Heidelberg, Univ., Diss., 2006.

Konstanz, Univ., Diss., 2006.

Egg activation is a series of highly coordinated processes that prepare the mature oocyte for embryogenesis. Typically associated with fertilisation, egg activation results in the resumption of the cell cycle, expression of maternal mRNAs and cross-linking of the vitelline membrane. While some aspects of egg activation, such as initiation factors in mammals and environmental cues in sea animals, have been well-documented, the mechanics of egg activation in many animals are still not well understood. This is especially true for animals where fertilisation and egg activation are unlinked. In order to elucidate how egg activation is regulated independently of fertilisation, I use Drosophila melanogaster as a model system. This insect provides extensive genetic tools, ease of manipulation for experimentation and is amenable for imaging. Through visualisation of calcium, Processing bodies and meiotic spindles, I show that osmotic pressure acts as an initiation cue for the calcium wave and downstream processes, including the resumption of cell cycle and the dispersion of the translational repression sites. I further show that aquaporin channels, together with external sodium ions, play a role in coordinating swelling of the oocyte in response to the osmotic pressure. I proceed to identify the requirement of internal calcium sources together with a dynamic actin cytoskeleton for a calcium wave to occur. Through co-visualisation of calcium and actin, I provide the first evidence that the calcium wave is followed by a wavefront of non-cortical F-actin at egg activation, which requires the calcium wave. Genetic analysis supports a model where changes in osmotic pressure trigger the calcium wave via stretch sensitive calcium channels in the oocyte membrane and the calcium wave is relayed by nearby channels via the actin cytoskeleton. My work concludes that the mechanism of egg activation in Drosophila is more similar to plants, compared to most vertebrates.

Freiburg i. Br., Univ., Diss., 2007.

Yersinia pestis is a human pathogen and the causative agent of bubonic plague, responsible for causing three massive pandemics, resulting in hundreds of millions of deaths in the 14th century alone. Yersinia’s virulence stems from its ability to overcome host immune defences by the injection of six Yersinia outer proteins (Yops) into the host cells via its Type III secretion system. One of these Yops, YopO specifically disables the actin polymerization machinery, leading to the crippling of phagocytosis. YopO consists of a GDI domain which sequesters Rac and Rho, and a kinase domain, the activity of which is dependent on host actin. Little is known about the targets of the kinase domain and the mechanism of function of YopO remains incomplete. In this work, YopO was crystallized in complex with actin, revealing that YopO binds to actin on subdomain 4, away from the 'hotspot’ between subdomains 1 and 3 which is involved in binding most actin-binding proteins. The structure reveals how recruitment of YopO-bound actin monomers stalls actin polymerization by steric hindrance. The structure also demonstrates how YopO uses actin for self-activation and suggests that actin is being used by YopO as bait for recruitment into actin machineries. Using SILAC mass spectrometry, actin cytoskeletal machineries within macrophages that recruit YopO are identified and these include, amongst others: VASP family proteins, gelsolin family proteins, formins and WASP. Of these, VASP, EVL, diaphanous1, WASP and gelsolin have been identified to be phosphorylated by YopO and were validated by in vitro phosphorylation. This work demonstrates that YopO uses actin as a scaffold for selection of kinase substrates, enabling targeted phosphorylation of the actin machinery and provides insight into the regulation of the actin cytoskeleton by phosphorylation under non-pathogenic conditions.