Δομική και λειτουργική ανάλυση του γονιδίου της parvin στη Drosophila

Abstract

The focus of my PhD thesis is the investigation of the molecular mechanism of parvin function. Parvin is a structural component of the integrin adhesome and genetic evidence has shown its essential role in the formation and maintenance of the integrin-actin link in the developing organism. Parvin has been shown to be involved in actin dynamics and processes like cell adhesion, migration and survival. Parvin is a Calponin Homology (CH)-domain protein and previous work has shown its ability to interact with F-actin. We engineered transgenic flies carrying truncated forms of parvin and expressed them in the mesoderm mainly using the GAL4/UAS overexpression system in Drosophila. We examined the localization of the truncated forms of parvin in Drosophila embryonic and larval muscles and we also tested their ability to rescue the loss of endogenous parvin. The results confirmed that CH2 domain is necessary for parvin localization at the muscle ends. This domain analysis also revealed that a second localization signal resides in the CH1 domain. This signal is blocked under normal conditions probably through an autoinhibitory mechanism. Finally it was shown that there is a genetic interaction between parvin and ILK through the CH1 domain of parvin and not only the CH2 as it was known so far. In order to investigate further the mechanism of parvin function we also followed another approach where the protein was overexpressed in several tissues. The resulted phenotypes were analyzed with immunofluorescence techniques. It was shown that high levels of parvin were toxic in mesodermal and epithelial tissues and resulted in lethality. Parvin toxicity was mediated through its CH2 domain and it could be rescued mainly when parvin was coexpressed with ILK or/and proteins like DIAP1 that are involved in survival pathways. The dominant negative phenotype of parvin overexpression was examined extensively in the wing epithelium. It was demonstrated that high levels of parvin in the wing imaginal disc led to high levels of apoptosis and activation of the JNK pathway. Parvin overexpression also caused actin misorganization at the basal side of the wing epithelium and deregulated intergrin adhesion. Finally in order to gain more insight into possible novel roles of parvin we conducted a modifier genetic screen in the Drosophila compound eye based on the fact that parvin overexpression in the eye caused a “rough eye” phenotype. On the other hand with pull down assays we tried to identify proteins that participate along with parvin in the same protein complex. The biochemical and genetic association of parvin with new proteins can reveal novel regulatory roles of parvin and its implication in pathways, that are not yet reported.