Cell, Vol. 63, 33-46,

October

5, 1990, Copyright

0 1990 by Cell Press

One of the Protein Phosphatase 1 lsoenzymes in Drosophila Is Essential for Mitosis J. Myles Axton: ViMor Dombr8di,tr’ Patricia T. W. Cohen,+ and David M. Glow&§ ‘Cancer Research Campaign Cell Cycle Genetics Group and f Medical Research Council Protein Phosphorylation Group Department of Biochemistry Medical Sciences Institute The University Dundee DDl 4HN Scotland

Summary Drosophila has four loci encoding type 1 protein serinehhreonine phosphatases (PPls). Here we describe mutations in one of these genes, at 87B on chromosome 3. Mutants die at the larval-pupal boundary with llttle or no lmaglnal ceil proliferation. Neuroblasts are delayed in progress through mitosis and show defective spindle organixation, abnormal sister chromatid segregatlon, hyperploldy, and excessive chromosome condensation. Germilne tranaformatlon of mutant flies with the wild-type PP1878 gene restores normal mitosis, vlabllity, and fertility. These results show that PPl activity is required for mitotic progression and that the other loci cannot supply sufficient activity to complement loss of expresalon of the PPf 878 gene. Alternatlvely, the Pf1 87B product may have a distinct specialized function in mitosis. introduction As cells enter mitosis, a number of specific proteins become phosphorylated (Davis et al., 1983). It is thought that the concerted phosphorylation of these substrates is required to initiate and coordinate the radical reorganization of the chromosomes, nuclear envelope, and cytoskeleton that occurs during this transition. A protein serine-threonine kinase crucial for this process is encoded by the essential cell division cycle gene cd@ of Schizosaccharomyces pombe, which has functional homoiogs in organisms as distantly related as budding yeast and humans (for review, see Norbury and Nurse, 1989). c&2+ is negatively regulated by we&, which is itself negatively regulated by niml+ (Russell and Nurse, 1987a, 1987b). Both weel+ and niml+ are protein kinase cognates, as is nimA+ of Aspergillus nidulans, a GP-specific activator of *Permanent address: Department of Medical Chemistry, University School of Medicine, Debrecen. H-4026, Hungary. 8 Former address: Cancer Research Campaign Cell Cycle Genetics Group, Department of Biochemistry, Imperial College, London, BW7 2AZ, England.

mitotic initiation (Osmani et al., 1987, 1988). The complexity of the systems of protein phosphoryiation in mitosis is evident from analysis of these genetically defined regulators of mitotic initiation. Although p34cdc2 kinase can phosphorylate many substrates, the identity of most of the proteins phosphorylated in vivo remains to be determined. Many workers have assayed p34cdc2 kinase by its ability to phosphorylate histone Hl in vitro (e.g., Labbe et al., 1988) and in vivo (e.g., Arion et al., 1988). The hyperphosphorylation of Hl on particular serine and threonine residues, observed in many organisms, is synchronous with chromatin condensation (reviewed by Hohmann, 1983; Matthews and Huebner, 1984; Wu et al., 1988). Condensation can be induced in late S or G2 phase Physarum plasmodia exposed to purified M phase-specific Hl kinase (Bradbury et al., 1974a, 1974b). The process of nuclear envelope breakdown is facilitated by the depolymerization of the underlying nuclear lamina, synchronous with the phosphorylation of nuclear lamins (Gerace and Blobel, 1980). However, Newport and Spann (1987) found that iamin depolymerization could occur without nuclear envelope breakdown. More recently it has been demonstrated that ~34~~~~ from starfish will cause solubilization of lamins B from chicken nuclei in vitro, phosphorylating the same amino acids that are modified in mitosis in vivo (Peter et al., 199Oa). This pMcdc2 will also phosphorylate nucleolar proteins on sites that become phosphorylated in M phase, and these authors have suggested that the mitotic reorganization of the nucleolus results directly from the action of p34CdC2kinase (Peter et al., 1990b). The p34cdc2 kinase may also play a major role in reorganizing the cytoskeleton as cells enter mitosis. Its association with centrosomes at the G2-M transition suggests it may influence the behavior of microtubules (Booher et al., 1989; Bailly et al., 1989). Verde et al. (1990) added starfish p34CdC2 to interphase extracts of activated Xenopus eggs and observed that the dynamics and steady-state length of centrosome-nucleated microtubules switch from an interphase state to those characteristic of a mitotic cell. Because protein kinases regulate many mitotic processes, we may expect an opposing regulatory contribution from protein phosphatases. ~3%~~ is phosphorylated on serine, threonine, and tyrosine residues in exponentially growing S. pombe cells (Gould and Nurse, 1989). Although dephosphorylation of tyrosine may be a critical step in p34cdc2 activation in mammalian cells (e.g., Mona et al., 1989) and S. pombe, threonine dephosphorylation is also noted (Gould and Nurse, 1989). Nlix et al. (1990) have demonstrated that the protein phosphatase(s) responsible for final activation of p34cdc2 protein kinase in extracts of activated Xenopus eggs is insensitive to okadaic acid, a potent inhibitor of protein serine-threonine phosphatases 1 and 2A (reviewed by Cohen et al., 1990) and

Cdl 34

PPl

Figure 1. The Cytology of the 978 Region of Salivary Gland Chromosomes The position of PP7 878 determined by in situ hybridization is indicated as an arrowed line above the conventional chromosome map drawn after Bridges (1941). Horizontal lines below the chromosome represent chromatin missing from deficiency chromosomes used in this study. Chromosome breakp&rts were originally published by Gauss et al. (1991). Dt(3R)Ker;v does not uncover WI 378 but is present on the cMshti chromosome.

I

B7A

I

B

I

c

I

VII I I l I D.I Df(3R)SI55 Df(3R)E-079 < I 1 I

I Df(3R)Kar3Q Df(3R)KarSZ-l2

thus distinct from these type 1 and 2A enzymes. A protein tyrosine phosphatase (reviewed by Hunter, 1989) capable of activating p34cdc* remains to be identified. In contrast, type 1 and/or 2A enzymes are required in regulating the extent of p34@jc2 kinase activity. Microinjection of okadaic acid or anti-PPl antibodies into starfish oocytes maintains a high level of p34MC2 protein kinase activity (Picard et al., 1989). Inhibition of PPPA by okadaic acid promotes the activation and decreases the rate and extent of inactivation of p34*2 kinase in “cycling” extracts of Xenopus eggs, whereas inhibition of PPl with the thermostable protein, inhibitor-2 (l-2) does not (Felix et al. 1990). Protein phosphatases are also required to return mitotic protein kinase substrates to their interphase levels of phosphorylation, events that must play an important part in mitotic steps after metaphase. Mutations in PPl genes have been identified that prevent sister chromatid disjunction and exit from mitosis. bimG+ of A. nidulans (Doonan and Morris, 1989) and dis2+ of S. pombe (Ohkura et al., 1988, 1989) both have a strikingly high level of sequence identity with the PPls of rabbit (Cohen, 1988) and Drosophila (Dombradi et al., 1989). Conditional mutations in both of these fungal genes have lethal effects in mitosis, but it seems that deletion of these genes is not lethal in either Aspergillus or fission yeast due to the presence of functionally equivalent homologs (for review, see Cyert and Thorner, 1989). We previously described the sequence and developmental pattern of expression of a gene encoding a PPl catalytic subunit at chromosomal position 878 in Drosophila melanogaster. We have also detected the presence of a family of highly conserved loci at chromosomal positions 9C, 1X, and 96A by in situ hybridization with a cDNA derived from this gene (Dombradi et al., 1989). Here we report that mutations in the gene for one of these isoenzymes result in abnormal mitosis in this multicellular organism, and in contrast to A. nidulans and S. pombe, the homologous Drosophila PPl genes do not provide sufficient activity to complement the loss of PP7 876.

> I

,

,

Results Mutatlons In PP7 876 Cormspond to Lethal Compiementation Gfwp c&79 D. melanogaster has four PPl cognates which we designate by their cytological location. PfJ7 876 lies within a region of chromosome 3 that has been systematically studied by saturation mutagenesis (Gausz et al., 1981). The PPI 878 gene hybridizes in situ to 876612, within an interval defined by the proximal breakpoint of Df(3ff)Kar;lo and the distal breakpoint of M(3R)E-079 (Figure 1). Three recessive lethal complementation groups, c/r/7-ck79, are uncovered by both of these deficiencies. If PI? 870 is an essential gene, it should correspond to one of these lethal complementation groups. For this reason, we examined these stocks for rearrangements of chromosomal DNA in the vicinity of the PPl locus. Blots of genomic DNA were prepared from heterozygous flies in which a wild-type allele of the lethal mutation is supplied by a TM6B balancer chromosome (Craymer, 1984). These were probed with 5’ and 3’ noncoding fragments of the PP7 876 gene (Dombradi et al., 1989). Restriction fragments differing from wild type were found associated with chromosomes carrying mutations 8277 and hs46 of the CM9 lethal complementation group (Figure 2). ckl9eo78 cklF@, cklin280, ~kl@~~, and the TM3 and TM% balancer chromosomes all showed the same EcoRl and BamHl fragments as the Oregon R wild type with the f/J7 87f3 probes (data not shown). EcoRl and BamHl fragments from the ck79hs4e chromosome are not homologous to the 5’ probe used, and the 3’probe detects bands generated by both enzymes which are some 500 bp smaller than their wild-type alleles. We therefore conclude that the cklw chromosome is deficient for r4Cl9 bp of the Vend of the P/V 876 gene, including the whole 170 bp homologous to the 5’ noncoding probe (Figure 3). Thus, this allele lacks the start point of translation and might be expected to be a null. The &Pa1 allele was cloned and compared with the wild-

PPl in Drosophila 35

Mitosis

5’ PROBE

hs46 --TMGB

+ +

3’ PROBE

e211 TMGB

hs46 --TMGB

+ +

e211 TMGB

B RB R B

RB R B RB R

10 8-

6-

A ckl9 Allele That Abolishes lkanscrlptlon tim PP1 978 To determine whether mutations in c/r79 affected PP7 876 expression we prepared RNA blots of poly(A)+ RNA from third instar larvae of ck79m, cI~IP”~, and ck79hs46, all

pm412 N

(R)B

I

RNA

Probes

Mutants

- - -

lkb

RNA blots probed with to PPl 978. The exact BamHl (B), EcoRl (R),

x

s

y

DNA from the indicated fly stocks was digested with BamHl (B), EcoRl (R), or a combination of the two enzymes (RB). Molecular size is indicated in kb. Southern blots of this DNA were hybridized to 32P-labeled 5’ and 3’ PP7 676 probes shown in Figure 3. The TM68 balancer and wild-type (+) chromosomes yield an identical pattern of bands. In the cklsh~/TMBB lanes note that the PPI 878 3’probe hybridizes to restriction fragments some 500 bp shorter than wild type. These are derived from the mutant chromosome and represent a small deficiency in the region homologous to the 5’probe (see Figure 3).

hemizygous with Df(3R)E-079. RNA blots were probed with PP7 878 3’ noncoding fragment and subsequently with pDmras84B (Mozer et al., 1985) as a control for loading (Figure 4). We previously demonstrated the presence of two RNA& of 1.8 and 2.5 kb, at all wild-type developmental stages (Dombradi et al., 1989). ck19@Jrs mutant larvae were found to contain both /VI 878 transcripts at wildtype levels. cM9e211 produced undetectable amounts of fP7 878 RNA. ck7Shw does not produce the 1.8 kb and 2.5 kb transcripts, but instead, two smaller, less abundant transcripts were observed. Transcripts of a similar size, ~2.0 and 1.1 kb, are also present at low levels in wild-type RNA, perhaps reflecting the activity of an alternative promoter or start of transcription. Thus, there are two explanations of the ck7ghm transcription pattern. First, the cklgh@ RNAs may be the wild-type minor transcripts, overexpressed in the absence of the 1.8 kb and 2.5 kb species. Alternatively, the ck79h848 RNAs may be truncated and poorly expressed versions of the 1.8 kb and 2.5 kb transcripts, which coincide in size with wild-type minor RNAs.

type gene by restriction mapping with enzymes Sall, EcoRI, and BamHl and Southern blotting with the P/J7 876 cDNAs and the 5’ and 3’ probes. While the c/r798211 restriction map is the same as the wild type from the Sal1 site in the coding region through to the SamHI site at the S’end of the pw813 clone, the restriction map of the mutant diverges from wild-type 5’ to the 5’ probe (Figure 3). This result strongly suggests that there is a DNA rearrangement at the 5’ end of the PP7 876 gene. No lesion was detected cytologically at 8788-12 in ckl9e211 or ck7ghm polytene chromosomes. From this analysis we expected that the DNA rearrangements found in the cklghm and cklgesll mutant alleles might interfere with transcription from the Pf7 878 locus.

pm13

Figure 2. Blots of Genomic DNA from Heten zygous Flies Show DNA Rearrangements in PPf 976 Mutants

7

s B(X)

Figure 3. Molecular Organization gion Flanking PP7 878

of the

Re-

The 6.5 kb wild-type genomic DNA fragment carried on pw313 (upper line) is sufficient to rescue PPI 878 mutants. The insertion of the _=1.6kb AM oligonucleotide to generate the disrupted ~2.5 construct pAAM results in the replacement of the Nrul site (N) by an Xbal site (X). The struc2.0 --ture of the transcription unit was deduced by 5' 3' comparison of PP7 978 cDNAs with the cloned genomic locus by mstrlction analysis and Southern blotting. Regions of cDNA homologous to PPl open reading frame are indicated by dou- - - - - $fj$::i ble horizontal lines. PPI 978~specific probes ( ) ck19e”‘8 corresponding to the 5’ end of the gene and a 3’untranslated region are indicated. DNApfesent in three ch79 mutant alleles is represented by the three solid lines at the bottom; the region where the ckWsn allele diverges is dashed. clones of the genomic locus reveal the presence of an adjacent transcription unit producing a 2.0 kb RNA with no homology position of this adjacent transcription unit was not determined (dashed line). Other restriction enzyme cleavage sites are: and Sall (S). Sites in brackets flanking the 8.5 kb fragment are within the vector sequence.

a

b

c

+

Table 1. Rescue PPl 878 Gene

PPl 3’

of Lethal ckl9

Alleles

by a Functional

A. Paternal

Gamete

-2.5 -1.6

Maternal

Gamete

w”‘W@R)E-079

w”‘~;7l466

Y. ckW*” Y;TMtN

WW

WW;Tb

WW;Tb

w”le WI”*

Qw+

embryo QCTfJ embryo

P[w+]cklsB*” P[w+]:TM88

Qw+;Tb

Tb

dies dies

B. Progeny

Phenotype

Paternal

X Insertion

P[w+PP7+]18 WW WW;Tb

Dmras64B I-

1.6

Figure 4. Transcripts of Mutant and Wild-Type Alleles of PPf 876 An RNA blot of polyadenyfated RNA from third instar larvae was first hybridized with PP7 978 3probe as previously daecribed by Dombradi et al. (198s). After autoradiography, the blot was stripped by boiling in wash buffer and reprobed with pDmras84B (Mozer et al., 1985) as a control for loading. The RNA was prepared from larvae heterozygous for Df@R)E-o79 and the following third chromosomes: (a) CM@? (b) CM* (c) c~IP”~; (+) Wild type.

Germllno lkansformatlon wlth PP7 878 Rescues c&l8 Mutants The co-localization of a lethal mutation with a DNA lesion that abolishes transcription of the PPl gene is not in itself sufficient evidence that PP7 878 is required for viability. Hence, the wild-type PP7 878 transcription unit was tested for its ability to complement the lethality of CM9 mutants by introducing a 6.5 kb BamHl fragment (Figure 3) into the germline by P element-mediated transformation (Rubin and Spradling, 1962). Appropriate stocks were constructed to generate mutant animals with and without the transposon that carries the additional wild-type PP7 878 transcription unit. Males bearing X-linked P elements and heterozygous for the PPl mutation and third chromosome balancer were crossed to balanced females heterozygous for the deficiency M(3R)E-o79 (Table 1A). These single-pair crosses generated female progeny with, and male progeny without, the P element construct. This could be confirmed in adults using the w+ eye color marker carried by the Pw6 vector (Klemenz et al., 1967). Table 1B shows that while mutant males generated in this way do not live to adulthood, their mutant sisters bearing the [PPF] construct do survive. In addition, these rescued females are fully fertile. As it remained possible that rescue resulted from another gene carried on the BamHl fragment, we inserted a linker containing translational termination codons in all three reading frames into the [PPF] gene of the p&l3 construct (see Experimental Procedures). This generated a synthetic mutant allele [PPW] in the control construct pAAM (see Figure 3). DNA sequencing of the pAAM con-

QW+ Q&in,

0 (0)

175 (178.4) 109 (89.2) 162 (178.4)

P[w+PPI~]Bl 0

(0)

245 (234.5) 0 (0) 224 (234.5)

(A) inheritance of P element bearing PPI 878 and the eye color marker &. The male parent carrtes the P element on the X chromosome and is hetemzyfpw for c&lVrnl and the 7’A#B batancer chromowme. The female parent is heterozy9ous for TMBBand a deMency for PPI 878, M(3R)E-o79. The phenotypes of the progeny are shown. (8) Numbers of progeny in each phenotypic c&s generated by crosses like that shown in (A). In one set of crwees the pamntal male X chromosome carries the wllbtype gene designated P[PPf 878+] 16. In the second set of crosses, the parental mate X bears the dirtrupted gene PIPPI 87@] 31. The numbers of progeny expected if [PPI +] but not [PPlAr4] rescues the ck19**11 mutation are in brackets.

struct confirmed that the linker insertion terminates the PPl open reading frame after the first 8 amino acid residues. Table 1B shows that unlike the [Ppl+] construct, [PplAM] fails to rescue ck79Bn1/M(3R)E-079 mutants. In a similar series of experiments, CMand c/r7p were also rescued by P element insertions of [PI?+] but not by [PPIAM]. To control for any effect of chromosomal position on expression of the PPl gene, independently derived transformants were employed (Spradling and Rubin, 1963). These independent insertions are denoted by a number or letter and number following the insert description in square brackets. pw613 at 5E4-7 (P[PPI 876+]16) and 66B (P[PP7 87@]15) did, and pAMl2 at lCtC9-Dl (P[PfY 87BAM]B1), 47C3-7 (P[PP7 87BAu]E1), and 368 (P[fP7 87BAM]F3) did not rescue the mutants. The mutant phenotype of ckl@t and ck19t@a alleles was also rescued by pw613 but not the disrupted construct. We are led to conclude that the ck79 complementation group is equivalent to the PP7 878 gene and that this gene is essential for viability. Loss of PPI 878 Function Results In Abnormal Mltoslr: Chm meCondenaatlon and Chmmatld SpamUon Mutant third instar larvae of genotype cklse2111Dy3R)E. 079 were superficially wlld type in size and behavior (they feed and crawl), but dissection revealed undev&ped imaginal discs, indicating failure of diploii cell protiiration. These larvae die either just before or just after pupar-

PPl in Drosophila 37

Mitosis

iation. The phenotypic combination of late lethality with nonproliferation of imaginal cells is common to many mutants exhibiting abnormal mitosis (Gatti and Baker, 1989; see Discussion). We therefore examined mitosis in the larval ganglia, which are the most suitable tissues for cytological analysis. Neuroblasts differentiate in the first instar ganglia and begin asymmetric stem cell divisions yielding smaller ganglion mother cells, which themselves divide symmetrically to form nondividing ganglion cells (Paulson, 1950; White and Kankel, 1978; Truman and Bate, 1988). Actively dividing giant neuroblasts remain at the surface of the developing brain hemispheres and ventral ganglia and may be studied in whole brains. Squash preparations of ganglia from late third instar larvae contain a mixture of these cell types. We found ganglia from such larvae of genotype cklP~VD~3R)E-O79 to be smaller than those of wild-type larvae of equivalent age and to have significantly fewer cells per microscope field in squash preparations (mean 77% of wild type). We conclude from this that cell division is impaired in mutant ganglia as well as in imaginal discs. Wild-type neuroblasts have by the crawling stage (108-112 hr posthatching at 25OC [Truman and Bate, 19883) produced more of their small progeny than their mutant counterparts. Mitoses were scored per microscope field and also as a proportion of the total number of nuclei in the preparation. Despite the difference in cell density between wild type and mutant, the following conclusions drawn from observations of the number of mitoses per field were not qualitatively different from those obtained by counting all cells.

Table 2. Frequencies

of Mitotic

Abnormalities

in Mutant,

Rescued,

The frequency of normal metaphase and anaphase mitotic figures was significantly reduced in squashes of mutant cklPa1/D~3R)E-079 ganglia (51% of wild type on a per field basis, 88% of wild type on a per cell basis). The ratio of metaphases to anaphases among these normal cells is about 3 in wild type and -4 in the mutant (Figure 2a). This difference is not, however, statistically significant. A chi-square test of homogeneity performed on the numbers of normal metaphases and anaphases in mutant and wild type found these to be consistent with the null hypothesis of no difference in ratio between mutant and wild type at the 99% confidence level. Therefore, the cells of this “normal” subpopulation in mutant ganglia undergo mitosis without impediment to the timing of chromatid separation. The proportion of all cells in mitosis was w2-fold higher in the mutant ganglia (190% of wild-type mitoses per field, 240% cell/cell). This significant increase in mitotic index was due to mutant cells showing various abnormalities (Table 2A), including extremely overcondensed chromosomes (Figures 5e-5h), extra chromosomes (hyperploidy: Figures 5b and 5c), or both abnormal features (Figures 5d and 5i). We infer that these aberrant cells are delayed in their progress through mitosis for lack of PPI 876 function. The presence of aneuploidy and polyploidy (4N,8N) suggests that the mutant is defective in chromosome segregation or cytokinesis. Overcondensed sister chromatids are seen that have disjoined but show no sign of anaphase orientation (Figure 5e). In some mitoses,

and Wild-Type

Larvae

A. Wild-Type Wild-type Wild-type

metaphases anaphases

Overcondensed Hyperploid Overcondensed

and hyperploid

Total figures Total animals

74.6 25.4

24.2 5.8

0 0 0

52.5 2.8 14.7

861 10

1241 10

Wild Type

ckl@‘V Df(3R)E-079

0.

Per Field Wild-type metaphases Wild-type anaphases Wild-type metaphasel wild-type anaphase Aberrant mitoses Mkoaes Cells Total fields Animals

2.05 (1.44) 0.60 (0.83) 3.42

1.10 (1.29) 0.25 (0.53) 4.40

0 2.65 (1.75) 146.00 (43.28)

3.66 (3.28) 5.01 (3.65) 112.00 (38.15)

70 7

a7 4

P[w+PPf+]lG; cklET2”l Lq3R)E-o?9 2.34 (1.44) 0.77 (0.96) 3.04 0 3.10 (1.70) 150.90 (34.43) 121 5

cklfln/ Df(3R)E-079 2.22 (1.77) 0.63 (0.97) 2.67 0.11 (0.35) 3.17 (2.16) 150.36 (46.47) 96 4

(A) Frequencies of mitotic figures in neuroblast squash preparations expressed as a percentage of total mitotlc figures scored. Statistical analysis is not shown. (6) Frequency of mitoses per microscope field. The numbers quoted in the upper pat-l are mean cells per field. The standard deviation 1s shown in brackets. Means that differ significantly from wild type (Student’s t test at gQ% level of confidence) are shown in bold-face type.

Figure

5. Acetic

Orcein-Stained

Ganglion

Squashes

Illustrating

the Range

of Mutant

Mitotic

Figures

Squashes were carried out as described in Experimental Procedures without colchicine or hypotonic treatment. (a) Chromosomes from a wild-type female larva whereas (b)-(i) are all from ck79 snr/Df(3R)E-079 mutant larvae. (b) A hyperploid XY cell with one extra metacentric chromosome but normal condensation. (c) A 4N polyploid with normal condensation, from XY larva. (d) An overcondensed hyperploid nucleus. (e) Strongly overcondensed, disjoined sister chromatids without anaphase orientation. (f) A late anaphase figure in which the lower chmmatid group is overcondensed. (g) and (h) Strongly overcondensed chromosomes of anaphase-like figures (see text). (i) Overcondensed hyperploid, chromatin of degenerate appearance.

The bar is 10 nm

PPI in Drosophila 39

Mitosis

Table 3. Rescue

of lmaginal

Disc Proliferation

and Mitotic

Phenotype

of ckl9

Mutant

Alleles

by a Functional

Disc Growth

PPl 876 Gene

Sex

lmaginal

w”‘*P[w+PPf+]18/w”‘s;ck79~~“/D~3R)E-079 ~“‘~N;ck19~~“lM(3R)E-07g

female male

+

Neuroblast +

w”‘BP[w+PPf+]16/w”‘B;ck79’2”/M(3R)s155 w”‘8N;ck19’2”lM(3R)S155

female male

+

+

w”‘8P[w+PP1M]Bl/w”‘B;ckf9e2”/Df(3R)E-079 w”‘8N;ck19~2”lM(3R)E-079

female male

w”‘BP[wCPP7AM]B1/w”‘B;ckf9e2’1/D~3R)S155 w”‘8N;ck19*2”~~3R~155

female male

Mitosis

Cytological examination of sibling larvae derived from crosses shown in Table 1A. A previously unpublished deficiency Df(3R)S155 that uncovers 1(3)ckl9 (kindly supplied by Janos Gausz [see Figure 11) was also used to uncover the lethal mitotic phenotype associated with the PPl mutations. Ganglia from ten larvae of each genotype were analyzed in squash preparations for the presence of overcondensed or hyperploid mitoses of the types shown in Figure 5. + denotes a result indistinguishable from wild type and - denotes a mutant.

anaphase separation has occurred but the chromatids in one of the resulting groups are overcondensed (Figure 5f). Many cells have overcondensed chromosomes that appear to be chromatids in anaphase-like groupings (Figure 5g). Commonly seen figures of the type shown in Figure 5h may result from successful telophase decondensation of only one polar group. However, given the extreme degree of chromatin condensation, this hypothesis must be considered with caution. Mutant cells arrested at metaphase in an equivalent state of pycnosis would be unlikely to display well-defined discrete chromatids. Mitosis in &79h~/Df(3R)E-079 neuroblasts did not differ significantly from ck79e~1/Df(3R)E-079 (data not shown). In contrast, less than 4% of mitotic figures in ck79eo78/M(3R)E-O79 were found to be abnormal, most being cells with overcondensed chromosomes without hyperploidy (Table 28). lmaginal disc development was nearly wild type. The mitotic phenotype of cklF/Df(3R)E-079 is not temperature sensitive: larvae kept at 29% for 12 hr prior to squashing showed no greater frequency of mutant figures than those at 18% (data not shown). Since ck19ea1, ck7ghm, and P[w’, PP7 87BAM]B1 fail to complement the lethality of this mutant, which is, however, rescued by P[w+ PP7 878+]16 (data not shown), we conclude that ck1980r8 is a hypomorphic mutant of PP7 876 with residual function nearly sufficient for normal mitosis. We analyzed neuroblast squashes from larvae generated by the rescue experiment described in Table 1A to ensure that the stocks used in that experiment exhibited the lethal mitotic phenotype. The results of this experiment (summarized in Table 3) demonstrate that the presence of the P element construct bearing the wild-type PPl gene rescues not only the adult viability of the ck19eal mutant (Table 1B) but also restores normal mitosis (see also Table 28). The disrupted construct PP7AM cannot effect rescue.

The Effect of WI 878 Null Mutation on Mltotlc Splndles While squashed the chromosome

preparations provide information about cycle, the mitotic spindles are destroyed

by fixation in acid, and even with alternative fixation, squashing cells distorts the spindle structure. To overcome these problems we examined mitotic neuroblasts in whole, formaldehyde-fixed ganglia using confocal indirect immunofluorescence microscopy. Serial optical sections of cells stained to reveal DNA and microtubules were superimposed in order to examine chromosome and spindle structure in three dimensions. Superficial giant neuroblasts of the medial region of the brain hemispheres of mid-third instar larvae are shown in Figure 6. Metaphase and anaphase spindles in the wild type have the characteristic elipsoidal shape shown in 6a and 6b. An optical section of the metaphase plate of a wild-type cell is shown in 6c. The majority of 36 ck7P211/Df(3R)E-079 mutant spindles in the brain were of the type shown in Figure 6d. These spindles show unusually little space between almost parallel bundles of spindle microtubules. Overcondensed metaphase chromosomes can be seen associated with such spindles of “collapsed” appearance. Strongly overcondensed chromatids in anaphase groups are found in whole neuroblasts (Figure Se) as well as in squashes (see above). The microtubule-free “hole” about the centrosome, which is typical of 50 wild-type giant neuroblast spindles examined, can be seen particularly well in Figure 6a. Such a centrosomal arrangement of microtubules was not seen in most mutant spindles. The ventral lobe of late (crawling) third instar ck7Pa1/ Df(3R)E-079 larvae is slim and poorly developed in comparison with wild type. In contrast to the brain hemispheres of earlier larvae, superficial giant neuroblasts at this stage and position were found to contain only overcondensed chromosomes. Of these cells, 34% were hyperploid, with no associated mitotic microtubules (Table 4). In 67 giant neuroblasts in ventral ganglia from 18 wild-type crawling third instar larvae processed in an identical manner, condensed chromosomes in prophase, metaphase, or anaphase configurations were invariably surrounded by a mitotic spindle. We suspect that hyperploid mutant cells without spindles are a terminal phenotype. In late third instar larvae languishing for several days without pupating, squashes revealed an excess of overcondensed hyperploid

Figure 6. Mitotic Spindles in Wild-Type and PPI 97!3-Deficient Neuroblasts Ganglia were prepared from third instar larvae for confocal indirect immunoffuorescence microscopy as described in the Experimental Procedures. Tubulin is stained blue, DNA red. Unless otherwise stated, the whole mitotic apparatus is shown, reconstructed from several 1 fun confocal optical sections. (a)-(c) are from wild-type larvae, showing: (a) a wild-type metaphase. (b) a wild-type anaphase, and (c) a single optical section of a wild-type metaphase plate with the spindle poles above and below the plane of focus. (d)-(f) are from ck7~snlDIy3R)E-079 mutant neuroblasts and show: (d) a mutant metaphase, 18 of 25 mutant metaphases showed this unusual “collapsed” spindle morphology, (e) a mutant anaphase with overcondensed chromatids, and (f) a single optical section of a cell with a tetrapofar spindb, one pole of which is above the plane of focus. The bar is 5 pm.

Table 4. Heterogeneity of ck19~2”lDf(3ff)E-079 Associated with Condensed Chromosomes

nuclei of degenerate appearance (Figure 5i), which we believe to be the same terminal cells. Multipolar hyperploid cells (Figure 6f, Table 4) are those in which centrosome replication has continued in the absence of cell division.

Spindles

Spindle Chromosomes

Bipolar

Multipolar

Disorganized

Absent

Overcondensed (ok) o/c and hyperploid

7 10

0 11

0 10

22

5

Data are based on confocal optical sectioning of 95 giant neuroblasts from whole mounts of the ventral thoracic region of the ventral ganglia of 19 fate third instar larvae. No normally condensed chromosomes were seen in this experiment.

IV7 878 Is Emmtial for Mitoslr In Droaophlla We have examined mutations in the PPl gene PP7 878. One, cklse21l, completely abolishes expression of this gene. Another, ck79-, which is phenotypically indistinguish-

PPl in Drosophila 41

Mitosis

able, eliminates the major PP7 878 transcripts, Such null mutants may still express PPl activity provided by one or more of the other PPl genes (Dombradi et al., unpublished data). We also identified a mutant allele, calm, which is also rescued by germline transformation with the wild-type PFY 878 gene but which has some residual function in that neuroblasts from larvae of genotype ck79BaTs/ Df(3R)E-879 are mitotically normal with well-developed imaginal discs. Thus, we conclude that the mitotic abnormalities seen in the null mutants cklge211 and c/09”prevent larval neuroblast and imaginal cell proliferation. If all of the Drosophila PPl isoenzymes are expressed in mitotically active tissue and differ only in relative abundance, then we may further conclude that there is a threshold level of activity required for normal mitotic progression. Even if the other PPl isoenzymes are expressed in mitotically active larval tissues, their contribution is insufficient to complement total loss of PPI 878 function. Recent biochemical analyses indicate that /J/J7 878 contributes 80% of the total PPl activity in whole larvae (Dombradi et al., unpublished data). This suggests that PP7 878 may contribute most of the activity in mitotic cells because PP7 878 transcription is not developmentally regulated (Dombradi et al., 1989) and transcripts are ubiquitously present in the third instar larva (C. H. Girdham, J. M. A., and D. M. G., unpublished data). However, it is still possible that the other PPl genes show differential developmental or tissue-specific expression. Tissuespecific expression of isoforms of other proteins is well documented in Drosophila. For example, mutations in the male-specific P-tubulin gene lead to defects in male meiosis and spermiogenesis even though functional isoforms of b-tubulin are found in other tissues (Kemphues et al., 1982; Fuller et al., 1987). Null mutations in the other PPl isogenes and assays of activity in isolated tissues will be needed to address this possibility experimentally. Despite the similarity of the predicted amino acid sequences of the D. melanogaster PPl isoenzymes (Dombradi et al., unpublished data), there also remains the possibility that the isoenzymes are not equivalent either in activity or in their ability to bind specific regulators. Although transformation of flies mutant in one PPl gene with chimeric PPl constructs or extra doses of another isogene may help to investigate this possibility, biochemical studies on null mutants or of isoenzymes expressed in vitro may prove to be more informative. The analysis of genes encoding essential cell cycle functions in Drosophila has been facilitated by the developmental biology of this multicellular organism (reviewed by Glover, 1989). The embryonic cleavage divisions are accomplished using maternally provided products, many of which remain stable and may persist until late larval stages. Progeny homozygous for a mutation in such a gene survive embryogenesis using wildtype products provided by their heterozygous mothers. Most larval cells do not divide but become polyploid or polytene and grow by cell enlargement. Thus, the zygotic effects of mutations in essential cell cycle genes may appear when the neuroblasts of the central nervous system

and the imaginal cells have to proliferate so that the death of the organism is deferred until the larval-pupal boundary (Gatti and Baker, 1989). Consequently, nonconditional null mutations may be analyzed in Drosophila, but some heterogeneity of terminal cellular phenotype is to be expected resulting from variation between cells in the residual level of maternal product. Mutations in the PP7 878 gene fall into this category of mitotic mutants. Although it is clear that a subpopulation of cells continues to cycle normally in late larvae bearing PP7 878 null mutations, some (hyperploid cells) have evidently been through one or more cycles with less residual PPl than that required for normal mitosis. It is at this stage in development in these cells, therefore, that we observe the earliest departure from normality caused by the mutations. Because cells of mutant larvae may undergo a number of mitotic cycles in the presence of diminishing PPl activity, it might be anticipated that the cumulative effects of the insufficiency of this enzyme in the regulation of a number of cellular processes would eventually prevent entry to mitosis, a major energy-requiring event. Surprisingly, we found a high mitotic index and cells with disjoined but highly overcondensed chromatids, both characteristic of defective regulation of the later stages of mitosis. It is possible that cells are rendered incapable of completing mitosis by having grown under conditions of reduced PPl activity. However, it is clear that in the processes of exit from mitosis, many substrates are dephosphorylated. It seems likely, therefore, that this period of the cell cycle demands a relatively high level of protein phosphatase activity, and that it is this requirement that leads to the defects seen in PPl-deficient mitotic cells. In contrast, genetic analysis of the cell cycle of fungi and in mammalian tissue culture cells has concentrated most fruitfully on conditional mutations, producing a characteristic terminal phenotype in largely homogeneous populations of cells within one cell cycle of the conditional shift. It is nevertheless possible to see some similarities between the mitotic phenotypes of null mutations in the Drosophila PP7 878 and conditional lethal mutations in the PPl genes of S. pombe and A. nidulans. PPl Mutatlono in Aspergillus, Schiwsaccharomyces, and Drosophila Disrupt a Subset of Mitotlc Pmcee8es The success of a mitotic division depends on the coordination in space and time of multiple serial and parallel molecular events. In all three species, PPl mutations result in an elevated mitotic index. We infer from this that mutant cells enter mitosis but spend longer in a mitotic state than the wild type. The dis2-77w mutation of S. pombe results in the initiation of mitosis at the nonpermissive temperature “apparently . . . with normal timing.” Cells with “strongly condensed” chromosomes then accumulate and progressively lose viability (Ohkura et al., 1988,1989). We found an accumulation of mitotic cells with overcondensed chromosomes to be the predominant cytological phenotype associated with mutations in Drosophila PP7 878. When A. nidulans bimG-77& mutant conidia are

Cdl 42

shifted to nonpermissive temperature, the chromosome mitotic index rises to a peak mlfl-fold higher than wild type but then declines as nuclei undergo at least one further cycle of chromatin decondensation and recondensation without completion of nuclear separation (Doonan and Morris, 1989). Abnormal mitotic spindle organization is seen in PPl mutants of all three species. dis mutant spindles in S. pombe lack the “blob-like” ends of the early spindle. Ohkura et al. (1988) suggest this might reflect an absence or malfunction of kinetochore microtubules. Also, in this mutant, parts of the spindle associated with the condensed chromosomes do not undergo the normal telophase disassembly. As in other animal cells, the spindle microtubules of Drosophila are structurally organized in a more complex way than those of fungi. The lack of PP7 878 in the Drosophila mutant results in a similarly complex set of unusual structures including multipolar spindles, disorganized microtubules surrounding overcondensed chromosomes, and unusually densely aggregated spindle microtubules bearing metaphase-like chromosomes. These latter spindles have an abnormal distribution of microtubules associated with the spindle pole. bimG-77 mutant spindles are abnormally bent and short, indicating that spindle elongation does not occur at the nonpermissive temperature. dis2 spindles do elongate but sister chromatids disjoin in fewer than 2% of mitoses studied. Without this elongation step, whole chromosomes are distributed unequally between the two ends of the cell in an anomalous anaphase. It is not clear whether chromatids disjoin in the bimG mutant mitosis. At the second mutant mitosis after temperature shift “two clumps of closely apposed, condensed chromatin” are observed, implying partial partitioning of the nucleus (Doonan and Morris, 1989). Although this is evidence that bimG mutant chromatids disjoin, it is not necessarily evidence that they do so during mitosis. Indeed, studies of acentric double minute chromosomes in mammalian cells provide an example of chromatids unable to disjoin in mitosis, which nevertheless do so in Gl before remplication (Bkayama and Uwaike, 1988). The presence of aneuploid and polyploid nuclei in the Drosophila mutant is indicative of defective chromosome segregation, which might result from the unusual spindle structures. The polyploid multipolar cells seen are evidence that prevention of nuclear separation by mutation of PP7 378 does not necessarily preclude the continuing replication of DNA and centrosomes. PPl Mutations Also Affect Other Vitsl Processes It is clear that the PPl mutations that have been studied to date are pleiotropic, disrupting mitotic processes but also affecting other cellular functions. This is not surprising given that PPl functions in multiple biochemical pathways in mammalian cells (for review, see Cohen, 1989). In addition to exhibiting abnormal mitosis, bimG-77 mutants do not establish developmental polarity at nonpermissive temperature (Doonan and Morris, lQ89). dis2-77 mutants even under permissive conditions are hypersensitive to caffeine, are unable to form homologous diploids, and lose a minichromosome at an elevated frequency (Ohkura

et al., 1988, 1989). We found that ck798~Wf(3R)E-O79 larvae were of superficially wild-type size and behavior. Salivary gland chromosomes in these mutant larvae appear normal with respect to banding pattern and degree of chromatid replication. The simplest inference is that PPl is not required in chromatid replication or that less activity is required for replication than for mitosis. This inference would be invalid were the isogenes differentially expressed in larval tissues. Mutations in PP7 878 probably do impair other vital processes at later developmental stages. There are two lines of evidence for this. First, while ck7V is undoubtedly a lethal mutation in Pf7 378 because it can be rescued by the wild-type gene P[w+ PP7 87St]18, ck1Q@Y Df(3R)E-079 larvae show little evidence of abnormal mitosis (see above). These mutants never complete pupation. Second, Reuter et al. (1988, 1987) have reported that several chromosomes bearing alleles of ck19 have the ability to suppress the variegation of euchromatic genes displaced by chromosomal rearrangements to a locus adjacent to a region of heterochromatin. The basis for heterochromatic position-effect variegation is not well understood but is likely to be a reflection of the higher order structure of interphase chromatin on gene expression (Tartof et al., 1984; Eissenberg, 1989). The number of mutations with the property of suppressing or enhancing variegation is large, and it is now clear that these genes may act in distinct pathways. For example, transformation with extra copies of the wild-type zinc finger protein gene Suuar(3)7’+ complements the suppression due to Suvar(3)7, Su-var(2)701, and Su-var(3)303 but not that due to Su-var(3)eol (Reuter et al., 1990). There is evidence for allelism of c/r79 and Suuar(3)6. Not only does the suppression of position-effect variegation cosegregate with ck19 lethality but also the lethality of tmns-heterozygotes between variegation-suppressing ck79 chromosomes and the semilethal Suuar(3)601 correlates well with the magnitude of the suppression of variegation in these flies. The possibility that PP7 878 is important in regulating interphase chromatin structure is intriguing given its role in mitotic chromosome condensation. However, the regulation of interphase and mitotic roles could be very different. It remains to be proven that it is mutations in fP7 878 itself which affect heterochromatic position-effect variegation. It will now be possible to use the PP7 878 transformants to attempt to influence variegation in wild-type, enhancer, and suppressor mutants, and thus to clarify this interpretation. Evolutionary Coneemtion of PPl Catalytic Subunits Refteds a Multipticity of Protein-Pmtein Intersdons PPls are among the most highly conserved homologous proteins known. Ninety-two percent of the amino acid residues are identical in the Drosophila and rabbit skeletal muscle enzymes (Cohen and Dombradi, lQ89). It is likely that the interaction of the enzyme with multiple regulatory subunits and substrates has placed a constraint on evolutionary sequence divergence. While the catalytic subunit has broad substrate specificity in vitro, the actual sub-

PPl 43

in Drosophila

Mitosis

strates dephosphorylated in vivo will be limited by the activity and location of PPl specified by regulatory proteins. The best characterized of these are the phosphoprotein inhibitors l-l and l-2 (for review, see Cohen, 1989). Inhibitory activity of l-2 oscillates in a cell cycle-dependent manner (Brautigan et al., 1990) which suggests a mechanism that could modulate the timing of PPl activity during the cell cycle. PPl is concentrated in the nucleus (Kuret et al., 1988; Ohkura et al., 1989; Cohen, 1989). The mechanism for this nuclear localization is unknown. However, G and M subunits have been identified that bind PPl catalytic subunits to glycogen particles and myofibrils, respectively. If analogous subunits localize PPl within the mitotic nucleus, then the structural state of the nucleus could determine the sites of phosphatase activity. It is important to note that the alleles of dis2 and bimG that affect mitosis in S. pombe and A. nidulans are not null mutations. Indeed, abolition of gene function by disruption of dis2+ or bimG+ is apparently not sufficient to produce abnormal mitosis because of functional complementation by other PPl genes. The conditional mutations studied by Doonan and Morris (1989) and Ohkura et al. (1989) are not dominant but nevertheless interfere with mitotic progression, perhaps by misregulation of PPl activity. This could result from an abnormal interaction of the catalytic and regulatory subunits (for discussion, see Cyert and Thorner, 1989). In contrast, a null mutation in Pf7 878 does cause abnormal mitosis, an observation most simply explained as a consequence of insufficient catalytic activity. However, it remains possible that loss of PP7 878 catalytic subunit permits excess regulatory subunits to interfere with the function of the other PPl isoenzymes. If so, then mutations such as c/&F1, which abolish the catalytic subunit, could be more deleterious than mere loss of Pf7 876 catalytic activity. What then is the role of PPI in mitosis? This phosphatase might be needed as one step in the inactivation of p34dc2 protein kinase after metaphase (see Introduction). Alternatively, the mitotic’arrest or delay found in PPl mutants could result from failure to dephosphorylate proteins that are phosphorvlated due to ~34~~~~ kinase activ. _ ity. Dynamic turnover of phosphate on specific proteins might also be important for some mitotic processes. For example, reorganization of interphase microtubules as a mitotic spindle in starfish embryos requires the activity of both ~34~~~~ protein kinase and the okadaic acid-sensitive PPl and/or PPPA (Picard et al., 1989). The balance of kinase versus phosphatase activity may also influence the density of chmmatin packing. If neuroblasts are blocked in a metaphase-like state, for example by treatment with colchicine, chromosomes continue to condense. The overcondensed chromosomes seen in the Drosophila PP7 878 mutants without drug treatment are, however, more highly condensed than those seen in wild-type neuroblasts blocked for 2 hr in colchicine (J. M. A., unpublished data) or in the metaphase-like arrested cells of abnormal spindle mutants (Ripoll et al., 1985). Other Drosophila mutants have been described in which chromosomes are found to be as extremely overcondensed as they are in PP7 878 mutants.

Mutants of this class include /(7)cLdeg3 and I(l)d.deglO (Gatti and Baker, 1989). Our analysis of the phenotype of neuroblasts from PP7 878 null mutant larvae leads us to the conclusion that phosphatase encoded by PP7 878 is required for the execution of steps in mitotic pathways subsequent to metaphase. It is quite possible that the protein phosphatase is directly required in the control of both chromatin packaging and spindle dynamics, or we may be observing the effects of an alteration in the timing or coordination of mitotic processes in the absence of this essential regulatory enzyme. Exparlmsntal Nauroblast

Procadums Squashes

Third instar larvae grown at 25OC on standard Drosophila medium were separated from food by floatation on 1.5 M NaCI, washed in distilled water, and placed in new food for at least 6 hr at 18OC. Mutants were selected using the larval marker Tb+; hetarozygotas expressed the dominant 7b marker of the 78466 balancer chromosome (Craymer, 1984). All cytological preparation and solutions were at 1Boc. Squashes were prepared by the method of Gonzalez et al. (1988) and examined using a Nikon Microphot-FX microscope equipped with a Zeiss 63x phase contrast objective. A “field” of nuclei was defined as the area covered by the photographic viewfinder at 63 x 10 magnification. Metaphases and anaphasas per microscope field ware recorded, as ware the total nuclei per field (mean = 146 for wild type). Tests of statistical significance were performed on tables of call numbers (chisquare test) and on mean figures per microscope field (Studanb t test).

Polytana

Chromosomes

Salivary glands from crawling third instar larvae were dissected in 0.7% NaCI, fixed for 30 s in 45% glacial acetic acid, and squashed in propionic lactic orcein (Ashburner, 1989) under an unsiliconizad 18 mm* cover slip by tapping with a pencil eraser through bibulous paper. The breakpoints of all deficiency stocks used were confirmed using Df/+ heterozygous chromosomes.

In Situ Hybrldlxstlon

to Polytana

Chromosomes

Salivary glands were fixed in 45% glacial acetic acid and squashed in 1:2:3 lactic acid:watar:acetic acid, left overnight to flatten, and processed according to the method of Ashburner (1989). Probes were prepared by random oligomer-primed labeling with biotin-1SdUTP (Soehringar Mannheim).

Isolation

of PP1 878 cDNA Clones A 1.2 kb cDNA derived from PPI 878 (Dombradi et al., 1989) was used to isolate cDNA clones from a O-2 hr embryonic library in the plasmid vector pNS-40 (kind gift of N. Brown [Brown and Kafatos, 19881). cDNAs were obtained that hybridized in situ to the Pf7 878 and PPl 964 loci (Dombradi et al.. unoublished data). Pp1 878 clones were identified using the 3’noncoding 160 bp probe described in Dombradi et al. (1989).

Ganomlc Clones of PPl 878 Wild-type clones in the CosPer cosmid vector were isolated using the fP7 878 3’probe from a library provided by John Tamkun (Department of MCD Biology, University of Colorado, Boulder). From one of these, pcos&‘Ba, a 6.5 kb BamHl fragment containing the PPI 878 coding region, was subcloned into the Pw8 vector (Klemanz at al., 1987) to generate the wild-type transformation construct p&13. pw813 was then oartiallv cleaved with Nrul. Full-lenath linear olasmid was ourified by gel eledrophoresis and ligated to-the dephosphorylated palindromic duplex. otigonucleotide Amber MURFI (AM) (5’ Cm TCTTAGACTAG 3’) as described by Perlman and Halvorson (1986). This oliFucleotide was kindly synthesized by Conrad Lichtanstain. The lioated DNA was used to transform E. coli XL-1 (Strataaana). Plasmids w&e then screened for replacement of the original Niul site at the 5’ end of the PP7 878 open reading frame by the Xbal site borne on the

Cdl 44

oligonucleotide. The structure of the disrupted construct, pAM12, was confirmed by Southern blotting. Insertion of the AM oligonucleotide introduces a frameshift and termination codons in all reading frames. A library of BamHlcut genomic DNA from cklCVTM3 flies was constructed in the lambda vector EMBL4 (Frischauf et al., 1963). Clones of an 11 kb BamHl fragment from the cklC”’ mutant and 6.5 kb from the balancer chromosome were obtained and subcloned into Bluescribe+.

lndlmct

Immuno?luofwcence

of Whole

Ganglla

Ganglia were dissected in 0.7% NaCI, fixed in 3.7% formaldehyde in PBS (0.16 M NaCI, 50 mM sodium phosphate [pH 6.91) for 1 hr in the presence of either 5 PM taxol or 1 mM GTP (GonzBlez et al., unpublished data). After blocking for 1 hr in 10% fetal calf serum, 0.3% Triton X-100 in PBS, gangliawere incubated for 12-16 hr at 4°C in a 15 dilution of rat anti-tubulin antibody YLV2 @era-Lab) and given two 15 min washes in PBS, 0.3% Triton x 100. They were then incubated in a lo-* dilution of fluoresceinconjugated goat anti-rat antibody (Jackson Laboratories) for 2 hr at 2VC or 12-16 hr at 4OC. After 2 x 15 min washes in PBS containing 0.3% Triton, ganglia were stained for 5 min in 1 ug/ml propidium iodide in PBS and mounted in 65% glycerol, 2.5% n-propyl gallate on slides coated with 1 @ml poly L-lysine. A siliconized cover slip was sealed in place with nail varnish. The inclusion of 2.5 pg/ml boiled RNAase A in the first antibody incubation was found to reduce the cytoplasmic staining by propidium iodide and improve visualization of the chromatin. No differences in spindle morphology were noted between neuroblasts fixed in the presence of taxol or GTP or when 0.7% NaCl was substituted for PBS in the fixation step. Spindles were only rarely preserved in ganglia fixed in the absence of taxol or GTP. Tubulin and chromatin were visualized simultaneously using the split-video display of an MRC 500 confocal microscope. Serial optical sections (3-5) were recorded differing in focal depth by 1 pm. Color images were permanently recorded using a Sony video printer. A detailed description of the morphology of mitotic cells in wild-type ganglia will be published separately (Gontilez et al., unpublished data).

P Element-Modlated

Germllnr

Tranaformatlon

of Flies

wI1ls embryos were manually dechorionated and placed on a line of Sellotape glue applied in n-heptane to a cover slip. Transformation was accomplished as described by Karess (1965). The Pw8 constructs were injected at 200 wg/ml. Transposase was provided in tfsns by defective helper element phsDelta2-3 (kind gift of Andrew Tomlinson) at 400 )rg/ml. Approximately 25% of embryos survived injection, 30% of these emerged as adults, 75% were fertile of which 5% (0.3% of injected embryos) gave transformed progeny bearing the W+ eye color marker. Nine independent transformed limes were isolated containing the wild-type PPI 876 construct pw613 and six lines with the disrupted construct p4MlP. GO (injected) flies and their transformed Gl progeny were crossed to wllls; 02 and 63 flies were crossed inter se to generate lines homozygous for the insertions. In situ hybridization with vector probe was used to detenine insertion sites. Lines with a single euchromatic insertion site were chosen for use in rescue experiments. We are currently investigating the effect of PP7 87Bc gene dosage on mitosis and relative viability using the mutants and transformed lines described here. Although the cklwl mutation is lethal in early pupae, the original, heavily mutagenized chromosome contains several other mutations. Consequently, much of this work was carried out using c&Iv’ and its alleles In the hemizygous state, that is, when heterozygous with a chromosome bearing an uncovering deficiency. The original cklPa’ chromosome was cw to the multiply marked rucuce (ru h fh St cu ST e ce) chromosome (for description of the genetic markers, see Lindsley and Grell, 1966; Lindsley and Zimm, 1965,1966,1967) and recombinants eetected that displayed the early pupal lethality and abnormal mitotic phenotype when homozygous. Even though these homorygous recomblnants were indistinguishable from &I~1 hemizygotes in larval phenotype and PPI activity, further mutations were still present on the chromosome. These were not detected until the mitotic lethality had been rescued by germline transformation. Thus, flies with such a recombinant third chromosome carrying the &IF1 allele and the rescuing P[Ppl 87B+] transpoeon were poorly viable, sterile,

and homeotically transformed with a bithorax phenotype. Flies carrying the ru h th st cklPnlce recombinant chromosome (stock “rf2”) were then cmswd to wild type (w?+) in the pmsence of pIw+ppl+] 16. Phenotypically wild-type, fertile recombinant lines were selected that required the rescuing transposon for viability. These latter recombinants were thus mutant in only one essential gene PPI 878.

Acknowledgments We are grateful to Jgnos Gausz for providing fly stocks. Philip Cohen, Chartes Girdham, Mike Goldberg, Cayetano GonzBlez, Roger Karess, Robert Saunders, and Will Whitfield all provided essential advice and encouragement. Alan Cheshire provided expert assistance with fly stocks and photography. J. M. A. received a research studentship from the Science and Engineering Research Council and subeequently a research assistantship from the Cancer Research Campaign. V. D. held an EMBO Long Term Fellowship. Work in the laboratory of P. T. W. C. was supported by a grant from the Wellcome Trust and Group Support from the Medical Research Council, London. D. M. G. is grateful to the Cancer Research Campaign for research group support. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 USC Section 1734 solely to indicate this fact. Received

May 25, 1990; revised

July IO, 1990.

Arion, D., Meijer, L., Brizuela, L., and Beach, D. (1966). c&2 ponent of the M phase-specific histone HI kinase: evidence tity with MPF. Cell 55, 3311-376.

is a comfor iden-

Ashburner, M. (1969). Dru.?opbi/e: A Laboratory Manual (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory), pp. 4449. Bailly, E., Do&e, M., Nurse, P., and Bornens, M. (1969). ~34~~~ is located in both the nucleus and cytoplasm: part is centmsomally associated at G2/M and enters vesicles at anaphase. EMBO J. 8, 39653995. Booher, R. N., Alfa, C. E., Hyams, J. S., and Beach, D. H. (1969). The fission yeast cdc2/cdcl3/sucl protein kinase: regulation of catalytic activity and nuclear localization. Cell 58, 465-497. Bradbury, E. M., Inglis, R. J., and Matthews, H. R. (1974a). Control of cell division by very lysine rich histone (Fl) phosphorylation. Nature 247. 257-261. Bradbury, E. M., (1974b). Molecular 249, 533-556.

Inglis, I? J., Matthews, H. R., and Langan, T. A. basis of control of cell division in eukaryotes. Nature

Brautigan, D. L., Sunwoo, J., LabbB, J.-C., Fernandez, A., and Lamb, N. J. C. (1990). Cell cycle oscillation of phosphatase inhibitor-2 in rat fibmblasts coincident with ~34~~ restriction. Nature 334, 74-76. Bridges, P N. (1941). A revision of the salivary gland 3Rchromosome map of Dmsophile melenogsster. J. Hered. 33, 299-300. Brown, N. H., and Kafatos. F. C. (1966). Functional Dmsophile embryos. J. Mol. Biol. 203, 425-437.

cDNA

libraries

Cohen, P T. W. (1966). Two isoforms of protein phosphatase-1 produced from the same gene. FEBS Lett. 233, 17-23. Cohen, P. (1969). The structure and regulation tases. Annu. Rev. Biochem. 58,453-508.

of protein

from

may be phospha-

Cohen, I? T. W., and Dombtidi, V. (1969). Three novel protein phosphatases identified by recombinant DNA techniques. In Advances in Pmtein Phosphatases, Mume 5, W. Merlevede and J. Di Salvo, eds. (Belgium: Leuven University Press), pp. 447-463. Cohen, I?, Holmes, C. F. 8.. and Tsukitani, Y. (1990). Okadaic new probe for the study of cellular regulation. Trends Biihem. 96-102. Craymer, 234.

L. (1964).

Third

Cyert, M. S., and Thorner,

multiple

six, b structure.

J. (1969). Putting

Dms.

acid, a Sci. 15,

Inf. Serv. 60,

it on and taking it off: phos-

PPI in Drosophila 45

Mitosis

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One of the protein phosphatase 1 isoenzymes in Drosophila is essential for mitosis.

Drosophila has four loci encoding type 1 protein serine/threonine phosphatases (PP1s). Here we describe mutations in one of these genes, at 87B on chr...
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