Browsing by Subject "CAP"

Epithelial cells form a barrier between the tissue and the external environment. Epithelial morphogenesis refers to the shaping of epithelial layers and is a key step in the development of organisms. The actin cytoskeleton provides the cell its form and during epithelial morphogenesis, produces force to shape the cells. To achieve this, the actin cytoskeleton is organized into protrusive and contractile networks. In a living cell, these actin networks are dynamic, as the filaments are constantly undergoing assembly and disassembly. Actin-binding proteins regulate the turnover of actin filaments, but in epithelial morphogenesis, the regulatory role of most of these proteins is still relatively unknown. In all multicellular organisms, actin disassembly is controlled by ADF/cofilin. ADF/cofilin activity is furthermore enhanced by other actin-binding proteins, one of which is cyclase-associated protein (CAP). CAP promotes actin turnover by accelerating ADF/cofilin mediated actin disassembly and in recycling actin monomers to sites of actin polymerization. Unlike ADF/cofilin that regulates actin disassembly throughout the whole cell, CAP could be subject to more specific spatial regulation, as loss of CAP leads to F-actin accumulation on the apical side of epithelial cells. However, the role of CAP in morphogenetic cell rearrangements remains poorly known. In addition, the in vivo role of the biochemical functions of CAP has not been elucidated. The aim of this master’s thesis is to describe the role of CAP in regulating the actin cytoskeleton in the follicular epithelium of the fruit fly Drosophila melanogaster. For this purpose, chimeric mutant flies with homozygous CAP loss of function mutation were generated. Subsequently, the effect of the CAP loss of function was observed in follicle cell populations undergoing morphogenetic changes. In addition, CAP loss of function was rescued with different transgenes producing mutant CAP proteins to identify the protein domains of CAP with in vivo significance. In addition, a Drosophila CAP specific antibody was purified to be used in immunostaining. The ovaries were imaged using confocal microscopy. In this thesis, it is shown that CAP loss of function caused accumulation of filamentous actin in all observed follicular cell populations. Surprisingly, the actin turnover was rescued by all of the used CAP rescue transgenes, but the mutant transgenes exhibited phenotypes resembling the CAP loss of function in other epithelial tissues. Moreover, CAP loss of function caused defects in the follicle cell movement and cell spreading. The loss of function also caused expression changes in other actin-binding proteins. The findings of these thesis support the current knowledge of CAP importance for functional actin turnover in the follicle cells, even though the protein domain necessary for in vivo function could not be deciphered. Moreover, this project provides indication that CAP has an indispensable role in dynamic morphogenetic processes in the epithelium. Together with other actin-binding proteins, CAP could regulate epithelial actin turnover in spatially directed manner, providing force for epithelial cell adhesions or protrusions.