Download Calmodulin-binding protein disrupts mitosis

3609
Journal of Cell Science 111, 3609-3619 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
JCS0044
The novel murine calmodulin-binding protein Sha1 disrupts mitotic spindle
and replication checkpoint functions in fission yeast
Randa Craig and Chris Norbury*
Imperial Cancer Research Fund, Molecular Oncology Laboratory, University of Oxford Institute of Molecular Medicine, John
Radcliffe Hospital, Oxford OX3 9DS, UK
*Author for correspondence (e-mail: [email protected])
Accepted 12 October; published on WWW 18 November 1998
SUMMARY
Entry into mitosis is normally blocked in eukaryotic cells
that have not completed replicative DNA synthesis; this ‘SM’ checkpoint control is fundamental to the maintenance
of genomic integrity. Mutants of the fission yeast
Schizosaccharomyces pombe defective in the S-M
checkpoint fail to arrest the cell cycle when DNA
replication is inhibited and hence attempt mitosis and cell
division with unreplicated chromosomes, resulting in the
‘cut’ phenotype. In an attempt to identify conserved
molecules involved in the S-M checkpoint we have screened
a regulatable murine cDNA library in S. pombe and have
identified cDNAs that induce the cut phenotype in cells
arrested in S phase by hydroxyurea. One such cDNA
encodes a novel protein with multiple calmodulin-binding
motifs that, in addition to its effects on the S-M checkpoint,
perturbed mitotic spindle functions, although spindle pole
duplication was apparently normal. Both aspects of the
phenotype induced by this cDNA product, which we term
Sha1 (for spindle and hydroxyurea checkpoint abnormal),
were suppressed by simultaneous overexpression of
calmodulin. Sha1 is structurally related to the product of
the Drosophila gene abnormal spindle (asp). These data
suggest that calmodulin-binding protein(s) are important
in the co-ordination of mitotic spindle functions with
mitotic entry in fission yeast, and probably also in
multicellular eukaryotes.
INTRODUCTION
Yamashita et al., 1990). In such cells the drug treatment results
in unscheduled entry into a mitosis-like state, characterised by
activation of Cdc2, chromatin condensation, the appearance of
mitosis-specific phosphoprotein epitopes and cell death.
Similarly, certain yeast mutants defective in components of the
intracellular S-M checkpoint signalling pathway are unable to
tolerate S phase arrest, instead undergoing catastrophic mitosis
when DNA synthesis is inhibited (Enoch and Nurse, 1990;
Enoch et al., 1992; Weinert et al., 1994; Carr, 1997). In several
instances such components have been shown to be inessential
for the completion of normal, unperturbed cell cycles,
suggesting that their primary function is to relay the cell cycle
arrest signal following inhibition of replication. A second class
of yeast mutants, lacking gene products encoding essential
components or regulators of the replication machinery itself,
fail both to initiate DNA replication and to inhibit progression
into mitosis (Kelly et al., 1993; Araki et al., 1995; D’Urso et
al., 1995; Navas et al., 1995; Sugimoto et al., 1996). For both
classes the mutant phenotype is characterised by mitosis
without prior chromosome replication. In the fission yeast S.
pombe the subsequent formation of a septum either generates
one haploid and one aploid daughter, or cleaves the
unreplicated nucleus in a random fashion, with consequent loss
of viability. Upon microscopic examination, such cells
The successful execution of mitosis requires the co-ordination
of multiple cellular processes including the completion of
DNA replication, reorganisation of the microtubular
cytoskeleton to form a bipolar spindle and the accurate
segregation of the chromosomes into the daughter nuclei. The
highly conserved M phase-promoting cyclin dependent kinase
Cdc2-cyclin B plays a central role in the co-ordination of these
processes, as activation of this kinase is thought to be directly
responsible for initiation of the major mitotic events (Nurse,
1990). In particular, a fraction of Cdc2-cyclin B has been
shown to localise to the microtubule organising centres in
diverse eukaryotes, suggesting that Cdc2 substrates important
for spindle organisation are also localised to the spindle poles
(Bailly et al., 1989; Alfa et al., 1990).
Checkpoint mechanisms that ensure mitosis is attempted
only after the completion of chromosomal DNA replication are
fundamental to the maintenance of genomic integrity, and the
importance of these ‘S-M’ checkpoint mechanisms is most
clearly illustrated by cells that lack them (Enoch and Nurse,
1991). In some mammalian systems, for example, exposure of
S phase-arrested cells to drugs such as caffeine or okadaic acid
overrides the S-M checkpoint (Schlegel and Pardee, 1986;
Key words: Calmodulin, Mitosis, Replication checkpoint, Fission
yeast, Spindle
3610 R. Craig and C. Norbury
therefore resemble those of strains defective in a variety of
aspects of chromosome segregation, collectively called cut
mutants (for cell untimely torn; Hirano et al., 1986).
The yeast spindle pole body (SPB) is a microtubule
organising centre, functionally equivalent to the centrosome in
more complex eukaryotes, that is responsible for the ordered
generation of the microtubular spindle in M phase (for a recent
review see Hagan, 1998). A recent report highlighted the
importance of molecular events at the S. pombe SPB in the coordination of spindle formation with activation of Cdc2-cyclin
B (Bridge et al., 1998). The gene defective in fission yeast
cut12 mutants was found to encode a SPB component (and
potential Cdc2 substrate) that is essential for the generation of
a bipolar spindle; cells lacking Cut12 are therefore unable to
segregate their replicated chromosomes and display the cut
phenotype. Significantly, a gain-of-function mutation in cut12
bypassed the requirement for the Cdc25 phosphatase, a
normally essential activator of Cdc2, while a temperaturesensitive mutation in cdc25 rendered the cut12 mutant
phenotype more severe. These data suggest either that the SPB
component Cut12 can influence the process of Cdc2 activation
or that Cut12 serves to determine the threshold level of Cdc2
activity required for spindle formation.
In this study we have sought to identify mammalian genes
involved in the S-M checkpoint pathway by expressing a
regulatable murine cDNA library in S. pombe. This strategy
has allowed us to identify SHA1, a murine gene encoding a
novel calmodulin binding protein which, when expressed in
fission yeast, both disrupts S-M checkpoint control and
prevents the formation of a bipolar spindle.
MATERIALS AND METHODS
cDNA library construction
A cDNA library (REP.Mm) was constructed in the fission yeast vector
pREP3X (Forsburg, 1993) using poly(A)+ RNA from mid-log phase
Swiss mouse 3T3 fibroblasts. The mRNA was used in a reverse
transcription reaction containing M-MuLV reverse transcriptase
(Stratagene), 5-methyl dCTP and the link-primer (5′)GAGAGAGAGAGAGGATCCGCGGCCGCTTTTTTTTTTTTTTTTTT(3′),
followed by second strand synthesis, end repair and ligation of a SalI
adapter consisting of the following pair of annealed oligonucleotides:
(5′)TCGACGTTAATTAAGC(3′) and (5′)GCT-TAATTAACG(3′), the
second of which was previously phosphorylated with polynucleotide
kinase. All manipulations were carried out as described elsewhere
(Ausubel et al., 1995) and, unless otherwise stated, all reagents were
obtained from Sigma. After size-selection by gel exclusion
chromatography (Sephacryl S-400, Stratagene) to enrich for cDNA
greater than 500 base pairs in length, the cDNA was digested with
BamHI and was ligated into pREP3X digested with SalI and BamHI.
The ligated mixture was used to transform E. coli PLK-F′
(Stratagene). Plasmid DNA prepared from approximately 200,000
independent transformants grown on LB agar plates was purified by
alkaline lysis and CsCl gradient centrifugation. Restriction digestion
of randomly selected plasmids showed that approximately 90% of the
library plasmids contained cDNA inserts.
Strains and yeast methods
All fission yeast manipulations were carried out as described in detail
elsewhere (Moreno et al., 1991; Norbury and Moreno, 1997) using
EMM2 minimal medium (Moreno et al., 1991), containing where
necessary leucine, uracil and adenine at 225 µg/ml. S. pombe leu1-32,
h− was used as the host strain for the cDNA library screen, following
transformation by electroporation (Bio-Rad Gene Pulser). To generate
the SL6Ai strain, pREP6X.SL6A was transformed into an ade6-704,
ura4-D18 h− strain, and after selection for ade+ transformants, integrant
strains were identified as white colonies after replica plating to minimal
agar containing 10 µg/ml adenine (Moreno et al., 1991). For the
calmodulin suppression experiments, a leu1-32, ura4-D18, h− strain
was transformed simultaneously with pREP3-HA3SL6A together with
either pREP4-lacZ or pREP4-cam1 (see below).
Plasmids
In order to generate pREP6X.SL6A, the SL6A cDNA was digested
with SalI and BamHI and was re-cloned into the vector pREP6X
(Forsburg, 1993), which contains the sup3-5 suppressor tRNA gene
and hence allows complementation of the ade6-704 mutation. The
plasmid pREP4-lacZ, containing the E. coli lacZ gene driven by the
nmt1 promoter and the ura4+ selectable marker, was the generous gift
of Susan Forsburg (Salk Institute, La Jolla). For regulatable
expression of a FLAG epitope-tagged version of S. pombe
calmodulin, a cam1 cDNA was amplified from a fission yeast cDNA
library (B. Edgar and C. Norbury, unpublished) using the primers
(5′)CCATGTCGACACCATGACGACGCGTAACCTTACAG(3′) and
(5′)GGTAGGATCCCTACTTGTCGTCATCGTCCTTGTAGTCCTGGAAGAAATGACACGAG(3′). Following digestion with BamHI
and SalI, the cDNA was cloned into pREP4 (Maundrell, 1993;
Forsburg, 1993) digested with BamHI and XhoI. A triple-HA epitope
tagged version of the SL6A cDNA was constructed by amplification
of the SL6A open reading frame and subsequent ligation into pREP3HA3, a derivative of pREP3X encoding a triple HA tag beginning with
an ATG codon between the XhoI and SalI sites (R.C., unpublished).
All plasmid constructions were confirmed by complete sequencing of
the inserts using an ABI 377 and ABI PRISM dRhodamine reagents
(Perkin Elmer). The sequence of the SL6A cDNA was determined on
both strands by primer walking, and was confirmed in part by database
(BLAST) searching of murine expressed sequence tags (dbEST).
Flow cytometry
2×106 cells per time point were collected by centrifugation (6,000 g,
5 minutes), washed once with phosphate-buffered saline (PBS) and
then fixed for 30 minutes in 2 ml 70% ice-cold ethanol, added while
vortexing. After fixation, cells were again collected by centrifugation
and rehydrated in 4 ml PBS before being resuspended in 0.5 ml of
PBS containing RNAse A (Sigma, 100 µg/ml). After incubation at
37°C for 2 hours, 0.5 ml of propidium iodide (Sigma, 4 µg/ml) was
added, and the samples were briefly sonicated. Red fluorescence
(DNA content) and forward light scatter (cell size) were measured by
flow cytometry (FACScan, Becton Dickinson) for 10,000 cells per
sample. Analysis of the data was performed using Lysys II and
CellQuest software.
Microscopy
Cells were fixed and stained for anti-HA or anti-tubulin
immunofluorescence using the combined aldehyde method (Hagan
and Hyams, 1988), with the modification that PBS, PBS-sorbitol and
PBS-BSA were substituted for PEM, PEMS, and PEMBAL. Cells
were stained with anti-tubulin (TAT-1) hybridoma supernatant (the
generous gift of Prof. K. Gull, University of Manchester) diluted 1:10
overnight at 4°C and goat anti-mouse IgG-Cy3 (Sigma) at a dilution
of 1:300 for 3 hours at room temperature. Anti-Sad1
immunofluorescence, subsequent processing of the samples and
microscopy were all performed as described elsewhere (Hagan and
Hyams, 1988; Moreno et al., 1991; Bridge et al., 1998). Images were
acquired using a Sony CCD camera and Kromascan software (Kinetic
Imaging) and were assembled using Adobe Photoshop.
Immunoblotting
Western blotting was performed according to standard techniques as
Calmodulin-binding protein disrupts mitosis 3611
described (Ausubel et al., 1995). Whole cell lysates (Moreno et al.,
1991) were separated by SDS-PAGE and transferred onto
nitrocellulose membranes (Hybond ECL, Amersham). Proteins were
detected using enhanced chemiluminescence (ECL, Amersham)
following one hour incubations at room temperature with the
respective primary and secondary antibodies.
RESULTS
Murine cDNAs that induce the cut phenotype in S.
pombe
In order to identify mammalian gene products capable of
disrupting normal mitotic controls we used a pREP3X-based
cDNA library designed for regulatable expression in fission
yeast. In this library, cDNAs from murine 3T3 fibroblasts are
cloned directionally to bring them under the control of the
A
regulatable library of cDNA
from Swiss mouse 3T3
fibroblasts
cDNA
LEU2
nmt
1
transform S. pombe leu1-32
promoter off
(+ thiamine)
AAAA
AA
AA
AA
AA
AA
replica plate
(+ phloxin
B)
promoter on
(- thiamine)
identify colonies with high
proportion of dead cells
(phloxin B stained)
microscopic screen to score
failure of chromosome
segregation
recover plasmid
AA
A
AA
A
A
AAA
AA
secondary screen to score
septation in hydroxyurea
thiamine-repressible nmt1 promoter (Maundrell, 1989). Library
plasmids also carry the Saccharomyces cerevisiae LEU2 marker,
which allows complementation of S. pombe leu1-32 mutant
strains. Expression of the cDNA is repressed when S. pombe
cells transformed with the library are grown in medium
containing 5 µM thiamine. Removal of thiamine from the
medium leads to the de-repression of cDNA expression once
intracellular pools of thiamine have been depleted,
approximately 12 hours after thiamine wash-out for cells grown
at 32°C. The cDNA library, which consists of approximately
200,000 independent clones, was transformed into S. pombe
leu1-32 and a total of approximately 185,000 transformants
selected for leucine prototrophy were screened by replica plating
(Fig. 1A). A primary screen was used to identify transformants
that died on replica plating to agar lacking thiamine.
Identification of colonies containing a high proportion of dead
cells was made more straightforward by the inclusion in the
medium of the dye Phloxin B, which stains dead cells dark red.
158 transformants were selected which gave a reproducibly
high level of cell death on re-streaking (from the replica plates
containing thiamine) and repeated replica plating to plates
lacking thiamine. The nuclear morphology of each of these
transformants was then examined by fluorescence microscopy
of DAPI stained, ethanol fixed cells from small-scale liquid
cultures grown for 24 hours in the absence of thiamine. From
this group of transformants, 26 were selected which showed
evidence of defects in chromosome segregation characteristic of
the cut phenotype (Fig. 1). Plasmids recovered from these 26
strains were checked for their ability to induce the cut phenotype
on re-transformation into S. pombe. The re-transformants were
then subjected to a final screen in which septation was scored in
liquid cultures that had been grown for 15 hours at 32°C in the
absence of thiamine, and to which 11 mM hydroxyurea (HU)
had been added for the final 3 hours of incubation. Ultimately,
two library plasmids designated c23 and SL6A were selected for
further study, as each appeared able to induce aberrant septation
in cell populations arrested in S phase with hydroxyurea. The
SL6A cDNA was studied in further detail in the work described
here; c23 will form the basis of a future report.
A murine cDNA that overrides the S-M checkpoint in
S. pombe
As plasmid maintenance in S. pombe is not under stringent
control, there is often significant variation in plasmid copy
Fig. 1. An expression screen in fission yeast identifies
murine cDNAs capable of inducing the cut phenotype.
(A) Overview of the cDNA library screen. (B,C) An
example of a fission yeast transformant containing a
murine cDNA scored as positive for the induction of
failed chromosome segregation. The transformant was
grown in liquid culture in the presence of 5 µM
thiamine (A) or in the absence of thiamine for 24 hours
(B). Cells were fixed, stained with DAPI to reveal DNA
and processed for fluorescence microscopy. cDNA derepression following thiamine removal resulted in the
appearance of septated cells with clear evidence of
defective chromosome segregation (arrows) as well as
apparently aploid cells (arrowheads). Bar, 10 µm.
3612 R. Craig and C. Norbury
Septation (% of all cells)
20
A
15
10
5
0
0
2
4
Time(hrs)
6
8
Fig. 2. The SL6A cDNA overrides the S-M checkpoint in S. pombe. (A) Liquid cultures of the fission yeast strain SL6Ai, containing a single
integrated copy of the nmt1-driven SL6A cDNA (see Materials and Methods) were grown in the presence or absence of 5 µM thiamine. After
12 hours, HU was added to 11 mM to some of the cultures. Samples were taken at the time of HU addition (0 hours) and at the times shown up
to 6 hours after HU addition. Cells containing two apparently normal nuclei, one on each side of a single septum (filled symbols) or abnormally
septated, cut cells (open symbols) were scored by fluorescence microscopy of fixed, DAPI-stained cells. Percentages of normally (filled
squares) and abnormally septated cells (open squares) in the culture grown in the presence of thiamine (promoter off) and HU indicate normal
operation of the S-M checkpoint. SL6A-induced abnormal septation in the absence of both thiamine (promoter on) and HU (open circles) is
largely unaffected by the addition of HU (open triangles). (B-G) SL6Ai cells from the experiment shown in A were processed for flow
cytometric determination of DNA content (propidium iodide fluorescence; arbitrary units, ordinate) and cell size (forward light scatter; arbitrary
units, abscissa). Data are represented as density contour plots for cells grown in the presence (B-D) or absence (E-G) of thiamine. (B,E) Cells
grown in the absence of HU (equivalent to time 0 in A above). Contour plots for cells collected 2 hours (C,F) or 4 hours (D,G) after the addition
of HU are also shown. The positions of cell populations with 2C (G2), 1C (G1) and <1C (hypo-haploid / aploid) DNA contents are indicated.
number among cells of the same transformed population. In
order to avoid this complicating factor, a strain (SL6Ai) was
constructed in which the nmt1-driven SL6A cDNA is
integrated at a single chromosomal locus (see Materials and
Methods). Removal of thiamine from liquid cultures of this
strain resulted in the appearance of cells with the cut phenotype
(scored as septated cells lacking two normal nuclei) beginning
at 12-14 hours after thiamine wash-out (Fig. 2A, circles; see
Calmodulin-binding protein disrupts mitosis 3613
also Fig. 5B). Addition of HU (11 mM) to parallel SL6Ai
cultures grown in the presence of thiamine (SL6A expression
repressed) led to early S phase arrest and consequent reduction
in the percentage of normally septated cells, which approached
zero by 5 hours after HU addition (Fig. 2A, filled squares).
Exposure to HU alone was not sufficient to induce the cut
phenotype to any significant level (Fig. 2A, open squares).
Addition of HU to SL6Ai 12 hours after thiamine wash-out had
no significant impact on the extent or timing of appearance of
cut cells (Fig. 2A, open triangles; see also Fig. 5B). Expression
Fig. 3. Expression of the SL6A cDNA induces mitotic spindle abnormalities.
SL6Ai cells were grown for 14 hours in the absence of thiamine (A-F,H), or
in the presence of thiamine (G) before being fixed and processed for antitubulin immunofluorescence (A-F) or dual staining with anti-tubulin and antiSad1 antibodies (G, H). Representative fields of DAPI-stained cells (A,C,E)
and the corresponding anti-tubulin images (B,D,F, respectively) are shown,
with arrows indicating cells with condensed or elongated chromatin (left
panels) and monopolar spindles (right panels). In G and H merged, falsecolour images are shown for DNA (DAPI; blue), anti-tubulin (green) and
anti-Sad1 (red) staining. The cell indicated by an arrowhead in H contains a
monopolar spindle despite having duplicated SPBs. Bar, 10 µm.
3614 R. Craig and C. Norbury
A
Sha1
Asp
Sha1
Asp
Sha1
Asp
Sha1
Asp
Sha1
Asp
Sha1
Asp
Sha1
Asp
Sha1
Asp
Sha1
Asp
Sha1
Asp
Sha1
Asp
B
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30
40
(NH2)MHFQGLNTAKQGRQQHGAAMITQKHFRAFKARRLMEAE-RGFQA--GC---|
||
| |
|||||| || |||||| || || ||
LVVQKRRRALLQMRKERQEYLHLREVTIKLQRRFHAQKSMRFMRAKYRGTQAAVSCLQMH
1250
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1270
1280
1290
1300
50
60
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80
90
------RKYKAKKYLSKVEAACRIQAWYRRWRAH-------KKYLTLLKAVNIIEGYLSA
|| | ||||| ||| || ||
||
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|
WRNHLLRKRERNSFLQLRQAAITLQRRYR---ARLNMIKQLKSYAQLKQAAITIQTRYRA
1310
1320
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1340
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1360
100
110
120
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140
QLARRR----FLKMRAAAIIIQRKWRATLSVRGARENLKRHRAACV-IQAHFRGYQ---| | ||
| ||| | | ||||||||| ||
| |||| | | || ||| |
KKAMQKQVVLYQKQREAIIKVQRRYRGNLEMRKQIEVYQKQRQAVIRLQKWWRSIRDMRL
1370
1380
1390
1400
1410
1420
150
160
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ARQSFLQQRSAVLIIQRHVRAMVAAKQERIKYIKLKKSTVVVQALVRG-WLVRKRVSEQK
| || | | | | |||| || | ||||| ||| ||| |||||||| ||||| | |
CKAGYRRIRLSSLSIQRKWRATVQARRQREIFLSTIRKVRLMQAFIRATLLMRQQRREFE
1430
1440
1450
1460
1470
1480
210
220
230
240
AKTR---LFHFTAAAYCHM------------CALKIQRAYRLHVTLRNAKKHMDSVIFIQ
| |
|||
| | |
|| ||| || | ||||||||| || |
MKRRAAVVIQRRFRARCAMLKARQDYQLIQSSVILVQRKFRANRSMKQARQEFVQLRTIA
1490
1500
1510
1520
1530
1540
250
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270
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300
RWFRKRLQRKRFIEQYHKILSTRREAHACWLQQDRAASVI-QKRYAAFSSAE--D--RKR
|||||| |||| | || || | |
| | || | | |||| || | | | |||
VHLQQKFRGKRLMIEQRNCFQLLRCSMPGF--QARARGFMARKRFQALMTPEMMDLIRQK
1550
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1570
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1590
310
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330
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SLAAPLEFRHYGEAILEKKNDHTEIKAIRRSLRAVSTTVEEENKLYRRTERALHHLLTYK
| || |||
|||||| | | ||| | |
|| ||| ||||||| |
RAAKVIQ-RYWRGYLIRRRQKHQGLLDIRKRIAQLRQEAKAVNSVRCKVQEAVRFLRGRF
1600
1610
1620
1630
1640
1650
370
380
390
400
410
HLSAILDALKHLEVVTRLSP----LCCENMAERGAVSTIFVVIRSCNRSVPCMEVVGYAV
| | |||||| ||| |
| | ||
| | || | ||
|||
IASDALAVLSQLDRLSRTVPHLLMWCSEFMS-----TFCYGIMAQAIRSEVDKQLIERCS
1660
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QVLLNVAKYDKTIAAVYEAENCVDTLLELLQVYREKPGDRVAEKSASIFTRTCCLLAVLL
|||||||||||| | ||| || | || |||
| |||| ||| | || ||
RIILNLARYNSTTVNTFQ-EGGLVTIAQMLL--------RWCDKDSEIFNTLCTLIWVFA
1720
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1760
480
490
500
510
520
---KTEQCAFDAQSRSKVTDRIYRLYKFTVPKHKVN---------TQGLFDKQKQNSCVG
| ||
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HCPKKRKIIHDYMTNPEAIYMVRETKKLVARKEKMKQNARKPPPMTSGRYKSQKINFT-1770
1780
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530
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570
FPCIPERTMKTRLVSRLKPQWVLRRDNVEEITNSLQAIQLVMDTLGISY(COOH)
||
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-PCSLPSLEPDFGIIRYSPYTFISSVYAFDTI--LCKLQIDMF(COOH)
1830
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of the SL6A cDNA therefore appeared sufficient to drive S.
pombe cells into mitosis, despite the HU-mediated imposition
of the S-M checkpoint.
In order to confirm that HU-treated cells expressing SL6A
were entering unscheduled mitosis without first replicating
their DNA, bivariate flow cytometry was performed on ethanol
fixed, propidium iodide-stained cells (Fig. 2B-G). In this type
of analysis red fluorescence is used to measure relative DNA
content, while forward light scatter provides an indication of
cell size. SL6Ai cells grown in the presence of thiamine (Fig.
2B) had the predominantly 2C DNA content expected of an
exponential population (the G2 phase occupies most of the
wild-type S. pombe cell cycle and cells replicate their DNA at
about the same time as septation is completed). Two hours after
the addition of 11 mM HU, these cells arrested in early S phase
with a 1C DNA content (Fig. 2C) and by four hours after HU
addition they had become elongated (Fig. 2D). Parallel analysis
of SL6Ai cells grown for 12 hours in the absence of thiamine
showed that they too had a 2C DNA content (Fig. 2E). Addition
of HU to this population resulted in the accumulation of 1C
cells within two hours (Fig. 2F), by which time significant
numbers of cells were entering SL6A-induced aberrant mitosis
(Fig. 2A). After a further two hours of HU treatment the
continued entry of cells with unreplicated DNA into mitosis
resulted in the appearance of small but significant numbers of
cells with less than the normal 1C DNA content (Fig. 2A,G).
We conclude that the SL6A cDNA product overrides the S-M
checkpoint and drives cells into aberrant mitosis even under
conditions where DNA replication is incomplete.
The SL6A cDNA disrupts mitotic spindle function
Progression into unscheduled mitosis in fission yeast can be
accompanied by the disassembly of the array of cytoplasmic
microtubules characteristic of interphase and the formation of
a relatively normal mitotic spindle (Enoch and Nurse, 1990).
In order to determine if either of these aspects of microtubular
reorganisation is executed faithfully in cells induced to express
the SL6A cDNA, SL6Ai cells were fixed and processed for
DAPI staining and anti-tubulin immunofluorescence 14 hours
after the removal of thiamine (Fig. 3A-F). At this relatively
early time after SL6A de-repression, many cells contained
condensed chromatin (Fig. 3A,C,E) and lacked an interphase
microtubular array (Fig. 3B,D,F). Despite these typically
mitotic features, such cells had not formed a normal mitotic
Fig. 4. Sha1, the predicted product of the SL6A cDNA, contains
multiple IQ motifs and is related to the product of the Drosophila
gene abnormal spindle. (A) The complete predicted amino acid
sequence of Sha1 (upper line) is shown aligned with the carboxylterminal 617 amino acids of the abnormal spindle product (Asp;
lower line). Amino acid identities are indicated by solid vertical bars,
conserved substitutions by shaded vertical bars. Matches to the
minimum core consensus IQ motif (I/V/LQXXXR) are underlined.
The Sha1 protein and DNA sequences have been submitted to the
GenBank database (accession number AF062378). (B) A dot plot of
the relationship between the two protein sequences generated with
the MegAlign program (DNAStar Inc.), using a window size of 38, a
threshold percentage of 14 and a filter setting of 61-154. The amino
acid residue numbers for Asp (horizontal axis) and Sha1 (vertical
axis) are indicated. Regions of similarity are represented on a colour
scale of red (most similar) to blue (least similar).
Calmodulin-binding protein disrupts mitosis 3615
B
spindle, but instead contained a monopolar, V-shaped spindlelike microtubular structure (Fig. 3B,D,F). Thus the SL6A
cDNA is capable not only of overriding the S-M checkpoint,
but also of disrupting normal spindle assembly.
Failure to form a bipolar spindle in fission yeast could be a
secondary consequence of failure to duplicate the SPB, failure
of one SPB to nucleate spindle microtubules, or some other
class of spindle dysfunction. To distinguish between these
possibilities, cells were doubly stained with anti-tubulin and
antibodies raised against the SPB component Sad1 (Fig.
3G,H). Mitosis in fission yeast is normally characterised by the
duplication of the spindle pole body at the nuclear periphery
and generation of a symmetrical, approximately linear spindle
having one SPB at each end (Fig. 3G). SPB duplication was
also apparently completed in cells containing monopolar
spindles resulting from expression of the SL6A cDNA, but
microtubules were nucleated from only one of the two SPBs
(Fig. 3H). The failure of SL6Ai cells to generate a bipolar
spindle would therefore seem to result from inactivity of one
of the duplicated SPBs.
The SL6A cDNA encodes Sha1, a putative
calmodulin-binding protein
The DNA sequence of the 1,978 base pair (bp) SL6A cDNA
revealed a single long open reading frame of 1,728 bp encoding
a predicted protein of 67 kDa (575 amino acid residues). We
propose to give this cDNA product the name Sha1 (for spindle
and hydroxyurea checkpoint abnormal) to reflect the
phenotype induced on expression in fission yeast. Database
(BLAST) searches revealed that the protein of known function
most closely related to Sha1 is the product of the Drosophila
gene abnormal spindle (asp). In addition the searches indicated
a significant relationship between the Sha1 sequence and a
those of a variety of myosin proteins. On closer examination
this similarity appeared limited to the ‘IQ’ motifs found in the
neck region of myosin family members and implicated in
myosin light chain binding (in the case of conventional
Fig. 5. Suppression of the Sha1-induced phenotype by co-expression
of fission yeast calmodulin. (A) A S. pombe leu1-32, ura4-D18 strain
was transformed with pREP3X-SL6A and with either pREP4X-cam1
(Sha1 + Cam1), allowing co-expression of S. pombe calmodulin, or
pREP4X-lacZ (Sha1 + LacZ). Liquid cultures were grown for 14
hours in the presence (+) or absence (-) of thiamine, at which time
1,000 cells from each culture were plated onto minimal agar
containing 5 µM thiamine. Photographs of 45 × 65 mm fields of each
plate were taken after three days incubation at 30°C. The fields (leftright) contain 656, 204 and 5 colonies, respectively. (B) Morphology
and DNA distribution in pREP3X-SL6A + pREP4X-lacZ cotransformants (Sha1 + LacZ) and pREP3X-SL6A + pREP4X-cam1
co-transformants (Sha1 + Cam1). Cells were grown for 16 hours in
the presence (+) or absence (-) of thiamine. Hydroxyurea (11 mM)
was added to half of each culture for the final 4 hours (+ HU).
Merged phase and DAPI (DNA) micrographs of representative fields
are shown. Bar, 10 µm. (C) Western blot showing the relative levels
of HA-tagged Sha1 protein (anti-HA; upper panel) in whole cell
lysates prepared from pREP3X-SL6A + pREP4X-lacZ cotransformants (lac) and pREP3X-SL6A + pREP4X-cam1 cotransformants (cam) after 14 and 18 hours growth in the absence (-)
or presence (+) of 5 µM thiamine. The filter was re-probed with an
anti-tubulin antibody (TAT-1; lower panel) to control for protein
loading.
3616 R. Craig and C. Norbury
myosins) or calmodulin binding (in the case of unconventional
myosins and other proteins containing this motif). Alignment
of the Sha1 sequence with the carboxyl-terminal region of
Drosophila Asp (Fig. 4A) revealed that the sequences share a
common pattern of IQ motifs, clustered in the amino-terminal
half of Sha1. Diagonal dot plot comparison of the two
sequences clearly identified the repeated sequence elements, as
well as a stretch of sequence similarity between the proteins
extending beyond the IQ repeats (Fig. 4B). The observed
similarity of Sha1 to Asp, and to a variety of comparatively
large myosin proteins, might be taken as evidence that the
SL6A cDNA is less than full-length, but two observations
suggest that this is not so. Firstly, an in-frame translation
termination codon is found upstream from the putative initiator
ATG and secondly, we were unable to generate longer cDNAs,
either by an anchored amplification approach (5′ RACE) or by
searching the available expressed sequence tag (EST)
databases (although multiple murine EST matches were found
that correspond to internal segments of the SL6A sequence).
We conclude that Sha1 is a novel murine Asp-related protein
with multiple potential calmodulin-binding motifs.
The Sha1-induced phenotype is suppressed by coexpression of calmodulin
On the basis of its sequence the Sha1 protein would be
predicted to interact with calmodulin in a functionally
significant manner. We therefore tested the possibility that the
phenotype induced by Sha1 expression in fission yeast might
be modulated by simultaneous overexpression of calmodulin.
To this end we transformed a S. pombe leu1-32, ura4-D18
strain with pREP3X-SL6A, to allow inducible expression of
Sha1, and with either pREP4X-cam1, allowing co-expression
of S. pombe calmodulin, or pREP4X-lacZ, directing
regulatable expression of E. coli β-galactosidase as a negative
control. These strains were grown in liquid culture for 14 hours
at 32°C in the presence or absence of thiamine at which point
1,000 cells of each were spread onto plates containing thiamine
(Fig. 5A). Examination of the plates after 3 days growth at
30°C showed that >99% of the (pREP3X-SL6A + pREP4XlacZ) co-transformants had lost viability by 14 hours growth
in the absence of thiamine, presumably because of the Sha1induced severe defects in chromosome segregation (Fig. 5B).
In contrast, co-expression of calmodulin in the Sha1expressing cells allowed 31% of the cells to retain long-term
viability (relative to the number of colonies formed when the
nmt1 promoter was repressed; Fig. 5A, + thiamine).
Furthermore the (pREP3X-SL6A + pREP4X-cam1) cotransformants, but not the (pREP3X-SL6A + pREP4X-lacZ)
co-transformants, were able to form colonies on minimal agar
lacking thiamine, albeit at reduced colony size in comparison
to those formed in the presence of thiamine (data not shown).
This suppression of the Sha1-induced phenotype at the
macroscopic level was reflected by restoration of relatively
normal cellular phenotypes as revealed by microscopy (Fig.
5B). Cells expressing Cam1 alongside Sha1 showed only low
levels of the chromatin segregation defects characteristic of
expression of Sha1, either alone or in combination with the
lacZ control. In addition, co-expression of Cam1 allowed
Sha1-expressing cells to respond relatively normally to HU, by
arresting as elongated cells with single nuclei. The observed
suppression was not due to a reduced level of expression of
Sha1, as cells co-expressing lacZ or cam1 contained equivalent
levels of Sha1 protein up to 18 hours after removal of thiamine
from the medium (Fig. 5C), after which time Sha1-induced
death in the lacZ cultures invalidated this comparison.
Interestingly, the low level of Sha1 expression in cells grown
in the presence of thiamine appeared sufficient to attenuate the
elongation of these cells when exposed to HU (Fig. 5B). This
effect, like the other aspects of the Sha1-induced phenotype,
was corrected on co-expression of cam1.
Sha1 relocalises from the nucleus to the cytoplasm
on cam1 overexpression
The subcellular localisation of Sha1 might provide further
clues to the likely mechanism of its action. With this in mind,
the distribution of epitope-tagged Sha1 protein was
investigated using immunofluorescent staining of S. pombe
leu1-32 cells transformed with pREP3-HA3SL6A (together
with either pREP4-lacZ or pREP4-cam1) and fixed 12 hours
after thiamine withdrawal (Fig. 6). This relatively early
Fig. 6. Sub-cellular localisation of HA-tagged Sha1. pREP3X-SL6A
+ pREP4X-lacZ co-transformants (A,B) and pREP3X-SL6A +
pREP4X-cam1 co-transformants (C-F) were fixed and processed for
anti-HA immunofluorescence after 12 hours growth in the absence of
thiamine. Paired images of single fields (A+B), (C+D) and (E+F)
correspond to DAPI (DNA) staining (A,C,E) and anti-HA
immunofluorescence (B,D). The control sample shown in E and F
was processed in parallel with the omission of the primary (anti-HA)
antibody.
Calmodulin-binding protein disrupts mitosis 3617
timepoint after the appearance of induced Sha1 protein was
used, as we reasoned that its subcellular localisation at later
times might reflect Sha1-induced secondary changes in cellular
architecture. Anti-HA-specific staining was apparently
restricted to the cytoplasm in some cells while, in the case of
the pREP4-lacZ co-transformants, others showed additional
staining in the region of the nucleus. Interestingly, coexpression of Cam1 prevented the appearance of this nuclear
fraction of HA3-Sha1. This suggests that the Cam1-mediated
suppression of the Sha1-induced phenotype might result from
cytoplasmic sequestration of the Sha1 protein, and implicates
the nuclear Sha1 pool both in S-M checkpoint disruption and
in induction of spindle defects.
DISCUSSION
We have used an expression library screen in fission yeast to
identify murine cDNAs that can disrupt the normal dependence
of mitosis on the completion of DNA replication. The relatively
low number of independent cDNA clones in the library used
(2×105), together with the practical limitations imposed by the
fission yeast methodology, mean that the screen has not yet
been saturated. Nonetheless, our use of this approach to isolate
two cDNAs with the required properties suggests that this type
of screen could usefully be extended in the future, either by
further studies using the murine library or by screening cDNA
libraries from other species. In this report we have focused on
one of the murine cDNAs isolated in this screen, SL6A, the
product of which, Sha1, is capable of disrupting both the S-M
checkpoint and normal function of the SPB.
Sha1 is a novel protein which, as it contains multiple IQ
motifs, would be predicted to bind the small, highly conserved
regulatory protein calmodulin. Calmodulin-binding proteins
are involved in a wide variety of biological processes (for
recent reviews, see Santella and Carafoli, 1997; Stull et al.,
1997; Yokokura et al., 1997; Santella, 1998). In many
instances, such as the calcium/calmodulin-dependent protein
kinases and the phosphatase calcineurin, the calmodulin serves
as a Ca2+ sensor and activation of the binding protein function
is calcium-dependent. The sequence of the IQ motifs in Sha1
might suggest calcium independence of calmodulin binding.
This cannot be taken as conclusive evidence, however, as some
‘calcium-independent’ calmodulin binding sites in myosins
require calcium ions for maximal binding (Rhoads and
Friedberg, 1997). By contrast, the affinity of other calmodulinbinding sites is actually decreased by the presence of Ca2+.
Clarification of this point will clearly require further work. It
will be interesting to determine, for example, whether or not
the Sha1-induced phenotype in fission yeast can be suppressed
by co-expression of a mutant calmodulin protein lacking
calcium-binding activity.
Calmodulin-binding proteins and the S-M
checkpoint
How might a calmodulin-binding protein influence the
dependence of mitosis on the completion of DNA replication?
Data linking calmodulin with DNA polymerases came from
studies in which specific calmodulin inhibitors were shown to
block either the activation of Polα and Polδ or entry into S
phase in cultured mammalian cells (Lopez-Girona et al., 1995;
Wang et al., 1996). Calmodulin inhibitors were also able to
induce a cell cycle checkpoint defect similar to that seen in
cells from ataxia-telangiectasia patients (Mirzayans et al.,
1995). One interesting possibility is that Sha1 might be the
murine homologue of a human 68 kDa calmodulin-binding
protein (CaM-BP68) identified biochemically in HeLa cell
extracts through its stable interaction with the DNA Polαprimase complex (Cao et al., 1995). Clarification of this point
will require further characterisation of CaM-BP68, for which
no sequence information is currently available. DNA
polymerases including Polα have been shown to play roles in
S-M checkpoint signalling in yeasts (D’Urso et al., 1995;
Navas et al., 1995), so it is possible that the Sha1-mediated
disruption of this checkpoint operates through modulation of
polymerase function. In this light, it is noteworthy that the
fraction of Sha1 protein implicated in S-M checkpoint
disruption is localised to the nucleus (Fig. 6).
Sha1 and spindle organisation
Fission yeast cells expressing Sha1 entered mitosis either in
the presence of HU or in its absence, as judged by apparently
normal SPB duplication, disassembly of cytoplasmic
microtubules and condensation of the chromatin. The mitotic
spindles generated in these cells were clearly defective,
however (Fig. 3), and bore a striking overall similarity to
those seen in cut12 mutant cells, or cells overproducing wildtype Cut12 protein (Bridge et al., 1998). Both Cut12 and
Drosophila Asp, the protein of known function most closely
related to Sha1, are localised to the spindle poles and are
required for normal spindle structure and function (Saunders
et al., 1997; Bridge et al., 1998). Sha1 production may
therefore interfere with SPB function through interaction
with endogenous Cut12 or an as yet unidentified fission yeast
Asp/Sha1 homologue.
As the S. pombe genome sequence is not yet complete, it is
not clear at this stage whether or not such a homologue exists.
In S. cerevisiae, for which the complete genome sequence is
available, the closest database match to Sha1 is the myosin
Myo2, a calmodulin-binding protein required for polarised
growth (Brockerhoff et al., 1994). In this case the protein
sequence similarity is largely restricted to the repeated IQ
motifs. Like Sha1/Asp, Cut12 is apparently not conserved in
budding yeast; thus modulation of spindle function through
Cut12 and Sha1/Asp may not be a feature of mitosis in all
eukaryotes. There is, however, a well documented involvement
of calmodulin and calmodulin-binding proteins in budding
yeast spindle function. Calmodulin is essential for budding
yeast viability (Davis et al., 1986), and performs an essential
function in mitosis (Davis, 1992). This function involves
interaction between calmodulin and Spc110, an essential SPB
component, and is required for normal chromosome
segregation (Geiser et al., 1993; Stirling et al., 1994; Sundberg
et al., 1996). A significant fraction of the cellular pool of
calmodulin is also localised to the SPB in fission yeast (Moser
et al., 1997) and this localisation appears necessary for normal
spindle function both in fission yeast and in budding yeast.
There is circumstantial evidence for conservation of such a
spindle-related function in vertebrate cells, where calmodulin
is spindle-associated in mitosis but restricted largely to the
cytoplasm during interphase (Welsh et al., 1979; Zavortink et
al., 1983).
3618 R. Craig and C. Norbury
Possible functions of Sha1
There are two general potential explanations for the observed
effects of Sha1 expression in S. pombe: the first is that
overexpression of Sha1 results in the sequestration of
endogenous calmodulin to the extent that potentially important
calmodulin functions in S-M control and spindle regulation are
lost. Alternatively, Sha1 could act autonomously, and be
negatively regulated by calmodulin. How might these
possibilities be distinguished? In addition to the welldocumented involvement in spindle organisation described
above, previous studies have identified positive requirements for
calmodulin at the G1-S and G2-M boundaries (Rasmussen and
Means, 1987, 1989; Wang et al., 1996) and a role for the
calcium-calmodulin protein kinase (Ca-CaMPK) in exit from M
phase (Lorca et al., 1993). These studies might have been
expected to reveal a positive requirement for calmodulin in the
S-M checkpoint, if such a requirement exists. There is, therefore,
no reason to believe that S-M checkpoint control would be lost
in fission yeast or any other organism following calmodulin
sequestration. Our observation that Sha1 is relocalised from the
nucleus to the cytoplasm on co-expression of calmodulin is also
consistent with the notion that Sha1 is negatively regulated by
calmodulin. We therefore favour the view that the observed
effects of Sha1 expression are not indirect consequences of
calmodulin depletion. Perhaps endogenous Cam1 is sufficient to
restrict comparatively low levels of Sha1 to the cytoplasm; at
higher levels of Sha1 expression this Cam1-dependent threshold
could be exceeded, allowing Sha1 to enter the nucleus and exert
its cytotoxic effects. Co-expression of Cam1 from the nmt1
promoter would appear to restore the Sha1/Cam1 ratio to a level
compatible with cytoplasmic retention. To our knowledge this is
the first suggestion that the nuclear-cytoplasmic distribution of
a calmodulin-binding protein could be regulated by the
availability of calmodulin. On the other hand, rapid and specific
relocalization of calmodulin from the cytoplasm to the nucleus
was recently implicated in activation of the mammalian
transcription factor CREB (Deisseroth et al., 1998); sub-cellular
relocalization of calmodulin and associated proteins may
therefore be a feature of diverse biological regulatory
mechanisms.
At this point, we can only speculate about the function of
Sha1 in murine cells. The observed effects of Sha1 expression
in fission yeast and the structural similarity to the Asp protein
suggest a likely role in mitotic spindle regulation and coordination of mitotic entry with completion of S phase. Sha1
could play a positive role in co-ordinating these processes in
murine cells, yet act in a dominant negative manner to perturb
them in S. pombe. Dominant effects of this sort are commonly
seen on expression of heterologous cell cycle regulators in
yeast, especially if the degree of structural conservation is not
very high (Norbury and Moreno, 1997). The recent description
(Bridge et al., 1998) of Cut12 as a SPB component that, when
mutated, can relieve the fission yeast cell of the normal
requirement for the Cdc2-activating phosphatase Cdc25 is of
particular interest, as Cdc2 mutants lacking the requirement for
Cdc25 are also defective in S-M checkpoint control (Enoch and
Nurse, 1990). We suggest that analogous mechanisms linking
spindle organisation and mitotic control are likely to exist in
multicellular eukaryotes, and that proteins of the Asp/Sha1
family are likely to participate in such mechanisms.
We thank Bruce Edgar for his collaboration on the construction of
the REP.Mm cDNA library, Karim Labib for his contribution to the
early stages of the cDNA library screen and identification of the c23
cDNA, Iain Hagan for affinity-purified antibodies against Sad1, Chris
Lehane and Paul Nurse for fission yeast strains, Jerry Hyams and
Karen May for their enthusiasm and suggestions, Susan Forsburg,
Trisha Davis and Karen May for reagents, and members of the
Molecular Oncology Laboratory for their advice and comments on the
manuscript. This work was supported by the Imperial Cancer
Research Fund.
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