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First published online 30 April 2003
doi: 10.1242/jcs.00411

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Drosophila securin destruction involves a D-box and a KEN-box and promotes anaphase in parallel with Cyclin A degradation

Department of Genetics, University of Bayreuth, 95440 Bayreuth, Germany

View larger version (149K): [in a new window]   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-CycA1-53, 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; CycAN), metaphase plates (arrows) in regions with stabilized Cyclin A are enriched in embryos that also express PIMdba (I,J; pimdba CycAN).

View larger version (55K): [in a new window]   Fig. 2. Stabilized Cyclin A does not inhibit PIM-myc degradation. Stabilized Cyclin A was expressed in alternating segments using prd-GAL4 and UAS-CycA1–170 (which results in a more extensive metaphase delay than UAS-CycA1-53, 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).

View larger version (123K): [in a new window]   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.

View larger version (136K): [in a new window]   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).