Regulation and Requirement of MCPH1 and Rough Deal during Drosophila Early Embryogenesis

Abstract

In a biochemical screen Anaphase-Promoting Complex (APC) substrates, we identified Drosophila MCPH1 isoform B (dMCPH1-B) as a substrate of APCCdh1. dMCPH1-B undergoes Cdh1-dependent degradation in Xenopus egg extract, which is mediated by an N-terminal Destruction (D)-box motif. dMCPH1-B is ubiquitinated by APCCdh1 in vitro and the D-box is required for ubiquitination. Additionally, an N-terminal dMCPH1-B peptide containing the D-box motif is sufficient for in vitro APCCdh1-mediated ubiquitination. dMCPH1 levels are also increased in Drosophila embryos derived from females with reduced APC activity, suggesting that it is an APC substrate in vivo. Human MCPH1, however, does not undergo Cdh1-dependent degradation in Xenopus extract and its levels do not fluctuate during the cell cycle. This suggests that APC-dependent regulation of MCPH1 is not conserved in humans. Furthermore, overexpression of either hMCPH1 or dMCPH1-B in Xenopus embryos results in cell division defects, suggesting regulation of their levels is required for cell-cycle progression. We hypothesize that APC–dependent regulation of dMCPH1-B is required for cell-cycle progression during Drosophila early embryogenesis In a genetic screen for regulators of Drosophila embryogenesis, we also identified rodZ3, a novel allele of rough deal (rod). Rod is a subunit of the Rod-Zw10-Zwilch (RZZ) complex, which is required for Spindle Assembly Checkpoint (SAC) activation in metazoans. rodZ3 encodes a G-E substitution in the Rod C-terminus. This mutation does not affect the levels of RZZ subunits or RZZ complex formation. rodZ3 females are sterile and produce eggs which undergo developmental arrest during syncytial embryogenesis. rodZ3 embryos contain large interphase-like polar bodies. These polar body defects are suppressed by increasing Cyclin B levels, suggesting that polar body condensation requires stabilization of Cyclin B. Additionally, rodZ3 embryos contain asynchronously dividing syncytial nuclei with aberrant centrosome attachments. They also do not have an active SAC response. Furthermore, GFP-Zw10 does not localize to kinetochores in rodZ3 embryos as it does in wild type embryos. We hypothesize that the Rod C-terminus mediates RZZ recruitment to kinetochores and is required to induce SAC activation, maintain polar body condensation, and regulate the progression of syncytial embryogenesis.