Analysis of the integrin cell junctions in Drosophila melanogaster

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PhD thesis (EN)

2011 (EN)
Κυτταρική ανάλυση των ιντεγκρινικών συνδέσεων στη Drosophila melanogaster
Analysis of the integrin cell junctions in Drosophila melanogaster

Ψαρρά, Ελένη

My thesis is focused on the functional analysis of the molecular mechanism of theintegrin-linked kinase (ILK) during development in Drosophila. We studied: a) thefunctional conservation of ILK in evolution, b) the possible role of specific aminoacid motifs in the subcellular localization and function of ILK and c) the functionalproperties of ILK, when covalently bound to the plasma membrane. Furthermore, wesought new functional roles for ILK during development: a) in other tissues besidesmuscle system and b) in oogenesis.ILK protein sequence shares 60% identity and 75% similarity with themammalian ILK. Based on these data, we tested the possible phylogeneticconservation of ILK function. For this purpose, we generated transgenic lines carryingthe coding sequence of either human ILK (hILK) or mouse ILK (mILK). Themammalian ILK has localizes similarly to the endogenous protein, in the muscle cellsof Drosophila. Both mammalian proteins can substitute for the ILK function inDrosophila. However, human ILK binds to Dparvin with reduced affinity comparedto the fly ILK.In order to investigate the molecular mechanism through which ILK regulatesand acts during development, we tested whether the phosphorylation on the aminoacids S176 and T180 contributes to the regulation of ILK function. It has been shown,in cell culture models, that the phosphorylation on these sites controls the subcellularlocalization of the protein in the nucleus. However, we proved that the possiblepgoshorylation of these highly conserved residues is dispensable for the ILKlocalization at the muscle attachment sites (MAS) as well as for the function of ILK.Another residue which is necessary to localise ILK at the focal adhesion sitesis F436. It is located on the last a helix of the carboxyl-terminal lobe of the kinase-likedomain. The subcellular localization and the ILK function are unaffected by the pointmutation F436A, in contrast to the experimental data on cell culture models. Thepoint mutation F436A affects the ability of ILK to bind to parvin.We examined, whether membrane-bound ILK, through palmytoylation orfarnesylation, is able to substitute the absence of the endogenous ILK, if ii can recruitproteins of the adhesome, independently of integrins. We generated two alternativeforms of membrane-bound ILK, GAP-ILK-GFP and ILK-GFP-HRAS, which bothlocalize successfully at the plasma membrane of the embryonic muscle cells. Also,GAP-ILK-GFP and ILK-GFP-HRAS can substitute for the endogenous ILKthroughout development. Moreover, GAP-ILK-GFP is able to recruit both PINCH andParvin, as well as talin at the MAS, in both wild type and aPS2 mutant embryonicmuscle cells.Furthermore, we studied, in genetic molecular level, the role of ILK in themorphogenesis of the egg chambers, the organization and the homeostasis duringoogenesis in Drosophila. We used two experimental approaches in order to silenceilk: a) we generated genetic mosaics for ilk and b) we used conditionally rescued ilk-/-flies. We observed that ILK is indispensable for the process of oogenesis in the fly.Loss of ILK disrupts the stalk cell formation and the separation of the successivenewly formed egg chambers (twin egg chambers).Also, our experiments revealed that ILK is essential for the organization of theactin stress fibers at the late developmental stages of oogenesis and for thehomeostasis of the actin cytoskeleton along apico-basal axis of the cell. ILK isindispensable for the organization and the maintenance of the baso-lateral celljunctions in the follicle cells, but not for the adherens junctions. Loss of ILK disruptsthe localization of integrins at the tips of the actin stress fibers of the follicle cells atlate developmental stages. Moreover, ILK participates in the regulation of the F-actindynamics by down-regulating Dia and up-regulating profilin. ILK is involved in thecontrol of the contractility of the acto-myosin fibers in the follicle cells at latedevelopmental stages, probably by affecting the subcellular localization of Rho1, andcausing ectopic accumulation of myosin (zipper).Finally, ilk interacts genetically with dpak in the follicular epithelium. ILK affectsdPAK localization in the follicle cells at late developmental stages. Furthermore,dPAK is essential for the localization of both integrins and ILK at the tips of actinstress fibers. Loss of dpak, similarly to ilk, disrupts the organization of actin stressfibers in follicle cells at late developmental stages.

Ινίδια τάσης
Stalk cells
Follicle cell
Stress fibers
Muscle system
Μυϊκό σύστημα
Μισχοειδή κύτταρα

Εθνικό Κέντρο Τεκμηρίωσης (ΕΚΤ) (EL)
National Documentation Centre (EKT) (EN)



Πανεπιστήμιο Πατρών
University of Patras

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