BIOLOGICAL OVERVIEW

Animals and plants use modified cell cycles to achieve particular developmental strategies. In one common example, most animals and plants have tissues in which the cells become polyploid or polytene by means of an S-G cycle, but the mechanism by which mitosis is inhibited in the endo cycle is not understood. The Drosophila morula (mr) gene, coding for APC2 subunit of the anaphase promoting complex/cyclosome, regulates variant cell cycles, because in addition to disrupting the archetypal cycle (G1-S-G2-M), mr mutations affect the rapid embryonic (S-M) divisions as well as the endo cycle (S-G) that produces polyploid cells. In dividing cells, mr mutations cause a metaphase arrest, and endo cycling nurse cells inappropriately reenter mitosis in mr mutants. mr encodes the APC2 subunit of the anaphase promoting complex/cyclosome. This finding demonstrates that anaphase promoting complex/cyclosome is required not only in proliferating cells but also to block mitosis in some endo cycles. The mr mutants further indicate that transient mitotic functions in endo cycles change chromosome morphology from polytene to polyploid (Kashevsky, 2002).

Degradation of protein substrates through the ubiquitin-proteasome pathway involves three components, E1, E2, and E3. E1 uses ATP to form a high-energy thiol ester with the C-terminal glycine of ubiquitin. This ubiquitin is then transferred to a cysteine residue in one of several E2s. Specific E2s assemble with appropriate E3s to initiate transfer of ubiquitin to an associated substrate. APC2 (Morula in Drosophila) is one of the core subunits of the anaphase promoting complex (APC) in Drosophila. The APC is a ubiquitin ligase complex, an E3. There are three known types of E3s (SCF, CBC, and APC/C complexes), and comparison of their domain structures reveals the core constituents and accessory proteins. All three types of E3s contain a core ubiquitin ligase composed of three subunits: (1) a cullin-homology domain (CHD) protein consisting of APC2 in the APC/C complex and Lin-19-like in the SCF complex; (2) a RING-H2 protein consisting of APC11 (Lemming in Drosophila) in the APC/C complex and Roc1a in the SCF complex, and (3) an adapter such as Skp1 in the SCF complex and whose identity is unknown in the case of the APC/C. The adaptor functions to facilitate interaction with substrate receptors [F-box proteins (see for example Drosophila Slimb), BC-box proteins, and Cdc20/Cdh1 proteins]. The RING-H2 finger assembles with the E2 (Harper, 2002).

The endo cycle is the modified cell cycle used throughout the plant and animal kingdoms to produce polyploid or polytene cells. In this cycle, DNA replication cycles with a gap (G) phase, but mitosis (M) does not occur. There is, however, variability in endo cycling tissues in the extent to which mitotic functions are repressed. In polytene cells, in which the replicated sister chromatids remain in tight association, it appears that no aspects of mitosis occur. In contrast, in mammalian megakaryocytes sister-chromatid separation and anaphase A movements occur, but anaphase B and cytokinesis are lacking. Oscillations in the levels and activity of cyclin E/cyclin-dependent kinase (CDK) complexes are crucial for endo cycles, but the mechanism by which mitotic functions are inhibited remains to be defined. Somehow, expression of mitotic cyclin proteins is shut off, and they may be destroyed in a regulated fashion. Variation in the control of the destruction of mitotic cyclins and other mitotic activators could explain the differences to which mitotic functions persist in distinct endo cycling cell types (Kashevsky, 2002).

A pathway for inactivation of mitotic regulators by targeted proteolysis has been delineated. Polyubiquitination of substrate proteins by a ubiquitin ligase, the anaphase promoting complex/cyclosome (APC/C), targets mitotic regulations for destruction by the 26S proteosome. The APC/C is composed of at least 11 subunits. In the yeast Saccharomyces cerevisiae mutations in the APC subunits cdc16, cdc23, and cdc27 were identified because they block cyclin ubiquitination and destruction. These mutations cause a failure of release of sister-chromatid cohesion, block the metaphase/anaphase transition, and prevent exit from mitosis. The APC/C is regulated in part by two associated proteins, Cdc20 (Fizzy in Drosophila) and Cdh1 (Fizzy-related in Drosophila), and these proteins both activate the APC/C with proper timing and provide substrate specificity. The APC/C is activated at the metaphase/anaphase transition by the Cdc20 protein and later in telophase and G₁ by the Cdh1 protein. Mutations in the Drosophila fizzy and fizzy-related APC/C regulators have been characterized. Embryos mutant for fizzy arrest in metaphase of mitosis, whereas embryos lacking fizzy-related fail to cease proliferation at the appropriate stage. Recently, mutations have been described in the Drosophila APC5 subunit gene and shown to affect mitotic divisions during larval stages (Bentley, 2002; Kashevsky, 2002 and references therein).

The failure of mitosis to progress beyond metaphase in mutants for APC/C subunits is caused by the failure to degrade substrates whose sequential destruction is needed for steps through mitosis. At the metaphase/anaphase transition the securin protein family members are ubiquitinated and proteolyzed. Members of this family include the Pds1 protein in S. cerevisiae, Cut2 in Schizosaccharomyces pombe, and Pimples in Drosophila. The securin proteins regulate the separase protease (see Separase) that targets the cohesin complex, and in yeast the Slk19 protein needed for mitotic spindle function. Thus, by indirectly activating separase, the APC/C causes the release of sister-chromatid cohesion and events needed for the completion of mitosis. Mitotic cyclins are also targeted for degradation by the APC/C; this shuts off the mitotic cyclin/CDK1 complex to inactivate mitosis-promoting functions and to also permit resetting of the replication origins for another round of DNA synthesis. Additional direct substrates of the APC/C as well as indirect substrates that are cleaved by separase are likely to be involved in the exit from mitosis (Kashevsky, 2002 and references therein).

The Drosophila morula (mr) gene is critical for the inactivation of mitotic functions throughout development in a variety of developmentally-modified cell cycles (Reed, 1997). The initial mr alleles, described in 1919 and 1937 by Bridges, are female sterile. In these mr¹ and mr² mutants, the endo cell cycle of the polyploid ovarian nurse cells is affected (Reed, 1997). The nurse cells initiate the endo cycle, but after several cycles return to mitosis, condensing their chromosomes, assembling mitotic spindles, and arresting in a metaphase-like state. Stronger alleles of mr cause lethality late in larval development. In these mutant animals, there is a failure to inactivate mitotic functions in proliferating cells. Dividing cells in the larval brain arrest in metaphase. The mr phenotypes indicate that mr is required to prevent mitosis in some endo cycling cells, but also for the inactivation of mitotic functions and exit from mitosis in dividing cells. These intriguing phenotypes make it important to define the molecular mechanism by which mr inhibits mitotic activities (Kashevsky, 2002 and references therein).

A positional cloning strategy was used to recover the mr gene. The gene is removed by the deficiency Df(2R)G10-BR27, but it is present in Df(2R)bw-S46. Quantitative Southern blots were used to map the position of the breakpoints of these two deficiencies, defining a minimal region of 40 kb that contains the gene. Within this region, the CG3060 is an ideal candidate for mr, because it contains a cullin domain, and cullin-domain proteins are involved in protein degradation during the cell cycle (Kashevsky, 2002).

The ability of a cDNA corresponding to this gene to rescue the mr mutant phenotypes was tested. To test for rescue of the female-sterile mr alleles, the Gal-4 transcriptional activator was expressed in the female germ line under the control of the nanos regulatory elements. Three independent cDNA transformant lines restored female fertility to mr¹/mr² transheterozygotes when Gal-4 was induced. Induction of Gal-4 by the ubiquitously expressed actin promoter also rescued fertility in these flies. The actin-Gal-4 driver was able to restore viability to transheterozygotes of two lethal alleles, mr³ and mr⁴. These experiments revealed differences in each of the transgenic lines, presumably reflecting different levels of expression; one complemented fully to restore both viability and fertility, a second restored viability and partial fertility, whereas a third rescued viability. The ability of the cDNA to rescue both strong mr lethal alleles and weaker female-sterile alleles demonstrates that it encodes the structural gene for mr (Kashevsky, 2002).

The identification of the Drosophila mr gene as APC2 demonstrates the essential role of the APC/C in developmentally modified cell cycles as well as the archetypal mitotic cycle. In particular, it is striking that APC/C function is crucial for endo cycles in which it appears that mitosis does not occur. The mr phenotypes reveal an unexpected and intriguing role for the APC/C in setting the parameters of the endo cycle that affects the chromosome structure of the replicated sister chromatids. These results are significant also in establishing an essential role for the APC2 subunit in metazoans (Kashevsky, 2002).

The endo cycle can produce polytene or polyploid chromosomes. In the former case, the replicated sister chromatids remain tightly associated, whereas they are dispersed in polyploid cells. The mr results provide clues into possible cell cycle differences in endo cycles leading to polyteny versus polyploidy. In Drosophila, most cells are polytene, and the nurse cells rarely become polyploid. No endo cycle failure was observed in any larval polytene tissue in mr mutants except the ring gland, which begins the endo cycle late in development. In polytene cells, APC/C activity may be required only at the initial transition from the mitotic cycle to the endo cycle, to remove any remaining mitotic regulators. The majority of larval tissues undergo the transition to the endo cycle late in embryogenesis. Once entrenched in the endo cycle with expression of mitotic cyclin genes shut off, the APC/C would be dispensable. Consistent with this hypothesis, in embryos homozyous for a deletion that removes the fzr gene, the onset of the first S phase of the endo cycle is inhibited in several tissues. These observations indicate that APC/C is required during embryogenesis, but it is likely that maternal stockpiles of Mr protein are present to permit the onset of the endo cycle. It remains possible that mr is essential not only for the onset but also for the maintenance of polytene endo cycles throughout development and that the maternal pools persist during larval development and into adult stages. The molecular identification of Mr permits the generation of reagents to distinguish whether this is the case (Kashevsky, 2002).

Even though the APC/C does not appear to be required for the maintenance of polytene endo cycles, it plays a critical role in the parameters of endo cycles that produce polyploid chromosomes. In polytene cells, APC/C may need to be inactive so that securin remains constitutively active and that the cohesin complex and sister-chromatid cohesion contribute to the tight alignment of replicated sister chromatids. In polyploid cells, degradation of securin by the APC/C could lead to the separation of sister chromatids as a result of separase activity. This activity would explain why the APC/C becomes crucial in the nurse cells when the transition from polyteny to polyploidy occurs. In addition, a low level of transient induction of cyclin B/CDK1 activity, so far undetectable by immunolabeling methods, could account for the chromosome condensation observed at this transition. This hypothesis is supported by the presence of cyclin B protein in mr mutant nurse cells at this time. Overexpression of cyclin B does not, however, induce the change from polyteny to polyploidy at an earlier developmental stage, and this is consistent with other mitotic activities such as the separase protease being necessary. Elimination of securin and separase activity in the nurse cells by making mutant clones might permit a test of this hypothesis (Kashevsky, 2002).

The requirement of APC/C activity for the endo cycle leading to polyploid chromosomes that is observed in Drosophila may be a characteristic feature of endo cycles in many organisms. In alfalfa the expression of a Cdh1-like gene (homolog of Drosophila Fizzy-related) is increased in nodules that have cells undergoing endo cycles. Overexpression of an antisense RNA reduces the ploidy of polyploid cells in the petioles, hypocotyls, and roots. These results are consistent with a role for the APC/C in the maintenance of the endo cycle in polyploid plant cells, though effects on the onset of the endo cycle were not addressed by these analyses. Elimination of mitotic cyclin protein is necessary for endo cycles in plants, because ectopic expression of cyclin B1;2 in Arabidopsis trichome cells causes these cells to undergo mitosis rather than endo cycles (Kashevsky, 2002 and references therein).

The mr mutant effects on the canonical G₁-S-G₂-M cycles are consistent with mutant phenotypes described for the budding yeast S. cerevisiae. An increased number of mitotic cells is seen in brains from mutant larvae, and the majority of these are arrested in metaphase (Reed, 1997). Interestingly, many of these are polyploid, revealing that the metaphase arrest is not indefinite and the cells re-replicate. It appears that sister-chromatid separation is occurring before this replication, because the extra chromosome copies are separate and not attached at their centromeres as in the pimples securin mutant. Thus, either sufficient mr function is present even in the lethal alleles (possibly from maternal pools) to allow eventual exit from mitosis, or an APC/C independent pathway for sister separation and resetting of replication origins may exist (Kashevsky, 2002).

The regulation of mitotic exit during the syncytial S-M cycles of early Drosophila embryogenesis requires localized degradation of mitotic cyclins in the vicinity of each nucleus. In mr mutant embryos the initial S-M cycles arrest in metaphase; this observation combined with the metaphase arrest seen in maternal-effect fzy alleles demonstrates that APC/C function is required for mitotic exit during the S-M cycles (Kashevsky, 2002).

Mutations in APC/C subunits in Caenorhabditis elegans have been demonstrated to block the metaphase I/anaphase I transition and completion of meiosis. In contrast in Xenopus oocytes, inactivation of the APC by injection of antibodies to either the Cdc27 APC subunit or the Fzr activator or injection of inhibitory peptides does not affect the completion of meiosis I but causes a metaphase II block. Both meiotic divisions are completed in the mr mutant eggs. This does not exclude a role for APC/C either in the separation of homologs in meiosis I or sister chromatids in meiosis II, because the mr mutations that produce eggs are weak alleles and residual activity may be sufficient for the completion of meiosis (Kashevsky, 2002 and references therein).

Analysis of APC function during metazoan development, here exemplified by the phenotypes of Drosophila mr mutants, defines the role of this ubiquitin ligase in cells undergoing an archetypal cell cycle but also illustrates its use in modified cell cycles. The role of the APC in meiosis requires further investigation, but its activity in the embryonic S-M cycles is clear. In addition to demonstrating a critical role for APC/C in endo cycles, the mr mutants uncover an intriguing use of mitotic activities to alter chromosome morphology in polytene and polyploid cells (Kashevsky, 2002).

GENE STRUCTURE

cDNA clone length - 1451

Bases in 5' UTR - 11

Exons - 8

Bases in 3' UTR - 90

PROTEIN STRUCTURE

Amino Acids - 449

Structural Domains

Morula protein is closely related to the APC2 subunit of the APC/C. APC2 contains a cullin domain, but Mr shows sequence conservation throughout the protein sequence, not solely within the cullin domain. Overall, Mr is 36% identical to human APC2 and shares 56% homology. Mr is more distantly related to the APC2 subunit from S. cerevisiae (Kashevsky, 2002).

date revised: 30 November 2003

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