Embryonic

The E2-C family of ubiquitin-conjugating enzymes contains two destruction boxes that at least in the case of the mammalian protein are required to mediate the destruction of the protein during mitosis. It was therefore of interest to determine whether the Vihar protein of Drosophila also undergoes cyclical proteolysis. Because it is very difficult to obtain synchronized material for biochemical studies of cell cycle progression in Drosophila, this question was approached in three different ways. First immunostaining was used to localize Vihar E2-C at different mitotic stages in both cultured S2 cells and in wild-type syncytial embryos (Mathe, 2004).

In cultured S2 cells, it was found that Vihar E2-C protein accumulates in nuclei as soon as the chromosomes start to condense and on the centrosomes while the daughter centrosomes have separated and are migrating. At metaphase, it shows strong association to centrosomes. The intensity of centrosome-associated Vihar E2-C staining is distinctly lower at anaphase, but it is not until telophase/cytokinesis that its degradation appears to be maximal. The cytoplasmic levels of Vihar E2-C appear to be similar during mitosis until telophase, when Vihar E2-C, initially excluded from the telophase nuclei, appears to accumulate in the equatorial region of the cell, where the cleavage furrow is to form. By the time cytokinesis is completed, almost all the cytoplasmic Vihar E2-C has been degraded. These characteristic patterns of Vihar E2-C immunolocalization are completely abolished following vih RNAi. A similar pattern of localization is observed in syncytial embryos. Taken together, these observations indicate that Vihar E2-C appears to undergo cyclical proteolysis initiated with either release of Vihar E2-C from the centrosome or the direct degradation of the centrosome-associated protein followed by a second wave of degradation of cytoplasmic protein that is maximal later in mitosis (Mathe, 2004).

In order to rule out the possibility that the diminution of Vihar immunostaining does not simply reflect its dispersal through the cell, attempts were made to demonstrate reduction in Vihar levels by Western blotting. To this end, cells were subjected to nocodazole treatment for 24 hr, causing them to accumulate in mitosis. The drug was washed away, and cells were analyzed at 1 hr intervals for the levels of Vihar E2-C and Cyclin B as a positive control. The majority of both Vihar E2-C and Cyclin B had been degraded by 2 hr, and both were scarcely detectable at 3 hr after removal of the drug. Thus, like Cyclin B, the E2-C enzyme is subject to proteolysis once nocodazole-arrested cells are released from their metaphase-like arrest (Mathe, 2004).

Effects of Mutation or Deletion

The vihar gene was first identified through a semilethal P-lacW insertion in which about 20% of individuals die as pharate adults. The mitotic index and proportion of cells at different phases of the mitotic cycle in the brains of vih¹/vih¹ larvae were not significantly different from those of wild-type. However, the mutant brains displayed aberrant mitotic figures in which the chromosomes were scattered apparently randomly through the cell and in which there was overall a significant increase in polyploid mitotic figures. The vihar locus was uncovered by chromosomal deficiencies that place the gene in the 69C-D region, consistent with the insertion site of the P-lacW element. The mitotic index and proportion of mitotic defects were increased in the larval brains of hemizygous mutant larvae, indicating the hypomorphic nature of the mutation. Both homozygous and hemizygous vih¹ females showed maternal effect lethality and produced syncytial embryos that failed to develop as a result of mitotic defects (Mathe, 2004).

It was possible to revert the vih¹ semilethal and maternal effect lethal phenotype under dysgenic conditions to restore full viability and fertility when homozygous or when heterozygous against the original vih¹ allele or an uncovering deficiency, Df(3L)iro-2. This experiment also generated one partial fully sterile revertant, vih². The P-lacW element from the vih¹ mutant was inserted 9 bp upstream of a Drosophila consensus sequence for the initiation of translation and 129 bp upstream of the start codon of the open reading frame CG10682. A germline transformant in which the CG10682 cDNA was expressed downstream of the polyubiquitin promoter fully restored the fertility of vih mothers. Thus, the vihar gene corresponds to CG10682 (Mathe, 2004).

To gain further insight into the mitotic defects, the mitotic spindles were examined of vih mutants. The central nervous systems of vih¹ larvae display characteristic mitotic defects at a modest frequency consistent with those in orcein-stained squashed preparations and are mirrored by only partial reduction of the Vihar protein. Defective mitotic spindles were characterized by chromosomes being scattered throughout the length of the spindle and/or by the absence of the core centrosomal antigen CNN from one or both poles. The spindle poles that lacked centrosomes are characteristically broad, and in all such cells the chromosomes appear to be overcondensed. Embryos derived from mothers carrying either the vih¹ or vih² alleles, whether homozygous, hemizygous, or transheterozygous, show similarly aberrant mitotic spindles and have a greater reduction in levels of Vihar protein. Thus, the P insertion would appear to affect maternally driven components of the vih promoter more strongly than zygotic ones. Such embryos rarely develop beyond the third nuclear division cycle, and all the mitotic figures show these defects. The mitotic figures in embryos at these stages frequently have a polyploid complement of chromosomes (Mathe, 2004).

Since members of the E2-C family of proteins in fission yeast UbcP4/Ubc11, clam E2-C, Xenopus UBCx, mouse E2-C, and human UbcH10 participate in the degradation of mitotic cyclins (Aristarkhov, 1996; Yu, 1996; Townsley, 1997; Yamanaka, 2000; Osaka, 1997), Cyclin B levels were followed in cultured cells subjected to vih RNAi. The development of the phenotype was followed over 3 days of treatment with vih dsRNA, during which time Western blots showed substantial elimination of Vihar protein that was associated with a corresponding increase in levels of Cyclin B. In the course of such experiments, there was a 7-fold increase in the mitotic index, such that 40% of the cells arrested in mitosis with a 3-fold increase in the frequency of metaphases at the expense of a similar decrease in the proportion of cells in cytokinesis. vih dsRNA was able to phenocopy the characteristic spindle defects observed in vih mutants, and the mitotic spindle never showed prominent lengthening as normally occurs in wild-type anaphase. Immunostaining identified three categories of defective bipolar spindles. The first class features spindles with aligned metaphase chromosomes having a Cyclin B-stained centrosome at only one pole. Such figures are also present in untreated control cells. The second class shows chromosomes scattered throughout spindles that usually have just a single centrosome. Finally, spindles of the third class have chromosomes in the equatorial region but are significantly shorter (by approximately 60%) than the metaphase spindles in untreated cells. These small spindles have centrosomes at each pole. The proportions of these last two classes of defective spindle increased during the course of the RNAi experiment. In contrast to untreated cells, where Cyclin B undergoes cyclical degradation, Cyclin B seems to accumulate at the centrosomes, at the spindle, and throughout the cell following vihar RNAi. This is consistent with the increase in Cyclin B seen in many cell types after APC/C inhibition, for example, following spindle integrity checkpoint arrest (Mathe, 2004).

The scattered distribution of chromosomes in cells with reduced levels of Vihar resembles the phenotype seen following the expression of nondegradable Cyclin B in Drosophila (Parry, 2001; Sigrist, 1995; Rimmington, 1994; Echard; 2003; Parry; 2003). In such circumstances, Parry (2003) showed that chromosomes undergo an oscillating behavior due to reestablishment of microtubule connections between kinetochores and both poles with concomitant reacquisition of checkpoint proteins. The kinetochore regions of the scattered chromosomes in cells with reduced amounts of Vihar are also associated with the BubR1 checkpoint protein, suggesting that, as in the case of cells expressing nondegradable Cyclin B, they are attempting chromosome congression (Parry, 2003). Consistently, the Aurora B kinase also remains associated with the kinetochore regions, and there is no sign in these cells of any attempt to organize the central spindle (Mathe, 2004).

Since both Cyclin B and Vihar E2-C have well-defined spatiotemporal profiles of localization and destruction on the mitotic spindle, it was of interest to determine the consequences of expressing a nondegradable form of Vihar E2-C upon mitotic progression. To this end, destruction box mutant variants of Vihar E2-C were cloned downstream of the GAL4 responsive element in pUASP. Germline transformants were then established that carried transgenes with mutations in each individual D box alone (vih^mD1or vih^mD2) or in both D boxes (vih^mD1D2). Crossing such lines with one in which the GAL4 protein is expressed from the maternal alpha-tubulin promoter activates female germline expression from the UAS-containing transgenes. Thus, the embryos derived from such mothers contain both wild-type and D box mutant Vihar E2-C protein (Mathe, 2004).

When wild-type Vihar E2-C protein was expressed from this maternal GAL4-driven system, the protein was fully able to rescue the vih¹ maternal effect mutant. In contrast, neither vih^mD1, vih^mD2, nor vih^mD1D2 is able to rescue this maternal effect mutant. This indicates that none of these constructs is fully functional. However, no significant change was seen in the embryonic phenotype above that of the background vih¹ mutant. When the three constructs were expressed in a wild-type background, flies were fully fertile and displayed no maternal effect. When, however, the gene dosage of wild-type protein was reduced to 50%, it was found that the expression of vih^mD1 has no effect, the expression of vih^mD2 results in some mothers failing to produce viable embryos, and the expression of vih^mD1D2 results in complete maternal effect lethality (Mathe, 2004).

Immunostaining experiments were carried out to determine whether expression of vih^mD1D2 results in any mitotic defects and, if so, whether they are associated abnormalities in Cyclin B degradation. In wild-type embryos, Cyclin B is degraded on the mitotic spindle in an apparent wave that spreads from the poles toward the chromosomes as mitotic figures progress through the metaphase-anaphase transition. This is most clearly visualized using GFP-tagged Cyclin B in cellularized embryos but can be seen with some difficulty in immunostained fixed preparations of syncytial stage embryos. The expression of vih^mD1D2 in embryos derived from hemizygous vih⁺/− mothers results in roughly equivalent levels of wild-type and mutant protein and leads to arrest of mitosis predominantly in a metaphase or anaphase state during the cleavage embryonic cycles in the absence of a proteasome inhibitor. It did not appear to prevent chromatid separation, because chromosomes were in either a metaphase or anaphase configuration. It was not possible to detect the His-tagged Vihar^mD1D2 protein at centrosomes in the arrested mitotic figures. In all cases, expression of the vih^mD1D2 mutant results in a reduction of Cyclin B staining at the spindle poles and an accumulation of Cyclin B in the central part of the spindle in a manner never seen in wild-type embryos across a whole field of mitoses. Thus, stabilization of the Vihar E2-C permits Cyclin B degradation in the region of the spindle poles, but not at the spindle equator, and this prevents completion of mitosis. This contrasts to loss of Vihar function when Cyclin B accumulates at the spindle poles (Mathe, 2004).

Reference names in red indicate recommended papers.

Aristarkhov, A., et al. (1996). E2-C, a cyclin-selective ubiquitin carrier protein required for the destruction of mitotic cyclins. Proc. Natl. Acad. Sci. 93(9): 4294-9. 8633058

Bastians, H., et al. (1999). Cell cycle-regulated proteolysis of mitotic target proteins. Mol. Biol. Cell 10(11): 3927-41. 10564281

Clute, P. and Pines, J. (1999). Temporal and spatial control of cyclin B1 destruction in metaphase. Nat. Cell Biol. 1(2): 82-7. 10559878

Criqui, M. C., et al. (2002). Molecular characterization of plant ubiquitin-conjugating enzymes belonging to the UbcP4/E2-C/UBCx/UbcH10 gene family. Plant Physiol. 130(3): 1230-40. 12427990

Echard, A. and O'Farrell, P. H. (2003). The degradation of two mitotic cyclins contributes to the timing of cytokinesis. Curr. Biol. 13: 373-383. 12620185

Gabellini, D., et al. (2003). Early mitotic degradation of the homeoprotein HOXC10 is potentially linked to cell cycle progression. EMBO J. 22(14): 3715-24. 12853486

Huang, J.-y. and Raff, J. W. (1999). The disappearance of cyclin B at the end of mitosis is regulated spatially in Drosophila cells. EMBO J. 18(8): 2184-2195.

Jiang, F. and Basavappa, R., et al. (1999). Crystal structure of the cyclin-specific ubiquitin-conjugating enzyme from clam, E2-C, at 2.0 A resolution. Biochemistry. 38(20): 6471-8. 10350465

Lin, Y., Hwang, W. C. and Basavappa, R. (2002). Structural and functional analysis of the human mitotic-specific ubiquitin-conjugating enzyme, UbcH10. J. Biol. Chem. 277(24): 21913-21. 11927573

Mathe, E., Kraft, C., Giet, R., Deak, P., Peters, J. M. and Glover, D. M. (2004). The E2-C vihar is required for the correct spatiotemporal proteolysis of cyclin B and itself undergoes cyclical degradation. Curr. Biol. 14(19): 1723-33. 15458643

Okamoto, Y., et al. (2003). UbcH10 is the cancer-related E2 ubiquitin-conjugating enzyme. Cancer Res. 63(14): 4167-73. 12874022

Osaka, F., Seino, H., Seno, T. and Yamao, F. (1997). A ubiquitin-conjugating enzyme in fission yeast that is essential for the onset of anaphase in mitosis. Mol. Cell Biol. 17(6): 3388-97. 9154838

Parry, D. H. and O'Farrell, P. H. (2001). The schedule of destruction of three mitotic cyclins can dictate the timing of events during exit from mitosis. Curr. Biol. 11(9): 671-83. 11369230

Parry, D. H., Hickson, G. R. and O'Farrell, P. H. (2003). Cyclin B destruction triggers changes in kinetochore behavior essential for successful anaphase. Curr. Biol. 13(8): 647-53. 12699620

Raff, J. W., Jeffers, K. and Huang, J.-y. (2001). The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the destruction of cyclin B in space and time. J. Cell Biol. 157: 1139-1149. 12082076

Rape, M. and Kirschner, M. W. (2004). Autonomous regulation of the anaphase-promoting complex couples mitosis to S-phase entry. Nature 432(7017): 588-95. 15558010

Rimmington, G., Dalby, B. and Glover, D. M. (1994). Expression of N-terminally truncated cyclin B in the Drosophila larval brain leads to mitotic delay at late anaphase. J Cell Sci 107: 2729-2738

Seino, H., Kishi, T., Nishitani, H. and Yamao, F. (2003). Two ubiquitin-conjugating enzymes, UbcP1/Ubc4 and UbcP4/Ubc11, have distinct functions for ubiquitination of mitotic cyclin. Mol Cell Biol. 23(10): 3497-505. 12724408

Sigrist, S., et al. (1995). Exit from mitosis is regulated by Drosophila fizzy and the sequential destruction of cyclins A, B and B3. EMBO J. 14(19): 4827-4838. 7588612

Townsley, F. M., et al. (1997). Dominant-negative cyclin-selective ubiquitin carrier protein E2-C/UbcH10 blocks cells in metaphase. Proc. Natl. Acad. Sci. 94(6): 2362-7. 9122200

Townsley, F. M. and Ruderman, J. V. (1998). Functional analysis of the Saccharomyces cerevisiae UBC11 gene. Yeast 14(8): 747-57. 9675819

Wagner, K. W., et al. (2004). Overexpression, genomic amplification and therapeutic potential of inhibiting the UbcH10 ubiquitin conjugase in human carcinomas of diverse anatomic origin. Oncogene 23(39): 6621-9. 15208666

Wakefield, J. G., Huang, J.-y. and Raff, J. W. (2000). Centrosomes have a role in regulating the destruction of cyclin B in early Drosophila embryos Curr. Biol. 10: 1367-1370. 11084336

Yamanaka, A., et al. (2000). Cell cycle-dependent expression of mammalian E2-C regulated by the anaphase-promoting complex/cyclosome. Mol. Biol. Cell 11(8): 2821-31. 10930472

Yu, H., King, R. W., Peters, J. M. and Kirschner, M. W. (1996). Identification of a novel ubiquitin-conjugating enzyme involved in mitotic cyclin degradation. Curr Biol. 6(4): 455-66. 8723350

date revised: 5 March 2005

Home page: The Interactive Fly © 2003 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.