Jovceva, E.; (2007) Functional proteomic and genomic analysis of cytoskeletal organisation in Drosophila. Doctoral thesis , University of London.

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Abstract

Phosphoinositide 3-kinase (PI3K) plays an important role in cellular signalling by generating phospholipid second messengers at the plasma membrane. A large repertoire of signalling and actin-binding proteins, which consistently regulate the dynamic assembly and spatial organisation of actin filaments, binds phospholipid second messengers, through their pleckstrin homology (PH) domains, and regulates changes in actin cytoskeleton dynamics and organisation in response to external stimuli. Thus, the actin cytoskeleton, which functions in the generation and maintenance of cell morphology and polarity, regulation of endocytosis and intracellular trafficking, contractility, motility and cell division, is considered as an integral part of the cell signal transduction system. PI3K-dependent actin cytoskeleton reorganisation has been the subject of intensive studies, as alteration in the cytoskeleton and thus in cell morphology and migration appear to be common signatures of malignancy where PI3K activation is significantly involved. PI3K- dependent regulation of actin cytoskeleton dynamics is proposed to be achieved by cross-talk with the Rho-family small GTPases, major regulators of actin cytoskeleton organisation. However, the molecular mechanisms behind PI3K-dependent actin reorganisation and their interaction with small GTPases in not yet clearly defined. The aim of this project was to investigate the role of the PI3K signalling in controlling actin cytoskeleton, and to explore possible common targets of PI3K and Rho-family small GTPase signalling pathways, as well as to search for new targets downstream of PI3K. Initially, the role of PI3K in the regulation of the actin cytoskeleton in Drosophila cells was defined. Furthermore, a "loss-of-function approach" based on RNA interference for genes involved in PI3K and small GTPase signalling was combined with quantitative differential protein expression analysis and mass spectrometry. The differentially expressed proteins, many of which were cytoskeleton proteins, metabolic and redox enzymes, were linked to signalling pathways and associated with the morphological phenotype of each knockdown. Finally, the research was focused on studying the regulation of phosphorylation of cofilin, an actin depolymerising protein. It has been established that cofilin phosphorylation and activity is not directly regulated by upstream signalling events, but by changes in the levels of filamentous actin itself, with slingshot, the cofilin phosphatase, being a key regulator in sensing the dynamic changes in F-actin levels. Thus, cofilin phosphorylation is a homeostatic sensor of actin polymerisation, which self-limits protrusive response to external stimuli.

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