Membrane-mediated cell biological communication mechanisms in reproduction

Phospholipid bilayers play a critical role in many fundamental aspects of cell biology. They provide the barrier that separates cells and the scaffold upon which many of the molecules that allow cell-cell communication are located. Furthermore, they delimit the boundaries of intracellular organelles, including those responsible for secretory processes, which communicate with the plasma membrane. My thesis focuses on two aspects of membrane-mediated cell-cell communication: cell-cell fusion and the establishment of dense-core granule (DCG) compartments, which package cargos directed for regulated secretion, for example in pancreatic β-cells.
In my first results chapter, I consider an important stage in the life cycle of the parasite Trypanosoma brucei (Euglenozoa: Kinetoplastea), the causative agent of African trypanosomiasis, which is transmitted by tsetse flies. Infectious forms of the parasite are found in the salivary gland of the fly, where genetic exchange between parasites has been shown to occur. While plasma membrane fusion between mating competent cells is likely to be the keystone process leading to this exchange, its cellular mechanisms have remained elusive. Bioinformatic analyses had shown that HAP2(GCS1), a gamete-specific factor related to sex cell fusion originally identified in plants, is conserved in trypanosomes. Here I further study the structure of HAP2(GCS1) in T. brucei and other organisms and identify new domains in this molecule. Furthermore, I present an analysis of ectopic and endogenous expression of TbHAP2(GCS1), which suggests that high levels of expression may be detrimental to the parasite. Overexpression can alter the nuclear content of parasitic forms, consistent with a role in fusion, while YFP tagging may inactivate the normal function of the protein. However, I was not able to set up an in vitro system to track this fusion process, making it difficult to take this work forward.
In the other two results chapters, I analyse the process of dense-core granule compartment biogenesis using a new in vivo genetic model to study this process, the Drosophila prostate-like secondary cell. In addition to the powerful genetics available in this system, including the ability to knockdown genes of interest specifically in these cells in adults, secondary cells have very large DCG compartments. In collaboration with another graduate student in the group, Benjamin Kroeger, I show for the first time in any in vivo system, that the formation of individual compartments can be visualised in real-time. I characterise a series of new live markers to follow this process and define the secretory trafficking pathways in these cells. I then test the function of some of the known conserved genes reported to be involved in DCG biogenesis by adult secondary cell type-specific knockdown. Using this approach, I not only show that the role of these genes is conserved, but also dissect out more specific roles for the AP-1 adaptor complex and the small G-protein Arf1, both of which are involved in important trafficking events. Furthermore, I test the function of new candidate regulatory genes and report a new association between the formation of intraluminal vesicles inside DCG compartments and core formation.
In summary, the second part of my thesis work reveals that the secondary cell has considerable potential to genetically dissect the processes of DCG biogenesis and secretion. In the future, it should be possible to use this system to find novel genes with key roles in these events, which can then be tested for similar functions in mouse models of human disease such as diabetes and neurodegenerative disorders.

Advisor: Wilson, C