Degradation of Drosophila PIM regulates sister chromatid separation during mitosis - PubMed (original) (raw)

Degradation of Drosophila PIM regulates sister chromatid separation during mitosis

O Leismann et al. Genes Dev. 2000.

Abstract

Drosophila Pimples (PIM) and Three rows (THR) are required for sister chromatid separation in mitosis. PIM accumulates during interphase and is degraded rapidly during mitosis. This degradation is dependent on a destruction box similar to that of B-type cyclins. Nondegradable PIM with a mutant destruction box can rescue sister chromatid separation in pim mutants but only when expressed at low levels. Higher levels of nondegradable PIM, as well as overexpression of wild-type PIM, inhibit sister chromatid separation. Moreover, cells arrested in mitosis before sister chromatid separation (by colcemid or by mutations in fizzy/CDC20) fail to degrade PIM. Thus, although not related by primary sequence, PIM has intriguing functional similarities to the securin proteins of budding yeast, fission yeast, and vertebrates. Whereas these securins are known to form a complex with separins, we show that PIM associates in vivo with THR, which does not contain the conserved separin domain.

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Figures

Figure 1

Figure 1

PIM and THR form a complex in vivo. Extracts (extract) prepared from embryos expressing either no transgene (+), or Cdk1–myc (Cdk1–myc), gpim–myc (pim–myc), gthr–myc (thr–myc), or pimdba–myc (pimdba–myc), as well as anti-myc immunoprecipitates isolated from these extracts (IP anti-myc), were analyzed by immunoblotting with antibodies against the myc epitope (myc), THR (THR), PIM (PIM), or Cyclin B (CYCB). (*) Crossreaction of the antibodies against PIM with an unknown protein.

Figure 2

Figure 2

The PIM destruction box variant can replace the Cyclin B destruction box. prd–GAL4, which directs UAS target gene expression in alternating segments starting before mitosis 15 of Drosophila embryogenesis, was used to express either Cyclin B with the myc epitope in place of the destruction box (UAS–CycB–dbm; A–C), or Cyclin B with the endogenous destruction box (UAS–CycB–dbCycB; D–F), or Cyclin B with the PIM destruction box (UAS–CycB–dbpim; G–L). Embryos (A,D,G) were fixed during the stage of mitosis 15 and double-labeled with antibodies against Cyclin B (CycB; A,B,D,E,G,H,J), tubulin (tub; C,F,I,K) and a DNA stain (DNA; L). Higher magnification views of the embryonic epidermis (B,C,E,F,H,I) are shown with the regions of UAS target gene expression to the right of the dashed vertical lines. UAS target gene expression is absent from the regions on the left of the dashed vertical line. These regions express only endogenous Cyclin B and serve as internal control for progression through mitosis 15. Progression through mitosis 15 is accompanied by degradation of Cyclin B protein when carrying a functional degradation box and occurs in a segmentally repeated pattern (Foe et al. 1993) first in the dorsal epidermis (above the horizontal dashed line) and only later in the ventral epidermis (below the horizontal dashed line). At the stage shown, mitosis 15 is largely completed in the dorsal epidermis and just starting in the ventral epidermis. Nondegradable Cyclin B with the myc epitope in place of the destruction box blocks exit from mitosis and results in an enrichment of mitotic figures (C, upper right) in cells that are labeled by anti-Cyclin B (B, upper right). In contrast, Cyclin B with the PIM motif in place of the destruction box does not block exit from mitosis and is degraded during late mitosis as illustrated in the regions shown at even higher magnification (J–L). Arrowheads mark a telophase cell that is not labeled with anti-Cyclin B (J) while the neighboring metaphase cells (right) are strongly labeled. The structure of the different _UAS_-transgenes is schematically illustrated above the panels with the corresponding results.

Figure 3

Figure 3

PIM with mutations in the destruction box motif is stable in mitosis and inhibits sister chromatid separation. nos–GAL4–GCN4–bcd3_′_UTR was used to express either wild-type PIM with carboxy-terminal myc epitopes (UAS–pim–myc; A–D) or PIM with carboxy-terminal myc epitopes and a mutant destruction box (UAS–pimdba–myc; E–H) in the anterior region of gastrulating embryos. Embryos (A,E) were fixed and labeled with antibodies against the myc epitope (myc; A,B,E,F), Cyclin B (CycB; C,G) and a DNA stain (DNA; D,H). Arrowheads in the high magnification views of a head region indicate normal anaphase and telophase figures (B–D), while arrows mark abnormal “metaphase” plates with decondensing chromosomes (E–H) in regions that lack anti-Cyclin B labeling and thus have progressed beyond the metaphase–anaphase transition. UAS–pimdba–myc I.1; UAS–pimdba–myc III.1 embryos for control (I) and UAS–pimdba–myc I.1/+; UAS–pimdba–myc III.1/da–GAL4 embryos (J), in which mitosis 15 is the first division affected by the expression of nondegradable PIM, were incubated in colcemid at the stage of mitosis 16 before preparation of mitotic chromosome spreads stained for DNA. Diplo-chromosomes (J) indicating the failure of sister chromatid separation during mitosis 15 were not observed in controls (I).

Figure 4

Figure 4

Overexpression of wild-type PIM inhibits sister chromatid separation. prd–GAL4 (A–D) or da–GAL4 (E,F) was used for overexpression of wild-type PIM with carboxy-terminal myc epitopes from either one (A,B) or two (C–F) UAS–pim–myc transgene copies. Embryos were fixed either after mitosis 16 (A–D) or during mitosis 16 (E,F) and labeled with antibodies against the myc epitope (myc; A,C), tubulin (tub; E) and with a DNA stain (DNA; B,D,F). Overexpression from one UAS–pim–myc copy does not affect progression through the sixteenth embryonic division. The normal nuclear density is therefore observed in the _prd–GAL4_-expressing segments (A,B, white horizontal bars). In contrast, overexpression from two UAS–pim–myc copies results in inhibition of sister chromatid separation during mitosis 16. Arrowheads in E and F indicate cells during telophase of mitosis 16 with unseparated chromosomes. As a consequence, cytokinesis fails as well, but exit from mitosis 16 occurs normally. This failure of sister chromatid separation and cytokinesis is evidenced by the lower density of interphase nuclei in the _prd–GAL4_-expressing segments (C,D, white bars) after mitosis 16.

Figure 5

Figure 5

PIM persists during the mitotic arrest caused by colcemid or lack of Fizzy. (A–D) prd–GAL4 was used to express one UAS–pim–myc copy in fizzy mutant embryos. Embryos were fixed at a stage where mitosis 16 is completed during wild-type development and labeled with anti-myc (A,C), a DNA stain (B,D), and anti-β-galactosidase for the identification of fizzy homozygotes (data not shown). High magnification views of the dorsal epidermis (A,B) illustrate that one UAS–pim–myc copy is sufficient to inhibit sister chromatid separation during mitosis 16 in fizzy mutants, leading to the reduced nuclear density in the _prd–GAL4_-expressing regions (white bar). High magnification views of the ventral epidermis (C,D) illustrate the persistence of PIM–myc in cells arrested in metaphase 16 because of lack of Fizzy (arrowheads), leading to the intense anti-myc labeling in the arrested cells within the _prd–GAL4-_expressing region (white bar). (E) Progeny from fizzy/CyO parents was aged to the stage where mitosis 16 is completed during wild-type development. fizzy homozygous embryos (fzy−) were sorted from sibling embryos (fzy+) and analyzed by immunoblotting with antibodies against PIM (PIM), Cyclin B (CYCB), Cdk1 (CDK1), FZY (FZY), and tubulin (TUB). (F–M) _nos–GAL4–GCN4–bcd3_′ UTR was used to express PIM with carboxy-terminal myc epitopes (UAS–pim–myc). Embryos at the stage of mitosis 14 were permeabilized and incubated for 25 min either in the absence (F–I) or presence (J–M) of colcemid before fixation and labeling with antibodies against the myc epitope (myc; H,L), Cyclin A (CycA; G,K) and a DNA stain (DNA; F,J). The merged panels (I,M) show labeling of DNA in blue, Cyclin A in red, and PIM–myc in green. (i) Interphase; (p) prophase; (m) metaphase; (a) anaphase; (c) colcemid-arrested cells.

Figure 6

Figure 6

Analysis of PIMdba–myc expression levels. Extracts from embryos with either the gpim–myc (PIM–myc) or the gpimdba–myc (PIMdba–myc) transgene were analyzed by immunoblotting with antibodies against the myc epitope (myc) or tubulin (TUB), which served as a loading control. In addition, to allow quantitative comparisons, we also analyzed an extract from embryos with a 3× higher gpim–myc transgene dose (3) as well as twofold serial dilutions of this extract (1.5; 0.75; 0.375).

Figure 7

Figure 7

Low levels of PIMdba–myc expression allow sister chromatid separation in pim mutants. pim1/ pim1 (A–C) or pim1/ pim1, gpimdba–myc (D–F) embryos at the stage of mitosis 15 were labeled with a DNA stain (DNA; A,D) and antibodies against Cyclin B (CycB; B,E). Regions of the dorsal epidermis are shown. In the merged panels (C,F) DNA labeling is shown in red and Cyclin B in green. Arrows in A–C indicate cells that have failed to separate sister chromatids even though they have progressed beyond the metaphase–anaphase transition as evidenced by lack of anti-Cyclin B labeling. Arrowheads in D–F indicate cells that have separated sister chromatids successfully after the metaphase–anaphase transition. (*, D–F) Cell with an anaphase bridge.

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