Sister chromatid separation during exit from mitosis requires separase. Securin inhibits separase during the cell cycle until metaphase when it is degraded by the anaphase-promoting complex/cyclosome (APC/C). In Drosophila, sister chromatid separation proceeds even in the presence of stabilized securin with mutations in its D-box, a motif known to mediate recruitment to the APC/C. Alternative pathways might therefore regulate separase and sister chromatid separation apart from proteolysis of the Drosophila securin PIM. Consistent with this proposal and with results from yeast and vertebrates, we show here that the effects of stabilized securin with mutations in the D-box are enhanced in vivo by reduced Polo kinase function or by mitotically stabilized Cyclin A. However, we also show that PIM contains a KEN-box, which is required for mitotic degradation in addition to the D-box, and that sister chromatid separation is completely inhibited by PIM with mutations in both degradation signals.

Introduction

Securins are regulatory proteins that associate with a cysteine protease called separase (Nasmyth,2002). Separase cleaves the Scc1/Mcd1/Rad21 subunit of the cohesin complex and thereby brings about the final resolution of sister chromatid cohesion during the metaphase-to-anaphase transition of mitosis. Securins from different eukaryotes are surprisingly divergent at the primary sequence level and their physiological importance varies. Deletion of the budding yeast(Pds1) and vertebrate (PTTG) securin genes results in increased rates of chromosome loss but is not necessarily lethal(Jallepalli et al., 2001; Wang et al., 2001; Yamamoto et al., 1996). By contrast, the securins of fission yeast (Cut2) and Drosophila (PIM)are essential. In the absence of Cut2 or PIM, sister chromatids are not separated (Funabiki et al.,1996a; Stratmann and Lehner,1996). These particular securins therefore provide a first function that is absolutely required for sister chromatid separation, perhaps by assisting in separase folding or localization. In addition, all of the securins also appear to function as inhibitors of separase activity and are degraded during the metaphase-to-anaphase transition(Ciosk et al., 1998; Cohen-Fix et al., 1996; Funabiki et al., 1996b; Leismann et al., 2000; Stratmann and Lehner, 1996; Uhlmann et al., 2000; Zou et al., 1999). Mitotic securin degradation therefore contributes to the temporal control of separase activity.

Mitotic securin degradation involves polyubiquitinylation by a special ubiquitin ligase known as anaphase-promoting complex/cyclosome (APC/C) (for a review, see Peters, 2002). During mitosis, APC/C activity is controlled by the mitotic spindle checkpoint pathway. This checkpoint assures that securin protein remains stable until all chromosomes have reached the correct bi-orientation within the mitotic spindle. The fact that securin stabilization by the mitotic spindle checkpoint prevents premature sister chromatid separation has been clearly shown in budding yeast (Ciosk et al.,1998; Yamamoto et al.,1996). By contrast, premature sister chromatid separation has not been observed in human cells lacking securin when arrested during mitosis(Jallepalli et al., 2001),suggesting that sister chromatid separation in these cells involves additional securin-independent pathways.

In addition to mitotic securin degradation, other levels of regulation have been implicated in the temporal control of separase activity and of sister chromatid separation. In budding yeast, phosphorylation of the Scc1 cohesin subunit by the Cdc5/Polo-kinase provides regulation at the substrate level(Alexandru et al., 2001). After phosphorylation, Scc1 is a better substrate for the yeast separase Esp1. Regulated Scc1 phosphorylation therefore provides sufficient temporal control of sister chromatid separation when the securin Pds1 is absent and Esp1 is constitutively active throughout the cell cycle. Cohesin phosphorylation by Polo-like kinase also controls the separase-independent dissociation of cohesin complexes from higher eukaryotic chromosomes during mitotic prophase(Losada et al., 2002; Sumara et al., 2002).

In higher eukaryotes, where separase is required for the removal of those cohesin complexes that remain on chromosomes until the metaphase-to-anaphase transition (Hauf et al.,2001), cyclin-dependent kinase 1 (Cdk1) has been proposed to inhibit separase activity in parallel to securin(Stemmann et al., 2001). Separase is phosphorylated by cyclin-Cdk1 complexes and thereby inhibited in cells arrested by the mitotic spindle checkpoint. High levels of non-degradable Cyclin B have been shown to inhibit sister chromatid separation in Xenopus egg extracts and in PtK1 cells(Stemmann et al., 2001; Hagting et al., 2002). In Drosophila embryos, expression of non-degradable Cyclin A, which is found exclusively in Cdk1(Cdc2) and not in Cdk2(Cdc2c) complexes, delays sister chromatid separation significantly(Jacobs et al., 2001; Kaspar et al., 2001; Parry and O'Farrell, 2001; Sigrist et al., 1995). Cdk1 inactivation resulting from the APC/C-dependent proteolysis of the mitotic cyclins at the metaphase-to-anaphase transition therefore presumably leads to separase activation, similar to securin degradation. Securin- and Cdk1-dependent separase regulation might be largely redundant, explaining why human cells display only very subtle defects in the absence of securin function.

In securin-expressing cells, mitotic sister chromatid separation is generally assumed to be strictly dependent on mitotic securin degradation. However, the corresponding evidence is from experiments involving overexpression of securin variants with mutant degradation signals. By contrast, our experiments in Drosophila embryos involving expression at physiological levels raised the possibility that sister chromatid separation might not depend on PIM degradation(Leismann et al., 2000). Therefore, we have further analyzed the role of PIM degradation.

We have previously shown that PIM contains a novel D-box variant that functions as a mitotic degradation signal(Leismann et al., 2000). D-boxes that form an RxxLxxxxN consensus sequence(Peters, 1999) were initially identified in B-type cyclins where they are required for mitotic destruction. The D-box variant identified in PIM starts with a K instead of an R. Apart from the D-box, a different destruction signal, the KEN-box, can also mediate APC/C-dependent degradation of various proteins(Pfleger and Kirschner, 2000; Peters, 2002). A KEN-box has recently been shown to contribute to the mitotic degradation of human securin(Hagting et al., 2002; Zur and Brandeis, 2001). Here,we show that PIM contains a functional KEN-box as well. Moreover, we show that physiological levels of PIM with mutations in both the D- and the KEN-box do not support sister chromatid separation, in contrast to our previous findings with D-box mutants. Sister chromatid separation in Drosophila,therefore, might well be strictly dependent on securin degradation. However,we also show genetic interactions arguing for the presence of additional,securin-independent regulation of sister chromatid separation.

Materials and Methods

Drosophila stocks

pim1, polo10, UAS-pim-myc,UAS-Cdk1-myc, UAS-CycA-Δ1-53 and UAS-CycA-Δ1170 (TF73) have been described previously (Donaldson et al.,2001; Jacobs et al.,2001; Kaspar et al.,2001; Leismann et al.,2000; Meyer et al.,2000; Stratmann and Lehner,1996). UAS transgenes were expressed with arm-GAL4,da-GAL4, prd-GAL4 or nos-GAL4-GCN4-bcd3UTR(Brand and Perrimon, 1993; Janody et al., 2000; Sanson et al., 1996; Wodarz et al., 1995). P{bTub85D-FLP} was used for testis-specific expression of FLP recombinase (Golic et al.,1997).

UAS-pimkena-myc lines were generated with a pUAST construct (Brand and Perrimon,1993). Its construction involved the introduction of the desired KEN-box mutations by enzymatic amplification using the plasmid pKS+gpim-myc as a template. pKS+gpim-myc was constructed by first ligating into the NotI and KpnI sites of pKS+ the regulatory, the coding and the 3′ region of pim+enzymatically amplified from the pKS+S2P4B1 plasmid containing a genomic pim+ fragment(Stratmann and Lehner, 1996)using the primer pairs RS37(5′-ATAAGAATGCGGCCGCAACAGCTCGCGCAGAAGAGG-3′) and RS61(5′-CGGGGATCCTTTTTGTTAGCATTTATTGG-3′), RS34(5′-GGATGGATCCGATTTTAAACAAGGAAA-3′) and RS35(5′-GAAGATCTATGCCTTCCAAGTTAGGCAATG-3′), or RS38(5′-GTAGATCTTCTATTTTAGATCCTTGTTTAAAA-3′) and RS39(5′-GGGGTACCGGGCGATCTTGGCCGAAC-3′), respectively, to yield pKS+gpim. These primers introduced a BamHI site immediately after the start codon, a BglII site just before the stop codon, and mutations (Q3P, V197L) that do not affect PIM function(Stratmann and Lehner, 1996). The construction of pKS+gpim-myc was then completed by amplifying a fragment encoding six copies of the myc epitope with the primers RS68(5′-GGATCCCATCGATTGGATCCCATGGAGCAAAA-3′) and RS51(5′-GAGAGGCTCGAGAAGATCTGAATTCAAG-3′) from pCS+MT(Rupp et al., 1994) and inserting it into the BglII site of pKS+gpim. Primers OL33(5′-GGAAAAAGGCGGCCGCGTTTAAAATCGGATCCAT-3′) and OL35(5′-GGAGGTACCAAGTGCCGCTCGTTTTCAG-3′) were then used for amplification of a first fragment from pKS+gpim-myc and OL32(5′-GGAAAAAGCGGCCGCCACCGGCATTAATTGTAAG-3′) and OL2(5′-GCATCTAGAAGTTTTTATAGTTGCTTTAATTC-3′) for a second fragment. Fragment 1 was digested with Asp718 and NotI, fragment 2 with NotI and XbaI. Both fragments were ligated into the Asp718 and XbaI sites of pUAST.

The transgenes gpim-myc and gpimdba-myc, in which expression is directed by the pim+ regulatory region, have been described previously(Leismann et al., 2000; Stratmann and Lehner, 1996). For the generation of gpimdba lines, we first enzymatically amplified a fragment from pKS+gpimdba-mycwith the primers RS33 (5′-TTCAATACGTAGGCGCC-3′) and OL59(5′-CAAGGAAAACACCGGCATTAATTG-3′). The resulting 300 bp amplification product with the D-box mutations was used as a primer, which was extended after hybridization to the plasmid pKS+S2P4B1. The newly synthesized strands were annealed and ligated, yielding pKS+S2P4B1dba. Its insert was excised by BamHI and transferred into the germ line transformation vector pCaSpeR 4 (Pirrotta,1988).

To generate the g>stop>pim,g>stop>pimkenadba, and g>stop>pimkenadba-myc lines, we first constructed pKS+5FRTgpim3′ by inserting a fragment with a FLP recombinase target sequence (FRT) amplified from the plasmid pKB345(kindly provided by K. Basler, University of Zurich) with the primers RS62(5′-GCGAGATCTACCGGGGGATCTTGAAGTTC-3′) and RS63(5′-CGCGGATCCATTTTTGTACCCAGCTTCAAAAGCGC-3′) after digestion with BglII and BamHI into the BamHI site of pKS+gpim. pKB345 contains a 2.4 kb Asp718 fragment with 3′UTR and transcriptional terminator sequences from the heat-shock protein gene, hsp70 (stop cassette) flanked by two FRT sites(Struhl and Basler, 1993). For the introduction of the stop cassette along with the second FRT site into pKS+5FRTgpim3′, we first transferred the Asp718 fragment from pKB345 into pKS+, in which the BamHI site had been destroyed by religation after filling the restricted site. The BamHI site within the insert fragment of the resulting plasmid was also eliminated. The plasmid was then used as a template for enzymatic amplification with the primers OL83(5′-GCCGGTGTGCTGACGCATGTGAAG-3′) and RS63(5′-CGCGGATCCATTTTTGTACCCAGCTTCAAAAGCGC-3′). The amplification product containing the 1500 bp stop cassette followed by the downstream FRT site was digested with BglII and BamHI and ligated into the BamHI site of pKS+5′FRTgpim3′. Ligation in the correct orientation resulted in pKS+>stop>gpim, a plasmid with an intact stop cassette flanked by FRT sites in front of the start codon. Its Asp718-NotI insert fragment was transferred into the corresponding sites of pCaSpeR 4. To introduce the KEN-box mutations,the corresponding pim region was amplified using the primers OL13(5′-CCATCTCTAGAAAAGTGCCGC-3′) and OL84(5-GCCGGTGGCAGCCGCGTTTAAAATCGGATC-3′) from the template pUASTpimkena-myc. To add the D-box mutations, the resulting product was used as a primer for an additional polymerase chain reaction in combination with a second primer OL8(5′-ATTAGTAGTACAAAGATACCTAGC-3′) and the template pKS+gpimdba-myc. The fragment with the kena and dba mutations was used to replace the wild-type sequence in pKS+>stop>gpim either by using BamHI and SnaBI (in the case of g>stop>pimkenadba) or BamHI and BglII (in the case of g>stop>pimkenadba–myc). The final pCaSpeR 4 constructs were again obtained by transposing Asp718-NotI fragments. All constructs were verified by DNA sequencing and used for Drosophila germ line transformation according to standard procedures.

Antibodies, immunoprecipitation and immunolabeling

Mouse monoclonal antibodies against a myc epitope (9E10), against Drosophila Cyclin B (F2) and against α-tubulin (Neomarkers,Fremont, CA) were used. Rabbit polyclonal antibodies against Drosophila Cyclin B, Three rows (THR), PIM and Separase (SSE) have been described previously (Jäger et al., 2001). Double labeling with mouse (Promega, Madison,WI) or rabbit antibodies against β-galactosidase (Cappel, Aurora, OH) in combination with blue balancers was used for the identification of embryo genotypes.

For co-immunprecipitation experiments, extracts were prepared with eggs collected from either gpim-myc III.1, or UAS-Cdk1-myc II.2; armGAL4 flies, or from a cross of pim1/CyO, P{w+, ftz-lacZ} females with pim1, g>stop>pimkenadba-myc II.1/CyO,P{w+, ftz-lacZ}; P{bTub85D-FLP}/+ males during 2 hours on apple-juice agar plates followed by aging for 6 hours at 25°C. Immunoprecipitations were performed as described previously(Leismann et al., 2000).

Fixation of embryos and immunolabeling was performed essentially as described previously (Leismann et al.,2000). Eggs were collected for 2 hours and aged at 25°C from the following crosses:

  • pim1/CyO, P{w+, ftz-lacZ} females with pim1, gpimdba II.1/CyO, P{w+, ftz-lacZ} males (aging 4.5 hours) or with pim1, gpimdba-myc II.5/CyO, P{w+, ftz-lacZ} males (aging 12 hours)

  • pim3/CyO, P{w+, ftz-lacZ}; prd-GAL4/+ females with pim1, UAS-pimkena-myc 2.4/CyO, P{w+, ftz-lacZ} males (aging 4.5 hours)

  • polo10/TM3, Sb,P{w+, Ubx-lacZ} females with gpimdba II.1, pim1/CyO,P(w+, ftz-lacZ); polo10/TM3, Sb, P(w+, Ubx-lacZ) males (aging 12 hours)

  • UAS-CycAΔ1-53 III.2 females with gpimdba II.1/CyO, P{w+, ftz-lacZ}; prd-GAL4/TM3, Sb,P{w+, Ubx-lacZ} males (aging 6 hours)

  • UAS-CycA-Δ170; gpim-myc III.5, gpim-myc III.6 females with gpim-myc III.5, gpim-myc III.6, prd-GAL4/TM3, Sb,P{w+, Ubx-lacZ} males (aging 6 hours)

  • nos-GAL4-GCN4-bcd3UTR females with either UAS-pim-myc III.3 or UAS-pimkena-myc III.3 males(aging 2 hours)

  • pim1/CyO, P{w+, ftz-lacZ} females with P{bTub85D-FLP}/+ males having a chromosome with pim1, g>stop>pimkenadba II.2 (or II.5) or with pim1, g>stop>pimkenadba-myc II.1 over the CyO, P{w+, ftz-lacZ} balancer chromosome or with pim1/CyO,P(w+, ftz-lacZ); g>stop>pimIII.2/P{bTub85D-FLP} males (aging 4 or 10 hours).

To analyze a potential synergism between gpimdba and reduced polo+ function, all embryos with a strong abnormal central nervous system (CNS) phenotype were first identified on the basis of the DNA labeling before genotypes were assigned on the basis of the anti-β-galactosidase labeling. Thereby 80% of the embryos with a strong abnormal CNS phenotype were found to be polo10 homozygotes with gpimdba II.1. An additional 10% were polo10 homozygotes without gpimdbaII.1, and 10% were polo+ siblings with gpimdba II.1.

To analyze a potential synergy between gpimdba and expression of mitotically stabilized Cyclin A, embryos with UAS-CycAΔ1-53 III.2 and prd-GAL4 at the stage of mitosis 16 were scored for the presence of gpimdba II.1 and for a strong enrichment of metaphase plates in prd-GAL4-expressing epidermal segments compared with the intervening segments. Although 82% of the embryos with gpimdba II.1 displayed a strong metaphase enrichment, only 25% of the embryos without gpimdba II.1 were comparably affected.

Results

Embryos homozygous for pim null mutations but equipped with a maternal pim+ contribution from pim heterozygous mothers progress normally through the initial embryonic cycles. Entry into mitosis 15 and progression to metaphase are still normal. Moreover, the transition from metaphase to anaphase is triggered as well, as evidenced by the degradation of the mitotic Cyclins A, B and B3. However, sister chromatid separation during mitosis 15 is completely inhibited(Stratmann and Lehner, 1996). This block of sister chromatid separation after the exhaustion of the maternal pim+ contribution is almost completely prevented when pim embryos inherit a gpimdba-myc transgene(Leismann et al., 2000). gpimdba-myc drives expression of PIM with C-terminal myc epitopes and a mutant D-box (AKPAGNLDA instead of KKPLGNLDN). PIMdba-myc is stable during mitosis according to confocal immunofluorescence microscopy, in contrast to PIM-myc with the wild-type D-box. gpimdba-myc expression is controlled by the normal pim regulatory region, and the resulting level of PIMdba-myc before mitosis 15 was found to be comparable to wild-type PIM levels. Because stabilized PIMdba-myc protein promoted sister chromatid separation in pim mutants, it appeared that sister chromatid separation is not dependent on degradation of the Drosophila securin PIM (Leismann et al., 2000). Analogous experiments with a gpimdba transgene driving expression of a D-box mutant PIM version without myc epitopes also revealed rescue of mitosis 15 in pim mutants (data not shown), excluding the possibility that sister chromatid separation in the presence of stabilized PIMdba-myc occurs simply because C-terminal myc epitopes specifically abolish the inhibitory PIM function.

Instead of being required during each mitosis, PIM degradation might be important to keep protein levels below a critical threshold. We have previously shown that already moderate overexpression of wild-type pim (about fivefold) is sufficient to block sister chromatid separation. Moreover, although gpimdba rescued sister chromatid separation during mitosis 15 and 16 in pim mutants, it did not allow later divisions (data not shown), perhaps because the levels of stabilized PIMdba had built up beyond the critical threshold.

If degradation of the securin PIM was not an obligatory process required during each mitosis, separase bound to securin would be expected to have sufficient basal activity to allow sister chromatid separation. In this case,premature sister chromatid separation during interphase and early mitosis would have to be prevented by securin-independent regulation. As securin-independent regulation at the level of Scc1 phosphorylation by Cdc5/Polo kinase has been described in yeast, we analyzed whether a reduction in polo function enhances the effects of stabilized PIMdba. Within the CNS of polo-mutant embryos, we observed many abnormal cells with very large polyploid nuclei, when these embryos also carried gpimdba (Fig. 1E,F). Similar abnormal cells were almost never observed in either polo+ sibling embryos with gpimdba(Fig. 1A,B) or in polo- sibling embryos without gpimdba(Fig. 1C,D). In the presence of stabilized PIMdba, therefore, the remaining level of maternal polo+ contribution is no longer sufficient to mask phenotypic abnormalities in polo-mutant embryos. Moreover, reduced polo+ function enhances the effects of stabilized PIMdba.

Fig. 1.

Polo kinase and stabilized Cyclin A modify the PIMdba phenotype.(A-F) DNA staining at stage 14 reveals the presence of many large polyploid abnormal nuclei in the CNS of embryos expressing the stabilized securin PIMdba if they are also polo mutant (E,F; pimdba polo-). By contrast, at this stage,cells in the CNS are hardly affected in polo mutants that do not express PIMdba (C,D; polo-) or in the polo+ siblings that express PIMdba (A,B; pimdba). B, D and F show high-magnification views with the CNS from the embryos displayed in A, C and E, respectively. (G-J) Using prd-GAL4 and UAS-CycAΔ1-53, stabilized Cyclin A was expressed in alternating embryonic segments. Expressing segments are indicated by arrowheads in G and I or by white lines in H and J, which display high-magnification views of epidermal regions. DNA staining indicates that the metaphase delay caused by stabilized Cyclin A is prolonged in embryos that also express the stabilized securin PIMdba under the control of the pim+ regulatory region. Compared with embryos without PIMdba (G,H; CycAΔN), metaphase plates(arrows) in regions with stabilized Cyclin A are enriched in embryos that also express PIMdba (I,J; pimdbaCycAΔN).

Fig. 1.

Polo kinase and stabilized Cyclin A modify the PIMdba phenotype.(A-F) DNA staining at stage 14 reveals the presence of many large polyploid abnormal nuclei in the CNS of embryos expressing the stabilized securin PIMdba if they are also polo mutant (E,F; pimdba polo-). By contrast, at this stage,cells in the CNS are hardly affected in polo mutants that do not express PIMdba (C,D; polo-) or in the polo+ siblings that express PIMdba (A,B; pimdba). B, D and F show high-magnification views with the CNS from the embryos displayed in A, C and E, respectively. (G-J) Using prd-GAL4 and UAS-CycAΔ1-53, stabilized Cyclin A was expressed in alternating embryonic segments. Expressing segments are indicated by arrowheads in G and I or by white lines in H and J, which display high-magnification views of epidermal regions. DNA staining indicates that the metaphase delay caused by stabilized Cyclin A is prolonged in embryos that also express the stabilized securin PIMdba under the control of the pim+ regulatory region. Compared with embryos without PIMdba (G,H; CycAΔN), metaphase plates(arrows) in regions with stabilized Cyclin A are enriched in embryos that also express PIMdba (I,J; pimdbaCycAΔN).

In addition to Scc1 regulation by Cdc5/Polo kinase, vertebrate Cdk1 has been shown to regulate separase independently of securin(Stemmann et al., 2001). The effects of stabilized Cyclin A in Drosophila embryos(Sigrist et al., 1995; Jacobs et al., 2001) are consistent with the finding that vertebrate Cdk1 phosphorylates and thereby inhibits separase. Mutant Cyclin A versions that cannot be degraded during mitosis delay progression through the embryonic cell divisions during metaphase before sister chromatid separation. Therefore, DrosophilaCyclin A-Cdk1 complexes might inhibit separase activity. Accordingly, the effects of stabilized Cyclin AΔ1-53 are expected to be enhanced by expression of stabilized PIMdba. Labeling with antibodies against tubulin (data not shown) and a DNA stain clearly revealed an increased number of metaphase figures in epidermal regions of embryos expressing both Cyclin AΔ1-53 and PIMdba (Fig. 1I,J), compared with embryos expressing only Cyclin AΔ1-53(Fig. 1G,H). The stabilized Cyclin AΔ1-53 therefore results in a more pronounced metaphase delay in the presence of the stabilized PIMdba.

In principle, stabilized Cyclin A might delay cells in metaphase because it results in an inhibition of PIM degradation during mitosis. However, cells delayed in metaphase by stabilized Cyclin AΔ1-170 no longer contained PIM-myc according to immunolabeling experiments, whereas metaphase cells that do not express Cyclin AΔ1-170 were always positive for PIM-myc(Fig. 2). We conclude,therefore, that the metaphase delay induced by stabilized Cyclin A does not result from delayed PIM degradation.

Fig. 2.

Stabilized Cyclin A does not inhibit PIM-myc degradation. Stabilized Cyclin A was expressed in alternating segments using prd-GAL4 and UAS-CycAΔ1170 (which results in a more extensive metaphase delay than UAS-CycAΔ1-53, shown in Fig. 1). In addition, the embryos expressed PIM-myc under control of the pim regulatory region throughout the epidermis. Epidermal regions with expression of stabilized Cyclin A on the right but not on the left side of the dashed line are shown at high magnification after labeling with a DNA stain (A, B; DNA, red in B),anti-Cyclin B (B,C; CYCB, green in B) and anti-myc (D, PIM-myc). Arrested metaphase cells in regions with stabilized Cyclin A were found to lack PIM-myc(see arrowheads).

Fig. 2.

Stabilized Cyclin A does not inhibit PIM-myc degradation. Stabilized Cyclin A was expressed in alternating segments using prd-GAL4 and UAS-CycAΔ1170 (which results in a more extensive metaphase delay than UAS-CycAΔ1-53, shown in Fig. 1). In addition, the embryos expressed PIM-myc under control of the pim regulatory region throughout the epidermis. Epidermal regions with expression of stabilized Cyclin A on the right but not on the left side of the dashed line are shown at high magnification after labeling with a DNA stain (A, B; DNA, red in B),anti-Cyclin B (B,C; CYCB, green in B) and anti-myc (D, PIM-myc). Arrested metaphase cells in regions with stabilized Cyclin A were found to lack PIM-myc(see arrowheads).

The phenotypic interactions between stabilized PIMdba and Polo or Cyclin A are consistent with the notion that separase complexed with non-degradable securin might have sufficient activity to allow sister chromatid separation and that the timing of this process is controlled by pathways other than securin degradation. observed However, the sister chromatid separation in PIMdba-expressing cells might also be supported by residual mitotic PIMdba degradation. A KEN motif,which is found close to the N-terminus in all of the securins(Fig. 3A), might allow some limited mitotic PIMdba degradation, escaping detection by confocal microscopy as applied in our previous experiments.

Fig. 3.

Mutations in the KEN-box of PIM result in mitotic stabilization and inhibit sister chromatid separation only at high-expression levels. (A) An alignment of the N-terminal regions of PIM with fission (SP CUT2) and budding (SC PDS1)yeast securins, as well as human securin (HS PTTG1), reveals the presence of KEN-boxes in addition to D-boxes. KEN- and D-boxes are underligned. (B-I)PIM-myc (B-E) or PIMkena-myc with a mutant KEN-box (F-I) were expressed in the anterior region of embryos during the stage of mitosis 14. Embryos were labeled with anti-myc (B,C,F,G; myc), anti-Cyclin B (D,H; CYCB)and a DNA stain (E,I; DNA). Whole embryos are shown in B and F, whereas high-magnification views from the anterior region are presented in C-E and G-I. Arrowheads indicate normal telophase figures in regions of PIM-myc-expressing embryos lacking anti-myc and anti-Cyclin B labeling (C-E),whereas arrows mark bridged telophase nuclei in regions of PIMkena-myc-expressing embryos that lack anti-Cyclin B but not anti-myc labeling (G-I). (J-L) Using prd-GAL4,UAS-pimkena-myc was expressed in alternating segments of pim-mutant embryos. DNA (J, red in L) and anti-Cyclin B labeling (K,green in L) indicated that PIMkena-myc can promote sister chromatid separation in pim mutants initially when expression levels are still low. The horizontal dashed line in the high-magnification view of the embryonic epidermis separates the upper dorsal epidermis, where cells have already progressed through mitosis 15 and re-accumulated some Cyclin B, from the lower ventral epidermis, where cells are in the process of mitosis 15 and therefore either still have high levels Cyclin B before metaphase or no Cyclin B after metaphase. The vertical dashed lines separate outer PIMkena-myc-expressing regions from a middle region without PIMkena-myc. Although the failure of sister chromatid separation in this middle region results in large undivided nuclei in the dorsal epidermis and in the absence of normal anaphase and telophase figures in the ventral epidermis, the outer PIMkena-myc-expressing regions display an almost normal nuclear density in the dorsal epidermis and normal late mitotic figures (anaphase indicated by white arrowhead) in the ventral epidermis.

Fig. 3.

Mutations in the KEN-box of PIM result in mitotic stabilization and inhibit sister chromatid separation only at high-expression levels. (A) An alignment of the N-terminal regions of PIM with fission (SP CUT2) and budding (SC PDS1)yeast securins, as well as human securin (HS PTTG1), reveals the presence of KEN-boxes in addition to D-boxes. KEN- and D-boxes are underligned. (B-I)PIM-myc (B-E) or PIMkena-myc with a mutant KEN-box (F-I) were expressed in the anterior region of embryos during the stage of mitosis 14. Embryos were labeled with anti-myc (B,C,F,G; myc), anti-Cyclin B (D,H; CYCB)and a DNA stain (E,I; DNA). Whole embryos are shown in B and F, whereas high-magnification views from the anterior region are presented in C-E and G-I. Arrowheads indicate normal telophase figures in regions of PIM-myc-expressing embryos lacking anti-myc and anti-Cyclin B labeling (C-E),whereas arrows mark bridged telophase nuclei in regions of PIMkena-myc-expressing embryos that lack anti-Cyclin B but not anti-myc labeling (G-I). (J-L) Using prd-GAL4,UAS-pimkena-myc was expressed in alternating segments of pim-mutant embryos. DNA (J, red in L) and anti-Cyclin B labeling (K,green in L) indicated that PIMkena-myc can promote sister chromatid separation in pim mutants initially when expression levels are still low. The horizontal dashed line in the high-magnification view of the embryonic epidermis separates the upper dorsal epidermis, where cells have already progressed through mitosis 15 and re-accumulated some Cyclin B, from the lower ventral epidermis, where cells are in the process of mitosis 15 and therefore either still have high levels Cyclin B before metaphase or no Cyclin B after metaphase. The vertical dashed lines separate outer PIMkena-myc-expressing regions from a middle region without PIMkena-myc. Although the failure of sister chromatid separation in this middle region results in large undivided nuclei in the dorsal epidermis and in the absence of normal anaphase and telophase figures in the ventral epidermis, the outer PIMkena-myc-expressing regions display an almost normal nuclear density in the dorsal epidermis and normal late mitotic figures (anaphase indicated by white arrowhead) in the ventral epidermis.

To determine whether the KEN motif of PIM functions as a degradation signal, we analyzed the mitotic stability of a myc-tagged PIM version with a mutant KEN-box (PIMkena-myc with AAA instead of KEN). PIMkena-myc, and PIM-myc for control, were expressed in the anterior region of embryos during cycle 14, as described previously(Leismann et al., 2000). Immunolabeling at the stage of mitosis 14 indicated that PIMkena-myc is largely stable throughout mitosis(Fig. 3F-I), in contrast to PIM-myc, which was detected before but not after the metaphase-to-anaphase transition (Fig. 3B-E). Progression beyond the metaphase-to-anaphase transition was monitored by the labeling of DNA and Cyclin B, which is rapidly degraded when cells enter anaphase. Our results show that the KEN-box is required and that the variant D-box (KKPLGNLDN), which is still present in PIMkena-myc, is not sufficient for normal mitotic PIM degradation.

Overexpression of PIMkena-myc resulted in mitotic defects. Normal anaphase and telophase figures were not observed in PIMkena-myc-positive cells that had progressed beyond the metaphase-to-anaphase transition according to the absence of anti-Cyclin-B labeling. Instead of pairs of well-separated telophase daughter nuclei, which were readily observed in Cyclin-B-negative regions in the PIM-myc control experiments (Fig. 3D,E,arrowheads), Cyclin-B-negative regions of PIMkena-myc-expressing embryos displayed decondensing metaphase plates or chromatin bridges between partially separated nuclei (Fig. 3H,I arrows). These abnormalities caused by PIMkena-myc were indistinguishable from those previously observed with PIMdba-myc which has been shown to inhibit sister chromatid separation (Leismann et al.,2000).

Sister chromatid separation is also inhibited by strong overexpression of wild-type PIM-myc (Leismann et al.,2000). By contrast, at low physiological expression levels,PIM-myc and, remarkably, also the stabilized versions PIMdba-myc(Leismann et al., 2000) and PIMkena-myc (Fig. 3J-L), can promote sister chromatid separation in pimmutants.

To analyze the function of PIM with mutations in both D- and KEN-box, we constructed additional transgenes (g>stop>pimkenadbaand g>stop>pimkenadba-myc), allowing the expression of PIMkenadba or PIMkenadba-myc under the control of the normal pim regulatory region. To establish chromosomal insertions of these potentially detrimental transgenes, we inserted a stop cassette flanked by FLP recombinase target sites (>stop>) into the 5′untranslated region. This stop cassette was eventually excised by transmitting the established insertions via males expressing FLP recombinase specifically in spermatocytes. Expression of the paternally recombined transgenes(g>pimkenadba and g>pimkenadba-myc) started at the onset of zygotic expression during cycle 14 of embryogenesis. Expression of g>pimkenadba and g>pimkenadba-myc in pim-mutant embryos did not allow sister chromatid separation during mitosis 15(Fig. 4M-O and data not shown). Instead of normal mitotic figures, which were readily apparent in pim+ sibling embryos(Fig. 4A, arrows), only decondensing metaphase plates were observed during exit from mitosis(Fig. 4M, arrowheads). Thus, pim-mutant embryos expressing g>pimkenadba and g>pimkenadba-myc displayed the same phenotype as pim mutants without transgene(Leismann et al., 2000) (and data not shown) or with the non-recombined g>stop>pimkenadba transgene(Fig. 4I-K).

Fig. 4.

PIMkenadba does not rescue sister chromatid separation in pim mutants. (A-P) Embryos were labeled with a DNA stain(A,D,E,H,I,L,M,P; DNA) and anti-Cyclin B (B,F,J,N; CYCB) at the stage of mitosis 15 (A-C,E-G,I-K, M-O) or after mitosis 15 (D,H,L,P). High-magnification views from epidermal regions, including merged views(C,G,K,O; DNA in red and anti-CycB in green), are shown. Mitosis 15 proceeds normally in pim+ sibling embryos (A-D; pim+) as well as in pim-mutant embryos with a recombined g>stop>pim transgene lacking the stop cassette (E-H; pim- g>pim), as evidenced by normal telophase figures(arrows) during mitosis 15 and normal nuclear counts after mitosis 15 (white numbers in D,H,L,P). By contrast, sister chromatid separation does not occur during mitosis 15 in pim-mutant embryos with either an unrecombined g>stop>pimkenadba transgene (I-K; pim- g>s>pimkenadba) or the recombined transgene lacking the stop cassette (M-O; pim-g>pimkenadba). Instead of normal late mitotic figures,these embryos contained decondensing metaphase plates (arrowheads) during mitosis 15 and a twofold lower nuclear count after mitosis. (Q-T) Expression of g>pimkenadba in pim+ embryos allows normal proliferation during the early mitotic divisions but not during the late divisions in the CNS. DNA staining at stage 14 reveals the presence of many large polyploid abnormal nuclei (arrowheads) in the CNS of g>pimkenadba embryos (R,T; g>pimkenadba), which are absent in control siblings(Q,S; pim+). S and T show high-magnification views with the CNS from the embryos displayed in Q and R, respectively. (U)Co-immunoprecipitation experiments show that PIMkenadba-myc associates normally with SSE and THR. Anti-myc immunoprecipitates (IP anti-myc) isolated from extracts (extract) of embryos expressing Cdk1-myc(Cdk1-myc), PIMkenadba-myc(pimkenadba-myc) or PIM-myc (pim-myc) were probed by immunoblotting for the presence of SSE (SSE), THR (THR), Cyclin B (CYCB)and tubulin (TUB).

Fig. 4.

PIMkenadba does not rescue sister chromatid separation in pim mutants. (A-P) Embryos were labeled with a DNA stain(A,D,E,H,I,L,M,P; DNA) and anti-Cyclin B (B,F,J,N; CYCB) at the stage of mitosis 15 (A-C,E-G,I-K, M-O) or after mitosis 15 (D,H,L,P). High-magnification views from epidermal regions, including merged views(C,G,K,O; DNA in red and anti-CycB in green), are shown. Mitosis 15 proceeds normally in pim+ sibling embryos (A-D; pim+) as well as in pim-mutant embryos with a recombined g>stop>pim transgene lacking the stop cassette (E-H; pim- g>pim), as evidenced by normal telophase figures(arrows) during mitosis 15 and normal nuclear counts after mitosis 15 (white numbers in D,H,L,P). By contrast, sister chromatid separation does not occur during mitosis 15 in pim-mutant embryos with either an unrecombined g>stop>pimkenadba transgene (I-K; pim- g>s>pimkenadba) or the recombined transgene lacking the stop cassette (M-O; pim-g>pimkenadba). Instead of normal late mitotic figures,these embryos contained decondensing metaphase plates (arrowheads) during mitosis 15 and a twofold lower nuclear count after mitosis. (Q-T) Expression of g>pimkenadba in pim+ embryos allows normal proliferation during the early mitotic divisions but not during the late divisions in the CNS. DNA staining at stage 14 reveals the presence of many large polyploid abnormal nuclei (arrowheads) in the CNS of g>pimkenadba embryos (R,T; g>pimkenadba), which are absent in control siblings(Q,S; pim+). S and T show high-magnification views with the CNS from the embryos displayed in Q and R, respectively. (U)Co-immunoprecipitation experiments show that PIMkenadba-myc associates normally with SSE and THR. Anti-myc immunoprecipitates (IP anti-myc) isolated from extracts (extract) of embryos expressing Cdk1-myc(Cdk1-myc), PIMkenadba-myc(pimkenadba-myc) or PIM-myc (pim-myc) were probed by immunoblotting for the presence of SSE (SSE), THR (THR), Cyclin B (CYCB)and tubulin (TUB).

Control experiments with g>stop>pim transgenes encoding wild-type PIM showed that expression after stop-cassette removal was sufficient to promote normal sister chromatid separation in pimmutants (Fig. 4E-G). Moreover,additional control experiments showed that the recombined g>pimkenadba-myc transgene was expressed as expected. Anti-myc immunoblotting clearly showed expression (data not shown), and co-immunoprecipitation experiments (Fig. 4U) indicated that the PIMkenadba-myc protein associates efficiently with Separase (SSE) and Three rows (THR), a Drosophila protein known to form trimeric complexes with SSE and PIM(Jäger et al., 2001). In addition, although g>pimkenadba-myc expression in pim+ sibling embryos had little effect during the initial embryonic cell divisions (mitosis 14-16), it resulted in a severe mutant phenotype in the CNS where additional cell divisions occur(Fig. 4T). Wild-type PIM therefore appears to protect cells from the effects of PIMkenadba-myc but only as long as the latter has not yet accumulated to high levels.

In summary, our experiments with g>pimkenadba and g>pimkenadba-myc in pim mutants show that sister chromatid separation does not occur in the presence of physiological levels of the double mutants PIMkenadba and PIMkenadba-myc, in contrast to our findings with the single mutants PIMdba, PIMdba-myc and PIMkena-myc.

Discussion

Sister chromatid separation during the metaphase-to-anaphase transition in Drosophila is strictly dependent on accumulation of the securin PIM(Stratmann and Lehner, 1996). Because we do not understand why Drosophila PIM has to accumulate to allow sister chromatid separation, we cannot evaluate whether PIM versions with mutations in both the D- and the KEN-box (PIMkenadba and PIMkenadba-myc) are still capable of providing this positive function. However, we emphasize that these versions still bind normally to the known partners SSE and THR.

Mutations in either the D- or the KEN-box result in significant stabilization of PIM protein during mitosis. Neither the D- nor the KEN-box,therefore, are sufficient for normal degradation during the embryonic cell divisions in Drosophila. Similar observations have been described for human securin (Hagting et al.,2002; Zur and Brandeis,2001). However, in contrast to Drosophila, mitotic degradation of human securin still occurs quite effectively when either only the D- or the KEN-box is intact. The D- and KEN-boxes of DrosophilaPIM, therefore, might function less independently than the corresponding motifs in human securin. Eventually, the understanding of D- and KEN-box function will require structural analyses of their interactions with Fizzy/Cdc20 and Fizzy-related/Cdh1, which recruit proteins with these degradation signals to the APC/C (Burton and Solomon, 2001; Pfleger et al., 2001). Fizzy and Fizzy-related are clearly both involved in PIM degradation, at least indirectly, as PIM is stabilized in both fizzy and fizzy-related mutants(Leismann et al., 2000) (data not shown).

Under the assumption that PIMkenadba and PIMkenadba-myc are still capable of providing the positive PIM function, our results with these stabilized mutants suggest that PIM must be degraded during each and every mitosis to allow sister chromatid separation. Although not detectable by confocal microscopy, the single mutants PIMdba and PIMkena might not be completely stable in mitosis. After low-level expression in pim-mutant embryos, residual mitotic degradation of single-mutant proteins might free some separase activity sufficient for sister chromatid separation. Similar results have been observed with the fission yeast securin Cut2, which is completely stabilized in a Xenopus extract destruction assay by mutations in either of the two D-boxes, and yet, low-level expression of single-but not double-mutant proteins is able to complement growth of cut2-ts strains at the restrictive temperature (Funabiki et al.,1997). We emphasize that even in wild-type cells, mitotic PIM degradation appears to be far from complete, and it can be speculated that it is the PIM protein of a special pool of separase complexes that is more efficiently degraded, perhaps on kinetochores or during transport on spindles towards kinetochores. At high expression levels of PIM with or without single mutations, free excess of this securin might rapidly re-associate and inhibit the activated separase, resulting in the observed block of sister chromatid separation.

Our results also point to alternative pathways that might regulate separase activity and sister chromatid separation independently of PIM degradation. As in yeast, the success of mitosis in cells with reduced separase function is dependent on Polo kinase in Drosophila embryos. Moreover, as expression of mitotically stabilized Cyclin A versions result in a metaphase delay without inhibiting PIM degradation, Cyclin A appears to contribute independently of PIM to the inhibition of premature sister chromatid separation. Even though it remains to be analyzed whether Polo kinase and Cyclin A-Cdk1 act during Drosophila divisions as proposed for Polo homologs (Alexandru et al.,2001) and vertebrate Cyclin B-Cdk1(Stemmann et al., 2001), our results indicate that separase and sister chromatid separation are unlikely to be regulated exclusively by securin degradation.

Acknowledgements

We thank D. M. Glover and F. Sprenger for fly stocks. The work was supported by grants from the Deutsche Forschungsgemeinschaft (DGF) (Le 987/2-1, 3-1 and 3-2).

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