with survivin
domains. Full-length (Met1-Asp142) or truncated
(Met1-Gly99 or Glu100-Asp142)
survivin were immobilized on plastic microtiter plates (1 µg/ml) and
incubated with the indicated dilutions of mAbs 8E2, 32.1, 58 or 60 to
survivin, or mAb 1G12 to ICAM-1. (B) Western blotting. Increasing dilutions of
recombinant survivin (r-Survivin) at 1, 0.2, 0.04, 0.008 µg were
immunoblotted with mAbs 32.1, 58 or 60 or rabbit polyclonal antibody BTD,
followed by chemiluminescence. Molecular weight (Mr) markers in kDa
are shown on the left. (C) Epitope mapping. Partially overlapping synthetic
peptides duplicating the indicated survivin sequences were immobilized on
plastic microtiter plates (5 µg/ml) and incubated with increasing dilutions
of mAbs 32.1, 58 or 60. For panels A and C, binding of the various antibodies
was determined by ELISA at A405. Data are representative of a
single experiment out of at least three independent determinations.
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Fig. 2. Sub-cellular distribution of immunochemically distinct survivin pools. (A)
Subcellular distribution of endogenous survivin. Equal volumes of nuclear (N),
cytosolic (Cy) and cytoskeleton (Csk) fractions were immunoblotted with pAb
NOVUS followed by chemiluminescence. (B) Differential antibody reactivity with
endogenous survivin. Aliquots (100 µg) of PNS (post-nuclear sedimentation,
comprising cytosolic and cytoskeletal extracts) and N (nuclear) fractions were
immunoblotted with mAbs 32.1, 58 or 60, or pAb NOVUS followed by
chemiluminescence. (C) Subcellular distribution of over-expressed survivin.
Aliquots (100 µg) of PNS or N extracts of HeLa cells transfected with
N-terminal FLAG-tagged survivin were immunoblotted with the indicated mAb 60,
pAb NOVUS or an antibody to FLAG.
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Fig. 3. Immunochemical characterization of centrosome- and microtubule-associated
survivin. (A) Differential antibody reactivity with cytosolic or
centrosome-associated survivin. PNS extracts (100 µg) or
centrosome-enriched fractions (Ce, 100 µg) were immunoblotted with mAbs
32.1, 58 or 60. (B) Differential mAb reactivity with microtubule-associated
survivin. Cellular microtubules were assembled through three successive rounds
of temperature-dependent polymerization/depolymerization, centrifuged through
a sucrose gradient and aliquots of input (I), supernatant (S) and pellet (P)
were immunoblotted with mAbs 32.1, 58, 60 or pAb BTD. Molecular weight markers
in kDa are shown on the left of each panel.
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Fig. 4. Differential subcellular localization of survivin pools. HeLa cells grown
on optical grade coverslips were fixed in MSB, and processed for dual
immunofluorescence labeling and confocal microscopy. (A) mAb 8E2 pattern. HeLa
cells were labeled with mAb 8E2 to survivin (FITC) and stained for DNA with
Hoechst 33342 in interphase (a), metaphase (b), anaphase (c) and telophase
(d). Dual immunofluorescence labeling with mAb 8E2 (FITC) and CREST antibody
(Texas Red, TR) is shown before (e) or after (f) microtubule depolymerization
by colchicine treatment. (B) mAb 32.1 pattern. Dual immunofluorescence
labeling of HeLa cells with mAb 32.1 to survivin (FITC) and CREST antibody
(TR) at metaphase (a), anaphase A (b), anaphase B (c) and telophase (d). DNA
was stained with Hoechst 33342. (C) Chromosomal localization. Chromosomes were
labeled with mAbs 32.1 (a) or 8E2 (d,e) to survivin (FITC), or with CREST
antibody (TR, b). (c) Merged image of reactivity of mAb 32.1 and CREST with
individual chromosomes. For all panels, DNA was labeled with propidium
iodide.
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Fig. 5. Simultaneous identification of both survivin pools within mitotic cells.
(A) Immunofluorescence. The experimental conditions are the same as in
Fig. 4A,B. Image merging of the
reactivity of pAb NOVUS (a-c) or BTD (d-f) (TR) and mAb 20C6 to tubulin,
(FITC) at metaphase (a,d), anaphase (b,e) and telophase (c,f). DNA was labeled
with DAPI. Arrows indicate labeling for survivin on the central spindle
midzone (mAb 32.1 pattern). (B) Epitope mapping. Increasing concentrations of
pAb NOVUS or BTD, or mAb 8E2 were analyzed for reactivity with the indicated
immobilized survivin peptides at A405 by ELISA.
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Fig. 6. Modulation of nuclear and cytosolic survivin pools during cell cycle
progression. (A) Sub-nuclear localization of survivin. Equal volumes of the
indicated sub-nuclear fractions were immunoblotted with pAb NOVUS. DRp, DNAse
released proteins; NMp, nuclear matrix proteins; NPp, nucleoplasmic proteins;
ONMp, outer nuclear matrix proteins; PNS, post nuclear sedimentation. (B)
Relative ratio between nuclear and cytosolic survivin. Equal volumes (5 µl)
of sub-nuclear PNS and NPp fractions were immunoblotted with pAb NOVUS
followed by chemiluminescence. (C,D) Differential modulation and
Thr34 phosphorylation of nuclear and cytosolic survivin during cell
cycle progression. Mimosine-synchronized HeLa cells were harvested at the
indicated time intervals after release and PNS (C) or nuclear (D) extracts
were immunoblotted with pAb NOVUS to survivin,  -survivinT34*
to Thr34 phosphorylated survivin, mAb 2E1 to the p80 subunit of Ku
or an antibody to ß-actin. (E) Mimosine-synchronized HeLa cells were
harvested at the indicated increasing time intervals after release and
analyzed for DNA content by propidium iodide staining and flow cytometry.
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Fig. 8. Multipolar mitoses induced by global intracellular antibody targeting of
survivin. (A) Immunofluorescence. Synchronized HeLa cells were loaded with pAb
NOVUS (a-f) or control rabbit IgG (g-i), and analyzed for tubulin staining by
confocal microscopy at metaphase (a-c,g-i) or telophase (d-f). DNA was stained
with DAPI. (B) Quantification of aberrant mitoses. The experimental conditions
are the same as in A. HeLa cells exhibiting multipolar mitoses were counted
after loading with pAb NOVUS or non-immune rabbit IgG. (C) Mitotic index. HeLa
cells were scored microscopically after intracellular loading with pAb NOVUS
or non-immune rabbit IgG. None, no antibody loading.
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© The Company of Biologists Ltd 2002
32 and 16.5 kDa. Western blotting of the
p34cdc2 immune complexes was carried out with mAb 32.1 or pAb
NOVUS. (E) Cell cycle analysis. Synchronized HeLa cells were harvested at the
indicated time intervals after taxol treatment and analyzed by propidium
iodide staining and flow cytometry. IP, immunoprecipitate; WB, western
blotting. Molecular weight markers in kDa are indicated on the left of each
panel.