Calcium and actin coordinate egg activation and the metaphase-anaphase transition in Drosophila oocytes

Egg activation is the process through which the mature oocyte is prepared for embryogenesis, consisting of key cellular changes including, but not limited to: i) Physical and chemical changes to the oocyte’s outer covering; ii) The release of meiotic arrest, enabling the formation of a haploid oocyte; iii) Large-scale changes in the translational landscape; iv) Cytoskeletal rearrangements for regulating downstream events of egg activation and supporting further growth of the zygote. Preceding these events in Drosophila is a single calcium transient observed in the form of a polar wave upon hydration and swelling of the oocyte. A model outlining how the initiation of such a wave is regulated and how it then enacts these downstream effects is not yet fully outlined. The original working model supported by previous research suggested that mechanical triggers during ovulation initiate calcium entry. How this mechanical stimulus is transduced into a calcium wave and what this then means for the source of calcium has not been explored. I first provide an in depth analysis of calcium entry dynamics at egg activation, exploring the significance of seemingly less regulated calcium events. I then investigate the source of calcium and identify an ion channel that is required for calcium entry. By utilising a combination of pharmacological and genetic analysis, I highlight the requirement of Trpm for calcium entry at egg activation. Taken together I demonstrate that calcium enters the oocyte from the peri- vitelline space (between the oolemma and vitelline membrane) through Trpm in the form of a wave. I next ask what mechanisms regulate calcium entry through Trpm channels. I provide detailed visualisation of the actin population in the mature oocyte both before and after egg activation, focusing on the cortical actin. I reveal a clear relationship between calcium entry and the cortical actin- In particular, reduction of the cortical actin density or level of cross-linking promotes the entry of calcium. In the mature oocyte I show that the Arp2/3 machinery and tandem-actin binding domain nucleators are required for maintenance of the cortical actin. I further demonstrate that actin-binding proteins (ABPs) play a role in regulating calcium entry, likely via mediation of cross-linking and density of the cortical actin. This data therefore supports a model in which polar waves are in part a result of a reduced cortical actin density at the poles of the oocyte. I then explore a specific downstream event of egg activation; the resumption of meiosis. I demonstrate the presence of a novel population of actin within the Drosophila oocyte that forms a spindle-like structure. Given this is such a recent discovery, key questions are highlighted: 1) What is the role of this spindle-like apparatus?; 2) How does the spindle-like actin regulate meiosis?; 3) Is there conservation of this population? I first highlight the requirement of Formins in production of this population. I further reveal that the spindle-like actin is required for regulating the formation and morphology of the spindle and therefore the accurate movement of chromosomes during meiosis, demonstrating remarkable conservation with mammals. Finally, I bring together concepts of calcium and actin signalling explored in the previous chapters, revealing an essential interplay between the two at the metaphase-arrested spindle.

BBSRC DTP