ABSTRACT
In a mammalian oocyte, completion of meiosis is suspended until fertilization by a sperm, and the cell cycle is arrested by a biochemical activity called cytostatic factor (CSF). Emi2 is one of the CSFs, and it maintains the protein level of maturation promoting factor (MPF) by inhibiting ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Degradation of Emi2 via ubiquitin-mediated proteolysis after fertilization requires phosphorylation by Polo-like kinase 1 (Plk1). Therefore, recognition and phosphorylation of Emi2 by Plk1 are crucial steps for cell cycle resumption, but the binding mode of Emi2 and Plk1 is poorly understood. Using biochemical assays and X-ray crystallography, we found that two phosphorylated threonines (Thr(152) and Thr(176)) in Emi2 are each responsible for the recruitment of one Plk1 molecule by binding to its C-terminal polo box domain (PBD). We also found that meiotic maturation and meiosis resumption via parthenogenetic activation were impaired when Emi2 interaction with Plk1-PBD was blocked by a peptidomimetic called 103-8. Because of the inherent promiscuity of kinase inhibitors, our results suggest that targeting PBD of Plk1 may be an effective strategy for the development of novel and specific contraceptive agents that block oocyte maturation and/or fertilization.
Subject(s)
Cell Cycle Proteins/chemistry , F-Box Proteins/chemistry , Peptidomimetics/chemistry , Protein Interaction Domains and Motifs , Protein Serine-Threonine Kinases/chemistry , Proto-Oncogene Proteins/chemistry , Animals , Binding Sites , Cell Cycle Proteins/metabolism , Cell Differentiation/drug effects , F-Box Proteins/metabolism , Fertilization/drug effects , Meiosis/drug effects , Mesothelin , Mice , Models, Molecular , Oocytes/cytology , Oocytes/drug effects , Oocytes/metabolism , Peptidomimetics/administration & dosage , Peptidomimetics/pharmacology , Phosphorylation , Protein Binding , Protein Conformation , Protein Serine-Threonine Kinases/metabolism , Proteolysis , Proto-Oncogene Proteins/metabolism , Spindle Apparatus/metabolism , Structure-Activity Relationship , Xenopus , Polo-Like Kinase 1ABSTRACT
Dynamic actin reorganization is the main driving force for spindle migration and asymmetric cell division in mammalian oocytes. It has been reported that various actin nucleators including Formin-2 are involved in the polarization of the spindle and in asymmetric cell division. In mammals, the formin family is comprised of 15 proteins. However, their individual roles in spindle migration and/or asymmetric division have not been elucidated yet. In this study, we employed a newly developed inhibitor for formin family proteins, small molecule inhibitor of formin homology 2 domains (SMIFH2), to assess the functions of the formin family in mouse oocyte maturation. Treatment with SMIFH2 during in vitro maturation of mouse oocytes inhibited maturation by decreasing cytoplasmic and cortical actin levels. In addition, treatment with SMIFH2, especially at higher concentrations (500 µM), impaired the proper formation of meiotic spindles, indicating that formins play a role in meiotic spindle formation. Knockdown of the mDia2 formins caused a similar decrease in oocyte maturation and abnormal spindle morphology, mimicking the phenotype of SMIFH2-treated cells. Collectively, these results suggested that besides Formin-2, the other proteins of the formin, including mDia family play a role in asymmetric division and meiotic spindle formation in mammalian oocytes.
Subject(s)
Asymmetric Cell Division/genetics , Carrier Proteins/genetics , Microtubule-Associated Proteins/genetics , NADPH Dehydrogenase/genetics , Oocytes/growth & development , Spindle Apparatus/metabolism , Thiones/pharmacology , Uracil/analogs & derivatives , Actins/metabolism , Animals , Carrier Proteins/metabolism , Cells, Cultured , Formins , Meiosis , Mice , Microtubule-Associated Proteins/metabolism , NADPH Dehydrogenase/metabolism , Oocytes/cytology , Oocytes/drug effects , RNA Interference , RNA, Small Interfering , Tropomyosin/metabolism , Uracil/pharmacologyABSTRACT
Tropomyosins are actin-binding cytoskeletal proteins that play a pivotal role in regulating the function of actin filaments in muscle and non-muscle cells; however, the roles of non-muscle tropomyosins in mouse oocytes are unknown. This study investigated the expression and functions of non-muscle tropomyosin (Tpm3) during meiotic maturation of mouse oocytes. Tpm3 mRNA was detected at all developmental stages in mouse oocytes. Tpm3 protein was localized at the cortex during the germinal vesicle and germinal vesicle breakdown stages. However, the overall fluorescence intensity of Tpm3 immunostaining was markedly decreased in metaphase II oocytes. Knockdown of Tpm3 impaired asymmetric division of oocytes and spindle migration, considerably reduced the amount of cortical actin, and caused membrane blebbing during cytokinesis. Expression of a constitutively active cofilin mutant and Tpm3 overexpression confirmed that Tpm3 protects cortical actin from depolymerization by cofilin. The data indicate that Tpm3 plays crucial roles in maintaining cortical actin integrity and asymmetric cell division during oocyte maturation, and that dynamic regulation of cortical actin by Tpm3 is critical to ensure proper polar body protrusion.
