RESUMO
An important feature of fertilization is the asymmetric inheritance of centrioles. In most species it is the sperm that contributes the initial centriole, which builds the first centrosome that is essential for early development. However, given that centrioles are thought to be exceptionally stable structures, the mechanism behind centriole disappearance in the female germ line remains elusive and paradoxical. We elucidated a program for centriole maintenance in fruit flies, led by Polo kinase and the pericentriolar matrix (PCM): The PCM is down-regulated in the female germ line during oogenesis, which results in centriole loss. Perturbing this program prevents centriole loss, leading to abnormal meiotic and mitotic divisions, and thus to female sterility. This mechanism challenges the view that centrioles are intrinsically stable structures and reveals general functions for Polo kinase and the PCM in centriole maintenance. We propose that regulation of this maintenance program is essential for successful sexual reproduction and defines centriole life span in different tissues in homeostasis and disease, thereby shaping the cytoskeleton.
Assuntos
Centríolos/metabolismo , Drosophila melanogaster , Oogênese , Óvulo/citologia , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Feminino , Fertilização , Deleção de Genes , Microtúbulos/metabolismo , Oócitos/citologia , Oócitos/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Centrioles duplicate once in each cell division cycle through so-called templated or canonical duplication. SAK, also called PLK4 (SAK/PLK4), a kinase implicated in tumor development, is an upstream regulator of canonical biogenesis necessary for centriole formation. We found that overexpression of SAK/PLK4 could induce amplification of centrioles in Drosophila embryos and their de novo formation in unfertilized eggs. Both processes required the activity of DSAS-6 and DSAS-4, two molecules required for canonical duplication. Thus, centriole biogenesis is a template-free self-assembly process triggered and regulated by molecules that ordinarily associate with the existing centriole. The mother centriole is not a bona fide template but a platform for a set of regulatory molecules that catalyzes and regulates daughter centriole assembly.
Assuntos
Centríolos/fisiologia , Centrossomo/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/embriologia , Embrião não Mamífero/fisiologia , Oócitos/fisiologia , Animais , Animais Geneticamente Modificados , Centríolos/ultraestrutura , Centrossomo/ultraestrutura , Drosophila/metabolismo , Proteínas de Drosophila/genética , Desenvolvimento Embrionário , Feminino , MitoseRESUMO
BACKGROUND: SAK/PLK4 is a distinct member of the polo-like kinase family. SAK-/- mice die during embryogenesis, whereas SAK+/- mice develop liver and lung tumors and SAK+/- MEFs show mitotic abnormalities. However, the mechanism underlying these phenotypes is still not known. RESULTS: Here, we show that downregulation of SAK in Drosophila cells, by mutation or RNAi, leads to loss of centrioles, the core structures of centrosomes. Such cells are able to undergo repeated rounds of cell division, but display broad disorganized mitotic spindle poles. We also show that SAK mutants lose their centrioles during the mitotic divisions preceding male meiosis but still produce cysts of 16 primary spermatocytes as in the wild-type. Mathematical modeling of the stereotyped cell divisions of spermatogenesis can account for such loss by defective centriole duplication. The majority of spermatids in SAK mutants lack centrioles and so are unable to make sperm axonemes. Finally, we show that depletion of SAK in human cells also prevents centriole duplication and gives rise to mitotic abnormalities. CONCLUSIONS: SAK/PLK4 is necessary for centriole duplication both in Drosophila and human cells. Drosophila cells tolerate the lack of centrioles and undertake mitosis but cannot form basal bodies and hence flagella. Human cells depleted of SAK show error-prone mitosis, likely to underlie its tumor-suppressor role.
Assuntos
Centríolos/fisiologia , Flagelos/fisiologia , Mitose/fisiologia , Modelos Biológicos , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/fisiologia , Animais , Células Cultivadas , Centríolos/genética , Centríolos/ultraestrutura , Drosophila , Flagelos/genética , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Proteínas Serina-Treonina Quinases/genética , Interferência de RNARESUMO
Cycles of protein phosphorylation are fundamental in regulating the progression of the eukaryotic cell through its division cycle. Here we test the complement of Drosophila protein kinases (kinome) for cell cycle functions after gene silencing by RNA-mediated interference. We observed cell cycle dysfunction upon downregulation of 80 out of 228 protein kinases, including most kinases that are known to regulate the division cycle. We find new enzymes with cell cycle functions; some of these have family members already known to phosphorylate microtubules, actin or their associated proteins. Additionally, depletion of several signalling kinases leads to specific mitotic aberrations, suggesting novel roles for familiar enzymes. The survey reveals the inter-digitation of systems that monitor cellular physiology, cell size, cellular stress and signalling processes with the basic cell cycle regulatory machinery.