Browsing by Subject "Actin"

Growth differentiation factor 15 (GDF15), a member of TGF-β super family is a soluble cytokine that is associated with different pathological conditions including cancer, cardiac and renal failure and obesity. Its high serum levels are linked with symptoms like cachexia/anorexia in cancer patients and can be used as a marker for these diseases. Its crucial role in weight regulation and energy homeostasis has been demonstrated by treating obese mice with GDF15, which results in weight lose along with improved glucose metabolism and increased insulin tolerance. It is now known that GDF15 exerts its metabolic effect by binding to a GDNF receptor -α-Like (GFRAL) receptor along with co-receptor RET. Interestingly, these two receptors co-localize only in the brain stem area of mice and humans indicating involvement of a neuronal circuit in GDF15 mediated effects. Despite its implications in major health disorders, little is known about the interaction of GDF15 with its receptors and how this interaction in turn modulates different cellular signalling and functions. The aim of the thesis was to study the mechanism and factors involved in endocytosis of GDF15. I employed high content imaging and flow cytometry techniques to visualize and analyse the internalization of ligand-receptor complex and investigate the role of actin, dynamin and phosphoinositide 3 kinase in the process. The results suggest that similar to the internalization of other cellular growth factors, the uptake of GDF15 is affected by disruption of the actin cytoskeleton. The role of dynamin is still unclear. I also discovered that the internalization of GDF15 was inefficient even in cells that expressed the receptor GFRAL, with large cell-to-cell variation. By following the intracellular localization of the receptor GFRAL, my results revealed that the receptor GFRAL is not efficiently exported to the plasma membrane and most of the protein is retained in the Golgi compartment of cells. This phenomenon was stronger in murine fibroblast cells, where the receptor was almost exclusively trapped in the secretory compartment, explaining why the uptake of the ligand GDF15 is so inefficient in these cells. The system developed during this project will now be used to analyse different factors involved in the uptake of GDF15 and eventually uncover the possible endocytic pathway. Moreover, the Golgi retention of the receptor opens up new questions to investigate like whether the physiological function of GDF15 is regulated by receptor export signals. This will help deciphering the complex and mysterious interaction of GDF15 with its receptor GFRAL.

Endocytosis is the process responsible for internalising membrane components and as such plays a key role in the biology of this structure. Mammalian cells have evolved various endocytic strategies, but Clathrin-Mediated Endocytosis (CME) is the most common type. Since the discovery of CME, around 50 years ago, the field has built a remarkable wealth of knowledge on the core CME components. In stark contrast, our understanding on the relationship between CME and the actin cytoskeleton, which is present throughout the process, is still in its infancy. In this thesis, I show the production and characterisation of recombinant, SpyCatcher tagged transferrin (TF), a canonical CME ligand. TF was expressed in E. coli and using an optimised protocol, successfully solubilised and refolded from inclusion bodies. The protein was then labelled with a fluorophore and purified to a high level of purity. Tests in mammalian cells showed that home-made TF has the same endocytic behaviour as TF purified from human plasma. Moreover, I could show that the SpyCatcher moiety attached to our home-made TF is capable to mediate its covalent linkage to its counterpart SpyTag. The successful production, refolding and functional characterization of recombinant TF in this study is an important first step to examine the participation of the actin cytoskeleton during CME.

Epithelial cells line the surfaces of organs and tissues in a continuous and tightly packed manner, thereby functioning as a protective barrier between the tissue and the external environment known as the epithelium. During development, the epithelium undergoes a series of morphogenetic events which alters the shape and size of epithelial cells, enabling them to perform tissue specific functions in mature tissue. During morphogenesis, cells sense the mechanical forces and establish polarity through cell proliferation and rearrangement according to morphogenetic signalling pathways. This manoeuvre is achieved by the underlying actin cytoskeleton network which enables cells to resist the tension and stresses of morphogenesis via alteration of filament dynamics and network architecture. In vivo, numerous actin-regulatory proteins generate various polymerized forms of straight, branched, or contractile actin-myosin filaments, regulating dynamic actin filament turnover. The robust actin cytoskeleton provides the cell with protrusive and contractile forces that enable cells to migrate, maintain, and change its shape and form during morphogenetic events. Actin filament depolymerization is accomplished by ADF/cofilin (Drosophila homolog twinstar) binding to actin monomers (G-actin) and actin filaments. However, ADF/cofilin alone is not very efficient in promoting disassembly of actin monomers, especially in subcellular regions where ADF/cofilin is highly concentrated. AIP1 (Drosophila homolog flare) then enhances actin depolymerization via preferential binding to ADF/Cofilin rich regions in vitro. The aim of my thesis was to study the localization and roles of AIP1 and cofilin in follicular epithelium during Drosophila oogenesis. My results showed that Actin-Interacting-Protein-1 (AIP1) was expressed throughout oogenesis. AIP1 expression was increased in cell type-specific manner and AIP1 showed spatiotemporal localization in follicular epithelium during oogenesis. Silencing of AIP1 led to accumulation of ectopic F-actin aggregates, localization of which may reflect the cellular sites of dynamic actin reorganization in the follicular epithelium. My results also indicate that AIP1 may be indirectly responsible for maintaining epithelial integrity as its silencing resulted in formation of epithelial gaps throughout follicular epithelium. Also delays in border cell migration were observed. Considering the above, understanding how AIP1 functions in Drosophila morphogenetic events would therefore pave the way for a greater understanding of how this protein works in other organisms. The knowledge gained may also be used to extend the current understanding of the role of actin binding proteins in diseased states.