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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 10 20 30 40 (NH2)MHFQGLNTAKQGRQQHGAAMITQKHFRAFKARRLMEAE-RGFQA--GC---| || | | |||||| || |||||| || || || LVVQKRRRALLQMRKERQEYLHLREVTIKLQRRFHAQKSMRFMRAKYRGTQAAVSCLQMH 1250 1260 1270 1280 1290 1300 50 60 70 80 90 ------RKYKAKKYLSKVEAACRIQAWYRRWRAH-------KKYLTLLKAVNIIEGYLSA || | ||||| ||| || || || ||| | ||| || | WRNHLLRKRERNSFLQLRQAAITLQRRYR---ARLNMIKQLKSYAQLKQAAITIQTRYRA 1310 1320 1330 1340 1350 1360 100 110 120 130 140 QLARRR----FLKMRAAAIIIQRKWRATLSVRGARENLKRHRAACV-IQAHFRGYQ---| | || | ||| | | ||||||||| || | |||| | | || ||| | KKAMQKQVVLYQKQREAIIKVQRRYRGNLEMRKQIEVYQKQRQAVIRLQKWWRSIRDMRL 1370 1380 1390 1400 1410 1420 150 160 170 180 190 200 ARQSFLQQRSAVLIIQRHVRAMVAAKQERIKYIKLKKSTVVVQALVRG-WLVRKRVSEQK | || | | | | |||| || | ||||| ||| ||| |||||||| ||||| | | CKAGYRRIRLSSLSIQRKWRATVQARRQREIFLSTIRKVRLMQAFIRATLLMRQQRREFE 1430 1440 1450 1460 1470 1480 210 220 230 240 AKTR---LFHFTAAAYCHM------------CALKIQRAYRLHVTLRNAKKHMDSVIFIQ | | ||| | | | || ||| || | ||||||||| || | MKRRAAVVIQRRFRARCAMLKARQDYQLIQSSVILVQRKFRANRSMKQARQEFVQLRTIA 1490 1500 1510 1520 1530 1540 250 260 270 280 290 300 RWFRKRLQRKRFIEQYHKILSTRREAHACWLQQDRAASVI-QKRYAAFSSAE--D--RKR |||||| |||| | || || | | | | || | | |||| || | | | ||| VHLQQKFRGKRLMIEQRNCFQLLRCSMPGF--QARARGFMARKRFQALMTPEMMDLIRQK 1550 1560 1570 1580 1590 310 320 330 340 350 360 SLAAPLEFRHYGEAILEKKNDHTEIKAIRRSLRAVSTTVEEENKLYRRTERALHHLLTYK | || ||| |||||| | | ||| | | || ||| ||||||| | RAAKVIQ-RYWRGYLIRRRQKHQGLLDIRKRIAQLRQEAKAVNSVRCKVQEAVRFLRGRF 1600 1610 1620 1630 1640 1650 370 380 390 400 410 HLSAILDALKHLEVVTRLSP----LCCENMAERGAVSTIFVVIRSCNRSVPCMEVVGYAV | | |||||| ||| | | | || | | || | || ||| IASDALAVLSQLDRLSRTVPHLLMWCSEFMS-----TFCYGIMAQAIRSEVDKQLIERCS 1660 1670 1680 1690 1700 1710 420 430 440 450 460 470 QVLLNVAKYDKTIAAVYEAENCVDTLLELLQVYREKPGDRVAEKSASIFTRTCCLLAVLL |||||||||||| | ||| || | || ||| | |||| ||| | || || RIILNLARYNSTTVNTFQ-EGGLVTIAQMLL--------RWCDKDSEIFNTLCTLIWVFA 1720 1730 1740 1750 1760 480 490 500 510 520 ---KTEQCAFDAQSRSKVTDRIYRLYKFTVPKHKVN---------TQGLFDKQKQNSCVG | || | || || | | |||| ||||| ||| | ||| | HCPKKRKIIHDYMTNPEAIYMVRETKKLVARKEKMKQNARKPPPMTSGRYKSQKINFT-1770 1780 1790 1800 1810 1820 530 540 550 560 570 FPCIPERTMKTRLVSRLKPQWVLRRDNVEEITNSLQAIQLVMDTLGISY(COOH) || | | | | | | | ||| | -PCSLPSLEPDFGIIRYSPYTFISSVYAFDTI--LCKLQIDMF(COOH) 1830 1840 1850 1860 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). 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