Abstract
BackgroundThe protein kinases Mps1 and Polo, which are required for proper cell cycle regulation in meiosis and mitosis, localize to numerous ooplasmic filaments during prometaphase in Drosophila oocytes. These filaments first appear throughout the oocyte at the end of prophase and are disassembled after egg activation.Methodology/Principal FindingsWe showed here that Mps1 and Polo proteins undergo dynamic and reversible localization to static ooplasmic filaments as part of an oocyte-specific response to hypoxia. The observation that Mps1- and Polo-associated filaments reappear in the same locations through multiple cycles of oxygen deprivation demonstrates that underlying structural components of the filaments must still be present during normoxic conditions. Using immuno-electron microscopy, we observed triple-helical binding of Mps1 to numerous electron-dense filaments, with the gold label wrapped around the outside of the filaments like a garland. In addition, we showed that in live oocytes the relocalization of Mps1 and Polo to filaments is sensitive to injection of collagenase, suggesting that the structural components of the filaments are composed of collagen-like fibrils. However, the collagen-like genes we have been able to test so far (vkg and CG42453) did not appear to be associated with the filaments, demonstrating that the collagenase-sensitive component of the filaments is one of a number of other Drosophila proteins bearing a collagenase cleavage site. Finally, as hypoxia is known to cause Mps1 protein to accumulate at kinetochores in syncytial embryos, we also show that GFP-Polo accumulates at both kinetochores and centrosomes in hypoxic syncytial embryos.Conclusions/SignificanceThese findings identify both a novel cellular structure (the ooplasmic filaments) as well as a new localization pattern for Mps1 and Polo and demonstrate that hypoxia affects Polo localization in Drosophila.
Highlights
The Drosophila ovary has proven to be a useful model system for studying the mechanisms by which the processes of oocyte maturation and chromosome segregation interact with or are controlled by the meiotic cell cycle
We reported that Mps1 and Polo filament formation appeared to initiate at the onset of nuclear envelope breakdown (NEB) [20], an observation that has been confirmed by analysis of subsequent oocytes that were fixed during the process of NEB (Figure 1)
The experimental data presented here demonstrate that Mps1 and Polo localization to the ooplasmic filaments is a transient response to hypoxic conditions, with the GFP-labeled proteins loading on and off of static filaments on a time scale of approximately 10 minutes
Summary
The Drosophila ovary has proven to be a useful model system for studying the mechanisms by which the processes of oocyte maturation and chromosome segregation interact with or are controlled by the meiotic cell cycle. In addition to the changes in the oocyte nucleus, numerous changes in the 16-cell cyst and its contents are taking place throughout oocyte development, including the growth and degradation of the polytene nurse and follicle cells [3], the kenotic dumping of nurse cell contents into the oocyte [4], the formation of a membranous sheath around the meiotic spindle [5], the growth of the dorsal appendages [6], and the maturation of the vitelline membrane and chorion [7] These processes produce the phenotypic landmarks that are used to divide oogenesis into 14 stages [8,9]. These filaments first appear throughout the oocyte at the end of prophase and are disassembled after egg activation
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