Journal of Cell Science 104,139-150 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
139
Microinjection of a monoclonal antibody against SPN antigen, now
identified by peptide sequences as the NuMA protein, induces micronuclei
in PtK2 cells
Markku Kallajoki, Jens Harborth, Klaus Weber and Mary Osborn*
Max Planck Institute for Biophysical Chemistry, Department of Biochemistry, W-3400 Goettingen, FRG
*Author for correspondence
SUMMARY
Several high molecular mass proteins which relocate
from the interphase nucleus to the spindle poles during
mitosis have been defined by antibodies. Microinjection
experiments have shown that at least the antigen defined
by SPN antibody plays a functional role during mitosis.
Recently the cDNA sequence for human NuMA antigen
was established and epitopes for antibodies to centrophilin, and to 1F1 and 1H1 antigens were found to
be included in the NuMA protein. Here we show that
immunoprecipitated SPN antigen reacts with an autoimmune human NuMA serum. In addition three peptides
derived from immunoprecipitated human SPN by
cyanogen bromide cleavage and covering more than fifty
amino acids show a perfect fit with the sequence predicted for NuMA protein. Thus SPN antigen and NuMA
are the same protein. Injection of SPN-3 antibody into
INTRODUCTION
NuMA protein was first described as a predominantly
nuclear protein which relocated at mitosis to the pole
regions. This protein with a molecular mass originally
reported as 300 kDa was present in several human cell types
but not in cells from other species (Lydersen and Pettijohn,
1980). It appeared to be autoantigen since NuMA antibodies were found in the sera of patients with rheumatic diseases (Price et al., 1984). Other high molecular mass proteins which relocate from the interphase nucleus to the poles
of the mitotic spindle have also been characterized by their
reactivity with monoclonal antibodies or with human
autoimmune sera. These include centrophilin (Tousson et
al., 1991), SP-H antigen (Maekawa et al., 1991), SPN antigen (Kallajoki et al., 1991) and the 1H1 and 1F1 antigens
(Compton et al., 1991). Recently the cDNA for NuMA has
been cloned from expression libraries by screening either
with a human autoimmune NuMA serum (Yang et al.,
1992) or with the monoclonal antibody 1F1 (Compton et
al., 1992). NuMA protein expressed in bacteria encoded by
the cDNA clone contained the epitopes for the 1H1 antibody, for the NuMA antibody 2E4 and for the centrophilin
interphase or mitotic PtK2 cells results in cells with
micronuclei. For cells injected in prophase,
prometaphase or metaphase 90%, 78% and 77% display defective cytokinesis or yield daughter cells with
micronuclei. In contrast only 16% of cells injected in
anaphase are abnormal. Thus SPN/NuMA antigen may
be required during early, but not during later, stages of
mitosis. Surprising parallels are seen between the effects
of microinjecting SPN-3 antibody and treatment with
colcemid and taxol of PtK2 and HeLa cells. Our results
identify an important role during mitosis for the
SPN/NuMA antigen.
Key words: micronuclei, microinjection, mitosis, nuclear matrix,
NuMA protein, SPN antigen
antibody 2D3 (Compton et al., 1992). Thus in spite of differences in the reported molecular mass, in staining patterns and in other properties reported in the literature,
NuMA, centrophilin, 1F1 and 1H1 seem to represent different names for the same protein.
SPN antigen was originally characterized using monoclonal antibodies raised against a urea extract of nuclei isolated from the human adrenal cortex carcinoma cell line
SW13 (Kallajoki et al., 1991). The SPN polypeptide had a
reported molecular mass of 210 kDa and was a nuclear
matrix component during interphase. During mitosis it relocated to the centrosome at prophase and accumulated at the
spindle poles in metaphase and anaphase. During telophase
it relocated to the reassembling nucleus. SPN antigen
extracted from mitotic HeLa cells bound microtubules stabilized in vitro with taxol (Kallajoki et al., 1992), a property it shared with SP-H antigen (Maekawa et al., 1991).
Treatment of mitotic cells with the microtubule disrupting
drug nocodazole resulted in the dispersal of SPN antigen
into many foci, which acted as microtubule organizing centers during recovery from the block (Kallajoki et al., 1991).
A similar result was found for centrophilin by Tousson et
al. (1991). In taxol-treated mitotic cells, SPN antigen was
140
M. Kallajoki and others
found at the center of the multiple microtubular asters
induced by the drug (Kallajoki et al., 1992) and similar
results have been reported with SP-H antigen (Maekawa et
al., 1992). When five SPN antibodies were microinjected
into the cytoplasm of HeLa cells, one antibody - SPN-3 caused a block in mitosis, spindle aberrations and resulted
in cells with micronuclei (Kallajoki et al., 1991). These
results suggest that SPN antigen acts as a microtubule
minus-end organizer in mitotic cells and also plays a critical role in mitosis.
The question of whether SPN antigen and the NuMA
antigen are identical is particularly important because the
experiments just described show that SPN antigen plays an
essential role in mitosis. Here we show that immunoprecipitated SPN antigen was recognized by a NuMA-type
human autoimmune serum. In addition, immunoprecipitation with SPN-3 antibody was used to isolate SPN antigen
for cyanogen bromide cleavage, while sequence analysis of
three of the resulting peptides covering more than 50
residues showed a perfect fit to the protein sequence predicted for the human NuMA protein. These results show
that the SPN antigen of HeLa cells is in fact NuMA. We
have also extended the microinjection experiments with
SPN-3 antibody on HeLa cells to PtK2 cells, so that we
could inject cells at identifiable mitotic stages. Injection into
the cytoplasm of interphase cells resulted after 24 hours in
>50% cells with micronuclei. Injection of mitotic cells led
to the formation of two micronucleated daughter cells in a
stage-specific manner. Thus injection of prophase,
prometaphase or metaphase cells resulted in the formation
of two daughter cells with micronuclei whereas injection
after the onset of anaphase resulted in the formation of
daughter cells with normal nuclei. When metaphase cells
were injected with SPN-3 and fixed after different times,
daughter cells with micronuclei were first observed after 90
minutes. The finding that SPN and NuMA antigens are
identical proteins, together with the results of the microinjection experiments, argue for a functional role of the
NuMA protein in mitosis.
MATERIALS AND METHODS
Cell culture
PtK2 cells were cultured in Minimal Essential Medium with Earle’s
salts, glutamine, non-essential amino acids, 1 mM sodium pyruvate and 10% fetal calf serum. Large scale cultures (2 liters) of
HeLa S3 cells were grown in spinner flasks in Joklik’s MEM supplemented with 10% fetal calf serum or 10% normal calf serum.
Cell synchronization
To obtain synchronous mitotic cells, cells were first grown in 2.5
mM thymidine for 18-22 h and then centrifuged for 5 min at 1500
revs/min in a Sorvall RC-5 centrifuge using the GSA rotor. The
cells were resuspended in fresh medium with 0.06 µg/ml of colcemid and cultured for a further 16-20 h.
Mitotic index determination
The mitotic index was determined from 10 ml of cells. Cells were
pelleted by centrifugation for 10 min at 800 revs/min, suspended
in 0.075 M KCl, and incubated for 10 min at 37°C. Then cells
were again centrifuged for 10 min at 800 revs/min and fixed in
methanol:acetic acid (3:1) for 10 min. The suspension was centrifuged for 10 min at 800 revs/min and the pellet was suspended
in 0.5 to 1.0 ml of fixative. Drops of this suspension were smeared
on microscope slides, air dried, and stained with Hoechst 33258
as described below. The mitotic index was >95%.
Immunoprecipitation of SPN antigen from
synchronized HeLa cells
HeLa S3 cells, grown in suspension and synchronized as described
above were harvested at a concentration between 3 × 105 and 5 ×
105 cells/ml and washed three times in PBS (137 mM NaCl, 7
mM Na 2HPO4, 1.5 mM KH2PO4, 2.7 mM KCl, pH 7.1) by centrifugation at 1000 revs/min for 5 min. The final pellet was suspended in ice-cold 0.5% Triton X-100 in PBS supplemented with
1 mM EGTA, 1 mM PMSF, 10 µg/ml aprotinin, 10 µM leupeptin,
1 µM pepstatin and 10 µM E-64 at a cell concentration of 2 ×
107/ml. Typically 30-50 ml were obtained from 2 liters of HeLa
cells. Cells were incubated on ice for 5 min and then homogenized with a Potter-Elvehjem homogenizer. Cell disruption
was confirmed by phase contrast microscopy. The suspension was
centrifuged for 5 min at 13,000 g. The mitotic cell supernatant
was aliquoted and stored at −70°C.
For large-scale immunoprecipitation of the SPN antigen, 10 ml
aliquots of the mitotic HeLa cell extract were thawed and clarified by centrifugation in a Beckman ultracentrifuge TL-100 using
the TLA-100.3 fixed angle rotor at 200,000 g for 20 min. Four
ml of SPN-3 antibody (Kallajoki et al., 1991) supernatant from
overgrown hybridoma cultures were added per 10 ml of cell
extract. The mixture was incubated on ice for 1 h. 120 µl of affinity-purified rabbit antibody against mouse immunoglobulins (1.9
mg/ml; Dako, Glostrup, Denmark) was added and incubation continued for 30 min. Then 1 ml of pre-swollen Protein A-Sepharose
beads (Pharmacia, Uppsala, Sweden) was added and the suspension was incubated for 1 h at 4°C with rocking. Beads were harvested by centrifugation at 1000 revs/min for 5 min and washed
three times with ice-cold 0.5% Triton X-100 in PBS. The final
pellet was suspended in 600 µl of 2× concentrated SDS-PAGE
sample buffer, heated to 85°C for 10 min and centrifuged at 13,000
g for 5 min. The SPN supernatants were frozen at −20°C, and
later used to isolate SPN antigen (see below).
Electrophoresis and immunoblotting
Proteins were separated by SDS-PAGE on 7.5% acrylamide 0.5
mm thick minigels. Each 4 mm well was loaded with 5 µl of
immunoprecipitated material or with 5 µl of supernatant fraction
before or after collection of the immunocomplexes. Gels were
either stained with Coomassie brilliant blue or used for
immunoblotting. Proteins were transferred electrophoretically to
nitrocellulose (0.2 µm pore size, Schleicher and Schuell Co.,
Dassel, FRG) in transfer buffer containing 25 mM Tris, 192 mM
glycine, 0.01% SDS and 20% methanol at 250 mA constant current for 16 h. Protein transfer was controlled by Ponceau S staining. The sheets were blocked with 4% bovine serum albumin
(BSA) in Tris-buffered saline (TBS: 20 mM Tris-HCl, pH 7.4,
0.15 M NaCl) overnight. Nitrocellulose sheets were incubated
without primary antibody or with SPN-3 as undiluted culture
supernatant, or with a NuMA-type human autoimmune serum (a
kind gift from Dr. H. Ponstingl, German Cancer Research Center,
Heidelberg, FRG) diluted 1:500 into 1% BSA, 0.2% Tween 20 in
TBS. Incubation was for 2 h at 37°C. Washes were with 0.2%
Tween 20 in TBS. The second antibody for SPN-3 was rabbit
anti-mouse immunoglobulins conjugated to peroxidase (Dako
Immunochemicals, Klostrup, Denmark) diluted 1:200. To detect
the human NuMA autoantibody, an affinity-purified, peroxidaseconjugated, sheep anti-human immunoglobulins diluted 1:500
(Amersham, UK)was used. After 1 h incubation the nitrocellulose
SPN/NuMA antibodies induce micronuclei
141
sheets were washed with TBS-Tween and the peroxidase was
detected using 4-chloronaphthol as the chromogen.
fied from ascites fluid with a Protein G column (Mab Trap, Pharmacia).
Isolation of SPN and microsequencing
Immunofluorescence analysis
The SPN supernatants in SDS-PAGE sample buffer described
above were unfrozen and immediately boiled. They were then subjected to preparative SDS-PAGE using 7.5% polyacrylamide gels
1 mm in thickness. Gels were stained with Coomassie brilliant
blue for 5 min and then destained in 7.5% acetic acid and 5%
methanol for approximately 5 min. As soon as the SPN band
became visible, the protein band was cut out. Gel pieces were suspended in equilibration buffer (0.125 M Tris-HCl, pH 6.8, 0.1%
SDS, 1 mM EDTA). After 1 h the buffer was changed and equilibration continued for 1 h on a rocking table. The buffer was then
removed and the gel pieces were stored at −20°C. When sufficient pieces had been collected, the SPN protein was electrophoretically eluted into dialysis tubing which had been washed
with electrophoresis buffer. Electrophoresis was in 25 mM Tris,
192 mM glycine, 0.1% SDS at 150 V for 48 h. The solution (1
to 1.5 ml) was removed from the tubing and concentrated to about
400 µl in the Speed Vac (Savant Instruments). The protein was
recovered by chloroform/methanol precipitation (Wessel and
Flügge, 1984) and suspended in trifluoroacetic acid (TFA). Water
was added to reach 70% TFA. Treatment with CNBr was in the
dark at room temperature for approximately 16 h. The reaction
was terminated by drying in the Speed Vac. The residue was
extracted with 100 µl of 50% TFA followed by centrifugation.
The soluble fragments recovered after drying were dissolved in
0.1 M Tris-HCl, pH 8.5, 6 M guanidine-HCl, 1 mM DTT and
subjected to reversed phase HPLC using a Vydac 214 TP52
column. Solvent A was 0.1% TFA and solvent B was 0.08% TFA,
70% acetonitrile. Gradient elution was from 10% to 90% solvent
over 90 min. Peak fractions were subjected to sequence analysis
using an Applied Biosystems gas phase sequenator (model A470)
or a Knauer sequenator (model 810). Both instruments were
equipped with an on line PTH-amino acid analyzer.
HeLa cells were fixed in −10°C methanol for 10 min. HeLa cells
were incubated with the human NuMA-type autoimmune serum
diluted 1:500 for 1 h. The second antibody was FITC-conjugated
goat anti-human IgGs (Miles Laboratories Inc., Kankaler, USA).
To identify microinjected PtK2 cells the cells were fixed in
−10°C methanol for 10 min and incubated for 1 h with rhodamineconjugated, affinity-purified goat anti-mouse IgGs (Dianova,
Hamburg, FRG). When detergent treatment was used, cells were
extracted in microtubule stabilizing buffer containing 0.5% Triton
X-100 for 15 s (see Kallajoki et al., 1991) prior to fixation with
methanol. For double labelling of SPN antigen and tubulin, cells
were fixed and incubated first with an affinity-purified rabbit-tubulin antibody at 20 µg/ml (Osborn et al., 1978). Then samples were
incubated simultaneously with affinity-purified, fluorescein-conjugated sheep anti-mouse IgGs (Amersham) to reveal the SPN
antibody distribution and with affinity-purified rhodamine-conjugated goat anti-rabbit IgGs (Dianova, Hamburg) to reveal the
microtubular distribution. Cells were then stained for 1 min with
Hoechst 33258 (20 µg/ml in 25% ethanol/75% PBS) and embedded in Mowiol 4.88 (Hoechst AG, Frankfurt, FRG). Microscopy
was with a Zeiss Axiophot microscope. Micrographs were taken
with Kodak T-Max 400 film processed at 1600 ASA.
Microinjection
Interphase PtK2 cells
These were grown on round 12 mm coverslips. Shortly before
microinjection the medium was changed to the same medium
without sodium bicarbonate but with 20 mM Hepes, pH 7.05.
Small colonies usually containing 10-15 cells were injected using
a semiautomatic microinjection apparatus (Eppendorf, Hamburg,
FRG). Usually 30-40 cells were injected in each experiment in a
marked area.The coverslips were placed in fresh medium without
Hepes and the cells were kept at 37°C for 24 or 25.5 h before
being fixed and subjected to immunofluorescence analysis.
Mitotic PtK2 cells
For microinjection of mitotic cells, PtK2 cells were grown on 25
mm × 25 mm coverslips with etched grid markings (Bellco, Vinland, NJ, USA). In a single grid square a particular mitotic cell
was identified and the stage of mitosis determined by phase contrast microscopy. This cell was then immediately microinjected.
On each coverslip 10-20 mitotic cells were localized and injected
within 20-30 min. After 3 h at 37°C cells were fixed and subjected to immunofluorescence analysis. In a second series of
experiments involving metaphase PtK2 cells, 10 metaphase PtK2
cells were microinjected with SPN-3 antibody. Only a single
metaphase cell was injected per coverslip. Cells were either fixed
immediately after microinjection or after 2, 5, 10, 20, 30, 60, 90,
120 or 180 min at 37°C.
Antibodies
The SPN-3 antibody and the SPN-5 control antibody were puri-
RESULTS
The HeLa SPN polypeptide is recognized by NuMA
antibodies
SPN present in mitotic HeLa cell extracts was purified by
immunoprecipitation using the monoclonal murine SPN-3
antibody followed by affinity-purified rabbit anti-mouse
immunoglobulins. The resulting immune complexes were
harvested by Protein A-Sepharose beads, extensively
washed, and dissolved in hot SDS-sample buffer. SDS-gels
of SPN antigen purified by immunoprecipitation were either
stained with Coomassie brilliant blue or electrophoretically
blotted on to nitrocellulose. Fig. 1A, lane 3, shows a
Coomassie brilliant blue-stained gel of the SPN-3 precipitated material. A band corresponding to a molecular mass
of 210 kDa was clearly visible and this band reacted when
tested in immunoblotting with SPN-3 antibody as shown in
Fig. 1B, lane 2. A faint band at ~180 kDa was also visible
in the immunoblots and is probably a breakdown product
of the SPN antigen. Fig. 1A also documents the complex
protein composition of the mitotic HeLa extract used for
immunoprecipitation (lane 1) and the resulting supernatant
fraction devoid of SPN antigen (lane 2). The SPN antigen
was clearly a minor component of mitotic cells since it was
not visible in lane 1.
Fig. 1B, lanes 3 and 4, shows that the polypeptide precipitated by the SPN serum was strongly decorated by a
human NuMA-type autoimmune serum. Again weak staining of a 180 kDa polypeptide was detected.
When the SPN antibody and the NuMA antibody were
used in immunofluorescence microscopy on HeLa cells,
identical staining patterns were seen (compare Fig. 2A and
C). Thus interphase nuclei were positive, and in addition
strong staining was seen at the pole regions of metaphase
cells.
142
M. Kallajoki and others
Fig. 1. (A) Coomassie brilliant blue-stained 7.5% SDS-PAGE gel
of immunoprecipitated material. Lane 1, supernatant fraction from
mitotic HeLa cells before addition of monoclonal SPN-3
antibody; lane 2, supernatant fraction after collection of immune
complexes with Protein A-Sepharose; lane 3, SPN antigen (arrow)
immunoprecipitated with SPN-3 antibody. The arrowhead shows
the position of the IgG heavy chain. Molecular mass standards are
given in kDa at the left. (B) Immunoblotting of material
immunoprecipitated with SPN-3 antibody. Lane 1, negative
control with peroxidase-conjugated rabbit anti-mouse IgGs only.
Lane 2, SPN-3 antibody followed by the same second antibody as
in lane 1. Lane 3, negative control with affinity-purified
peroxidase sheep anti-human antibody. Lane 4, NuMA type
human autoimmune serum with the same anti-human antibody
used in lane 3. Note that the 210 kDa polypeptide precipitated
with SPN-3 antibody is stained both by the SPN antibody (lane 2)
and by the NuMA type autoimmune serum (lane 4). The faint
band at around 180 kDa in lanes 2 and 4 may be a degradation
product of the SPN antigen. The rabbit anti-mouse second
antibody reacts with the mouse immunoglobulin chains in the
immunoprecipitate (lanes 1 and 2).
HeLa SPN polypeptide sequences are contained
in the predicted NuMA sequence
Although the SPN polypeptide could be electrophoretically
transferred from SDS-PAGE to nitrocellulose or
polyvinyldifluoride membranes and subsequently detected
by SPN antibodies, the efficiency of transfer was poor. Several variations of the electroblotting procedure of the SPN
polypeptide present in the immunoprecipitate did not yield
enough protein on the blot to allow subsequent microsequencing procedures. Therefore the immunoprecipitate was
processed by preparative SDS-PAGE. After a short staining and destaining of the gel the 210 kDa band was excised.
Gel fragments were collected and subjected to electroelution using the procedure described in Materials and methods. The SPN polypeptide was recovered and treated with
cyanogen bromide (CNBr). Soluble fragments were separated on a C4 reversed phase HPLC column.
Although the elution profile obtained for the CNBr fragments was very complex, three fragments were pure upon
sequence analysis. These were peptide 1-GDILQTPQFQ-,
peptide 2-GNELERLXAALMESQ- and peptide 3-
Fig. 2. Comparison of the immunofluorescence staining patterns
of HeLa cells with SPN-3 monoclonal antibody (a) and NuMA
autoimmune serum (c). SPN antibody (a) and the same cells
visualized in phase contrast (b). NuMA antibody (c) and the same
cells visualized in phase contrast (d). Note the metaphase cells in
(a) and (c) (arrow), the prophase cell in (a) (arrowhead) and the
anaphase cell in (c) (arrowhead). Note the staining of interphase
nuclei and the mitotic spindle pole regions by both antibodies.
×600.
LKKAHGLLAEENRGLGERANLGRQF LEVE. When the
cDNA sequence of human NuMA became subsequently
available (Yang et al., 1992; Compton et al., 1992), the
three peptide sequences we obtained on HeLa SPN showed
a perfect fit with the predicted protein sequence of human
NuMA protein. Peptide 1 occupies residues 206 to 215 and
lies directly before the putative helical domain. Peptide 2
covers residues 978 to 992 located in the helical domain.
The unassigned residue X is an arginine in both NuMA
sequences. Peptide 2 is an overlapping fragment which
arose due to incomplete cleavage at methionine 989. Peptide 3 spans residues 1440 to 1468 and is located towards
the C-terminal end of the helical domain. In agreement with
the known specificity of CNBr cleavage, all three peptide
sequences are preceded by a methionine in the proposed
protein sequence of NuMA. Our peptide sequences covering a total of more than 50 amino acid residues show that
the SPN antigen of HeLa cells is NuMA.
Microinjection of SPN antibodies into PtK2 cells
Since rat kangaroo PtK2 cells stay relatively flat during
SPN/NuMA antibodies induce micronuclei
143
Table 1. The effect of SPN-3 antibody microinjected
into the cytoplasm of interphase PtK2 cells
Experiment no.
Incubation time (h)
No. cells injected
No. antibody positive cells*
No. cells with micronuclei†
1
25.5
36
57
44(77%)
2
25.5
35
54
36(66%)
3
24
30
46
27(58%)
SPN-3 antibody was injected at a concentration of 5.4 mg/ml.
*Microinjected cells were identified by staining with a rhodaminelabelled anti-mouse antibody after the incubation times shown. Note the
increase in number of antibody positive cells over the number of cells
originally injected due to cell division.
†Cells with micronuclei were identified with Hoechst staining. The
percentage of antibody positive cells that have micronuclei is shown in
parenthesis.
mitosis, it is possible to inject mitotic cells. Here we document the results of injecting SPN-3 antibody (a) into interphase PtK2 cells, and (b) into mitotic PtK2 cells at defined
mitotic stages. All experiments used SPN-3 antibody at a
concentration of ~5 mg/ml. As a control SPN-5 antibody
was used at the same concentration, since this antibody does
not affect mitosis of HeLa cells (Kallajoki et al., 1991).
Interphase PtK2 cells
In three experiments, SPN-3 antibody was injected into
interphase cells and the cells were fixed and stained after
24 or 25.5 hours (Table 1). 58-77% of the antibody positive cells had developed micronuclei (Fig. 3 a-b). The other
antibody positive cells showed only a single nucleus.
Rounded cells, arrested in mitosis in a prometaphase-like
state as reported for HeLa cells (Kallajoki et al., 1991),
were not seen when of PtK2 cells were injected with SPN3 antibody. In two control experiments, 75 interphase PtK2
cells were injected with SPN-5 antibody, and when examined one day later only a single cell had micronuclei.
Mitotic cells injected at defined mitotic stages
Single cells in different stages of mitosis were injected with
SPN-3 antibody. The stage of mitosis of a particular cell
was determined by phase contrast microscopy and the cell
was immediately injected with SPN-3 antibody. The positions of injected cells on the grid marked coverslip were
noted. 10-20 such cells were injected on each coverslip and
the cells were incubated at 37°C for 3 hours before fixation and immunofluorescence analysis. Results are summarized in Table 2 and Figure 4. In PtK2 cells, injection of
SPN-3 antibody did not seem to cause mitotic arrest, but
instead resulted in daughter cells that had micronuclei. The
results depended on whether cells were injected before or
after the onset of anaphase. Thus injection of prophase (Fig.
4a, b), prometaphase (Fig. 4c, d) or metaphase (Fig. 4e, f)
cells resulted in formation of micronucleated daughter cells
for 90%, 65% and 61% of the cells injected in each of these
mitotic stages (Table 2, Fig. 4a-f). When injection was performed after the onset of anaphase, only 5% of the resulting daughter cells had micronuclei, while almost all other
cells yielded two daughter cells with apparently normal
nuclei (e.g. Fig. 4g, h). Very occasionally, injection of SPN3 led to defective cytokinesis, resulting in one or three
daughter cells with one, two or multiple nuclei (see Table
Fig. 3. Microinjection of SPN-3 antibody into interphase PtK2
cells. Interphase PtK2 cells were injected with SPN-3 antibody.
After 24 hours the cells were extracted with a Triton X-100 buffer
to remove unbound antibody and then stained with goat antimouse IgGs (a) and with Hoechst (b). Injected cells are positive in
both a and b, while microinjected cells are positive only in b. Note
the micronuclei visible in 4 of the 5 injected cells. ×600.
2). As a control, 20 prophase, prometaphase and metaphase
cells were injected with SPN-5 antibody at an equivalent
concentration (Fig. 5). This led to defective cytokinesis in
one cell, resulting in a single cell with two nuclei. Two
normal daughter cells were formed from each of the other
19 injected cells, regardless of the stage of mitosis used for
injection. Examples are shown for cells injected with the
control SPN-5 antibody in prophase (Fig. 5a, b),
prometaphase (Fig. 5c, d) and metaphase (Fig. 5e, f).
In a second set of experiments, metaphase cells were
identified and injected with SPN-3 antibody. They were
incubated for different times, and then fixed and stained to
visualize the injected SPN-3 antibody, the mitotic spindle
and the chromosomes. Cells fixed immediately (Fig. 6a-c),
and at 2, or 5 (Fig. 6d-f) or 10 minutes after injection
showed normal mitotic spindles and normal chromosome
arrangements as judged by staining with tubulin antibody
and with Hoechst. The injected SPN-3 antibody was distributed diffusely throughout the cell with spindle pole
regions detectable above the background of free antibody
144
M. Kallajoki and others
Fig. 4. The effect of injecting SPN-3 antibody into PtK2 cells at different mitotic stages. A single cell microinjected in prophase (a, b)
prometaphase (c, d) metaphase (e, f) or anaphase (g, h), respectively, is shown. After injection cells were incubated for 3 hours, fixed and
stained with second antibody. Left panels show the distribution of the injected SPN-3 antibody and the right panels show the DNA
staining of the same cells with Hoechst 33258. Note the micronucleated daughter cells in a to f and the single normal nuclei in the cells in
g, h. All, ×540.
SPN/NuMA antibodies induce micronuclei
145
Fig. 5. Microinjection of SPN-5 control antibody into PtK2 cells in different mitotic stages. Cells were injected in prophase (a,b), in
prometaphase (c,d,) or in metaphase (e,f ). Left panels show the distribution of the injected SPN-5 antibody and the right panels show the
Hoechst 33258 staining for DNA of the same cells. After 3 hours two normal daughter cells with a single nucleus were found. ×540.
Table 2. Results of microinjecting of SPN-3 antibody into PtK2 cells at different mitotic stages
Microinjected cells forming
Mitotic
stage
No
inj.
No
found
Daughter
cell pairs with
normal nuclei
Prophase
Prometaphase
Metaphase
Anaphase
10
29
39
30
10
23
31
19
1 (10%)
5 (22%)
7 (23%)
16 (84%)
Defective cytokinesis
Daughter
cell pairs with
micronuclei
Total
1N
2N
Micronuclei
Normal
(%)
Abnormal
(%)
9 (90%)
15 (65%)
19 (61%)
1 (5%)
3 (13%)
5 (16%)
2 (11%)
−
1
1
1
−
−
2
4
1
10
22
23
84
90
78
77
16
Cells in mitosis were identified by phase-contrast microscopy and immediately injected with SPN-3 antibody. After 3 hours they were stained with
rhodamine-labelled anti-mouse antibody and with Hoechst dye to determine whether they had normal nuclei or had micronuclei. During the 3 h time
interval almost all cells completed mitosis and formed daughter cell pairs. The first column shows the number of cells at each mitotic stage that were
microinjected. The second column shows the number of injected cells that could be relocated after fixation and staining. The difference between the
numbers in columns 1 and 2 reflects the loss of injected cells. Microinjected cells are further subdivided depending on whether microinjection resulted in
daughter cell pairs with normal nuclei (column 3), daughter cell pairs with micronuclei (column 4) or defective cytokinesis (column 5).
146
M. Kallajoki and others
Fig 6. Metaphase PtK2 cells microinjected with SPN-3 antibody and fixed after different incubation times. Cells were fixed immediately
after injection (a-c), after 5 min (d-f), after 20 min (g-i), after 60 min (j-l), or after 120 min (m-o). Left panels show the distribution of the
microinjected SPN antibody, panels in the middle show tubulin staining and the right panels show staining with Hoechst 33258. (a and d)
show that the injected SPN-3 antibody binds to spindle pole regions. Tubulin staining shows intact mitotic spindles in (b and e) and
normal distribution of microtubules in (h, k and n). Note the micronucleated daughter cells in (m-o), 120 min after microinjection. ×540.
SPN/NuMA antibodies induce micronuclei
147
(Fig. 6a, d), demonstrating that the antibody recognized its
native antigen inside the living cell. Cells injected with
SPN-3 antibody in metaphase appeared to proceed through
anaphase normally as judged by cells fixed 20 minutes after
injection (Fig. 6g-i). In telophase the injected cells showed
normal organization of microtubules in the intercellular
bridge (Fig. 6k, n), but the decondensing chromosomes
appeared strangely clumped in cells fixed 60 minutes after
injection (Fig. 6j-l). Micronucleated cells were first seen 90
minutes after injection of metaphase cells, and showed a
normal organization of the interphase microtubule network
(Fig. 6n).
Comparison of the effects of microinjected SPN-3
antibody and drug treatment of interphase PtK2
and HeLa cells
When SPN-3 antibody is injected into interphase PtK2 cells,
after 24 hours 58-77% of the cells had micronuclei, while
the remaining cells had apparently normal nuclei (Table 1;
Fig. 3). In contrast, when the same experiment was performed with HeLa, approximately half of the cells had
micronuclei while most of the remaining cells were rounded
and arrested in a prometaphase-like state. These cells
showed distorted mitotic spindles (Kallajoki et al., 1991).
Micronuclei can also be induced by mitotic inhibitors
such as colchicine, colcemid, vinblastin and griseofulvin as
well as by X-irradiation (Ringertz and Savage, 1976). The
percentage of cells that develop micronuclei depended on
the mitotic inhibitor, the dose of the drug and the time of
exposure. Cells from different species also displayed different sensitivities to the same mitotic inhibitor. Drugs such
as colcemid also induced mitotic arrest.
We therefore compared the response of interphase PtK2
and HeLa cells to colcemid and taxol treatment. A striking
difference in response of the two cell types to the drugs
was observed. After 24 hours in either colcemid (0.5 µg/ml)
or in taxol (20 µg/ml), ~80% of the PtK2 cells had micronuclei while the others were flat, still attached to the coverslip and had only a single nucleus (Fig. 7a-c). In contrast,
after 24 hours in either drug the vast majority of HeLa cells
(>95%) were rounded, and arrested in a prometaphase-like
state (Fig. 7b, d). The majority of flat spread cells attached
to the coverslips had normal nuclei while a minority had
micronuclei.
Thus PtK2 cells respond to both SPN-3 antibody injection, and to drug treatment, by preferentially forming
micronucleated cells (~70% micronucleated after SPN-3
injection vs 80% after drug treatment). In contrast HeLa
cells respond to SPN-3 antibody injection and to drug treatment in a qualitatively similar but quantitatively different
manner. Thus 50% of the cells are micronucleated after
SPN-3 injection vs 1% after drug treatment, and ~50% of
cells are arrested in a prometaphase-like state after SPN-3
injection vs 95% after drug treatment.
DISCUSSION
Our results show that SPN antigen is in fact NuMA protein. Immunoprecipitated SPN antigen is recognized on
immunoblots by a human autoimmune NuMA antiserum
Fig. 7. (a, b) Phase contrast micrographs of PtK2 (a) and HeLa (b)
cells treated for 24 hours with colcemid at 0.5 µg/ml. Note the
different appearance of the two cell lines after colcemid treatment.
(c, d) Hoechst stain of PtK2 (c) and HeLa (d) treated as in (a, b).
Note the micronucleated cells present in (c) and the cells arrested
in prometaphase in (d). Many of the round cells visible in (b) have
been lost during the staining because they are not firmly attached
to the coverslip. a, b, × 95; c, d, × 240.
(Fig. 1B). In addition, sequence analysis of three peptides
derived from HeLa SPN show identity with the predicted
amino acid sequence of human NuMA protein (see Results).
Antibodies 1F1 and 1H1 (Compton et al., 1991), the centrophilin antibody 2D3 (Tousson et al., 1991) and the
NuMA antibody 2E4 (Lydersen and Pettijohn, 1980) all
react with recombinant NuMA protein expressed in E. coli
(Compton et al., 1992). Thus it is clear that at least NuMA
protein, the 1F1 and the 1H1 antigens, centrophilin and SPN
antigen are the same protein. SP-H antigen (Maekawa et
al., 1991), characterized with a human autoantibody, shows
very similar behaviour during the cell cycle and a similar
molecular mass to the antigens listed above and also shares
other properties with SPN and the other antigens (Kallajoki
et al., 1992; Compton et al., 1992). Thus it seems probable that all six different antigens represent different names
for the same protein. Alternatively, since a functional role
148
M. Kallajoki and others
for SPN antigen has been directly demonstrated by microinjection experiments, the same function can now be assumed
to have been shown for NuMA/centrophilin/1F1/1H1 and
probably also for SP-H antigen.
A total of 108 cells in different stages of mitosis were
injected. Injection of SPN-3 antibody into PtK2 cells in
prophase, prometaphase or metaphase resulted in abnormal
mitosis in 90%, 78% and 77% of cells, respectively. Usually
each injected cell yielded two daughter cells with micronuclei (Table 2 and Fig. 4). In contrast, injection into PtK2
cells in anaphase resulted in abnormal mitosis in only 16%
of the injected cells while the other daughter cells had
normal nuclei. Anaphase and/or telophase are not particularly short when compared to other mitotic stages. Thus for
PtK 2 cells prometaphase is about 12 minutes (range 5-15
minutes), metaphase about 16 minutes (range 7.5-18 minutes), anaphase about 7 minutes (range 5-10 minutes) and
telophase 4-7 minutes (De Brabander et al., 1986). Thus it
seems unlikely that the results with anaphase cells could be
explained by the rate of SPN-3 antibody binding. Instead
our results suggest a central role for SPN/NuMA beginning
in early prophase, and ending in early anaphase. Thus the
critical time interval, in which SPN/NuMA has to be functional, starts with the relocation of SPN/NuMA to the centrosomes at the beginning of prophase. In taxol-treated cells
the striking redistribution of SPN (and SP-H) antigen into
multiple foci which act as organizing centers for the multiple microtubule asters also occurs during the same time
interval (Maekawa et al., 1991; Kallajoki et al., 1992). That
injection of SPN-3 antibody into PtK2 cells does not cause
abnormal spindles but does prevent taxol-induced aster formation in cells injected in early mitotic stages (Kallajoki et
al., 1992) may perhaps be explained by the different structures of the spindle pole and the taxol-induced asters. For
example taxol-induced asters contain neither centules nor
pericentular components such as the 5051 antigen. Thus
taxol-induced asters may be more labile than spindle poles
in PtK2 cells
One day after injection of SPN-3 antibody into the cytoplasm of interphase PtK2 cells, 58-77% of cells had
micronuclei. Presumably the antibody gains access to the
SPN antigen as the nuclear membrane breaks down at the
onset of prophase. Daughter cells with micronuclei would
then be formed in a manner analogous to that discussed
above for cells injected in the early mitotic stages. Injection of the same antibody into interphase HeLa cells also
resulted in 38-56% of the cells being micronucleated one
day after microinjection (Kallajoki et al., 1991).
In HeLa cells it was relatively easy to demonstrate that
injection of SPN-3 results not only in micronuclei formation but also in abnormal spindle formation, since a substantial fraction (up to 60%) of the cells were found arrested
in a prometaphase-like state. As judged by tubulin staining,
mitotic spindle microtubules were present, but the spindles
were abnormal and multipolar, and the chromosomes were
widely scattered (Kallajoki et al., 1991). In the current study
with PtK2, cells arrested in prometaphase with abnormal
spindles were not seen. In addition, again at the light microscope level, abnormalities in spindle structure were not seen
by tubulin staining when metaphase cells were injected with
SPN-3 antibody and followed for different times (Fig. 6).
Our results show that interphase HeLa and PtK2 cells also
behave differently when treated for long times with mitotic
drugs such as taxol and colcemid. Thus at 24 hours the vast
majority of HeLa cells became arrested in prometaphase.
Mitotic spindle microtubules were not formed, and only a
small minority of the cells contained micronuclei. In contrast, under the same conditions, ~80% of PtK2 cells had
micronuclei, and cells arrested in mitosis were not found.
Thus the differences seen after microinjection of SPN-3
antibodies into the two cell types is paralleled by the differences seen after drug treatment of the two cell types.
Several functions have been proposed for NuMA protein.
Originally it was suggested that NuMA plays a role in postmitotic nuclear assembly (Lydersen and Pettijohn, 1980;
Pettijohn et al., 1984; Price and Pettijohn, 1986). NuMA
proteins relocate to the nucleus before nuclear lamins, again
suggesting a role in nuclear assembly (Yang et al., 1992).
Although the cDNA sequence of NuMA protein did not
show significant homology to any other protein in the data
bank, the central domain of NuMA is similar to coiled-coil
forming regions of structural proteins such as myosin and
intermediate filament proteins (Yang et al., 1992; Compton
et al., 1992). Thus like these proteins NuMA may form filaments. Recently a nuclear skeleton with 10 nm diameter
filaments and with a 23 nm axial repeat has been described
(He et al., 1990; Jackson and Cook, 1988). These observations and the association of NuMA (Lydersen and Pettijohn, 1980) and SPN antigen (Kallajoki et al., 1991) with
the nuclear matrix suggest that NuMA may be a structural
component of interphase nuclei.
A role for NuMA in spindle function is supported by the
localization of NuMA proteins to mitotic spindle pole
regions (Lydersen and Pettijohn, 1980; Tousson et al., 1991;
Kallajoki et al., 1991, 1992; Maekawa et al., 1991). NuMA
proteins also located to sites of microtubule nucleation
during recovery or after treatment with drugs such as
nocadazole and taxol (Tousson et al., 1991; Maekawa and
Kuriyama, 1991; Kallajoki et al., 1991, 1992). In cells
treated with drugs in prophase or prometaphase, the NuMA
protein is invariably found at the centers of multiple microtubule asters (5-20/cell) (Tousson et al., 1991; Maekawa
and Kuriyama, 1991; Kallajoki et al., 1991, 1992). Injection of SPN-3 antibody into taxol-treated PtK2 cells prevented the formation of such asters (Kallajoki et al., 1992).
In addition SPN or SP-H antigen from mitotic HeLa cells
bound to microtubules in vitro (Maekawa and Kuriyama,
1991; Kallajoki et al., 1992), suggesting that the NuMA
protein may function as a microtubule minus end organizer
in vivo. However we note, as did Yang et al. (1992) and
Compton et al. (1992), that the sequence of the NuMA protein does not reveal known microtubule binding motifs.
Our microinjection experiments with the SPN-3 antibody
(Kallajoki et al., 1991, 1992, and this paper) are currently
the strongest argument for a direct role of NuMA in mitosis. Clearly the target in such injection experiments is the
SPN/NuMA protein. Thus inactivation of this protein can
lead to micronuclei in both HeLa and PtK2 cells. Mitotically arrested cells with abnormal spindles have been
demonstrated after injection of HeLa, but not of PtK2 cells.
Of particular interest is the unexpected similarity in the
effects of antibody injection and of treatment with antimi-
SPN/NuMA antibodies induce micronuclei
totic drugs such as colchicine or taxol. These drugs bind
directly to tubulin or microtubules. Drug treatment led to
micronuclei in low numbers in HeLa cells and in large numbers in PtK2 cells. Treatment of HeLa cells with colcemid
resulted in large numbers of cells which are mitotically
arrested and which do not contain microtubules in the
mitotic spindle. Thus although SPN-3 antibody injection
and colcemid target different proteins, the net result in HeLa
cells was the same, i.e. micronucleation and mitotically
arrested cells due to an inactive spindle in SPN-3 antibody
injected cells or to a non-existent spindle in colcemidtreated cells. In PtK2 cells, both SPN-3 antibody injection
and drug treatment led to micronuclei formation. The mechanism proposed by Ringertz and Savage (1976) for
micronuclei formation supposes that the drug initially
blocks cells in prometaphase, resulting in cells with widely
scattered chromosomes, which cannot form a metaphase
array because of microtubule disturbances. Micronuclei are
formed as the nuclear envelope re-forms around individual,
or groups of chromosomes. Thus the difference between
HeLa and PtK2 cells with both SPN antibody injection and
drug treatment may be that while many HeLa cells are
blocked in prometaphase, PtK2 cells are able to progress
further through mitosis and form micronuclei. Major differences in the response of human and rodent cell lines to
drugs such as colcemid have been noted (Kung et al., 1990;
Rieder and Palazzo, 1992). Kung et al. (1990) have shown
that differences exist in the ability of different cell lines to
progress into the next cell cycle in the absence of mitosis.
They have further suggested that such differences may be
linked to different levels of cyclin B and cdc2 kinase found
in arrested HeLa and CHO cells.
Antibodies to other spindle components have also been
injected into cells, and their effects can be compared and
contrasted to those of the SPN-3 antibody. When injected
in concentrations higher than 6 mg/ml into interphase cells
a rat monoclonal antibody YL 1/2 reacting specifically with
the tyrosylated form of alpha-tubulin, prevented disassembly of the cytoplasmic microtubule complex and therefore
spindle formation, so cells were arrested in mitosis
(Wehland and Willingham, 1983). 80% of the PtK2 cells
injected in prophase with YL 1/2 did not divide and showed
abnormal spindle structures after 2 hours. Polyclonal antibodies with high affinity for beta-tubulin were found to disrupt cytoplasmic microtubules after microinjection of interphase PtK2 cells, whereas mitotic microtubules were
resistant to even high antibody concentrations and cells were
able to proceed through mitosis and divide normally (Füchtbauer et al., 1985). The effects of injecting antibodies against
a variety of non-tubulin spindle associated components have
been summarized by McIntosh and Koonce (1989). Antibodies to CENP-A, -B, -C and -E (Bernat et al., 1990; Yen
et al., 1991) and to the CHO-1 antigen (Nislow et al., 1990)
have recently also been injected into cells. Looking at the
effect of injection of antibodies to such proteins only antibodies to p13suc1 are reported to cause mitotic abnormalities and micronuclei formation (Riabowoi et al., 1989). The
effects seen when p13suc1 antibodies are injected into
fibroblasts are very similar to those seen with the SPN-3
antibody. However the distribution of SPN/NuMA antigen
and p13suc1 during the cell cycle seem different.
149
Finally, NuMA/SPN antigen could also have dual or multiple functions. Thus it could have a structural role in the
interphase nuclei and it could act as a microtubule minus
end organizer during mitosis. Further experiments, e.g. with
in vitro microtubule nucleation model systems, with in vitro
systems for studying nuclear assembly, or with transfected
cells are necessary to get a deeper insight to the function
of the NuMA protein.
The data in this paper were presented at the International Cell
Biology meeting in Madrid in July 1992. We thank Uwe Plessmann, Susanne Isenberg and Monika Dietrich for expert technical assistance, and Claudia Hake and Jaoko Liippo for their photographic expertise. M.K. was supported by a long-term EMBO
fellowship. This work was supported in part by a grant from the
Deutsche Forschungsgemeinschaft to M.O. (Os 70/2-1).
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(Received 26 August 1992 - Accepted 11 November 1992)