The precise structure of the adult Drosophila eye results from a coordinated process of cell sorting, differentiation and selective cell death in the retinal epithelium. Mutations in the gene echinus cause supernumerary pigment cells due to insufficient cell death. This study reports the identification of echinus and the characterization of its role in Drosophila retinal development. Using a combination of deletion mapping, gene expression analysis and genomic sequencing, echinus was cloned and several alleles were sequenced. echinus encodes a ~180kDa protein containing an ubiquitin hydrolase domain at its N-terminus and a polyglutamine tract at its C-terminus. echinus is expressed in the retina during pupal development and mutants of echinus have decreased levels of apoptosis during several stages of retinal development. Defects in the cell sorting process that precedes cell death are also observed in echinus loss-of-function mutants and echinus overexpression can cause defects in ommatidial rotation and the morphology of cone cells. echinus is a positive regulator of DE-cadherin and Enabled accumulation in adherens junctions of retinal epithelial cells. Genetic interactions were observed between echinus and the genes wingless, enabled and expanded. An immunofluorescence assay in Drosophila S2 cell cultured demonstrated that Echinus localizes to intracellular vesicles that do not appear to be endocytic in nature, and the C-terminal region of Echinus was shown to be necessary for this association. A protein interaction screen using an immunoprecipitation and mass spectrometry approach identified interactions between Echinus and the vesicle coat protein Clathrin, the scaffolding protein RACK1 and the casein kinase I epsilon (Dco). Co-immunoprecipitation additionally identified an interaction between Echinus and Enabled. This work has revealed echinus to be an important regulator of cell sorting and adherens junction formation in the developing retina and has identified multiple interactions between echinus and enabled, a regulator of the actin cytoskeleton.

The seven-pass transmembrane atypical cadherin, Flamingo (also known as Starry Night) is evolutionally conserved in both structure and function in vertebrates and invertebrates. It plays important roles during the establishment of planar cell polarity (PCP) of epithelial tissues and during the development of axons and dendrites in both peripheral and central neurons. / This thesis looks at the role of Flamingo/Starry Night in axon growth and guidance in the embryonic abdominal peripheral nervous system (PNS) of Drosophila. It describes the expression pattern of Flamingo in the PNS and its environment. A combination of single cell labelling and immunohistochemical techniques was used to define the effect of mutations in flamingo as well as several genes coding for potential Flamingo interaction partners. Rescue- and over-/mis-expression experiments featuring targeted expression of either a wild type version or mutant versions of flamingo provide information on the cellular and molecular mechanisms by which Flamingo regulates sensory axon development. Loss of Flamingo function results in a highly penetrant axon stall phenotype. Both sensory and motor axons frequently halt their advance early along their normal trajectories. Flamingo appears to mediate an axon growth promoting signal upon contact of sensory growth cones with specific early intermediate targets. Expression of Flamingo in sensory neurons is sufficient to rescue the mutant sensory axon phenotype. This rescue is at least partially independent of most of the extracellular region of the Flamingo protein. While Flamingo was previously found to have homophilic adhesion properties in vitro and appears to function by a homophilic mechanism during the neurite development of several types of neurons, this study supports a heterophilic signalling mechanism by which Flamingo fulfils its role in abdominal sensory axon growth promotion.

Cadherin-based cell-cell contacts are prominent sites for phosphotyrosine signalling, being enriched in tyrosine-phosphorylated proteins, tyrosine kinases and phosphatases. The functional interplay between cadherin adhesion and tyrosine kinase signalling, however, is complex and incompletely understood. In my thesis I have tested the hypothesis that c-Src contributes positively to cadherin biology by functioning as part of an adhesion activated cell-signalling pathway. I found that c-Src is active at both established and reforming cell-cell contacts, and c-Src can be activated by homophilic ligation of the adhesion receptor. However, c-Src has a biphasic impact on cadherin function, exerting a positive supportive role at lower signal strengths, but inhibiting function at high signal strengths. Inhibiting c-Src under circumstances when it is activated by cadherin adhesion decreased several measures of cadherin function. This suggests that the cadherin-activated c-Src signalling pathway serves positively to support cadherin function, while quantitative changes in signal strength may result in qualitative differences in functional outcome. Finally, my data implicated PI3-kinase signalling and cortactin as potential targets for cadherin-activated c-Src signalling. By inhibiting protein tyrosine phosphatases with pervanadate, I found that tyrosine phosphatase activity and not just protein binding was required to stimulate Src activity in response to cadherin ligation. I identified the tyrosine phosphatase RPTPα as a possible regulator of cadherin-activated Src signalling. RPTPα localises to cell-cell adhesions and it is found in a complex with E-cadherin and c-Src. Furthermore, knockdown of RPTPα disrupted the integrity of cadherin-based contacts and the activity of Src at these cell-cell contacts. This suggests that in response to cadherin-homophilic ligation PTP activity is required to stimulate Src signalling. Finally, I identified a novel pathway by which aberrant growth factor signalling could be downregulating cadherin function and promoting the invasion of epithelial cells. Stimulating cells with high levels of EGF revealed that aberrant epidermal growth factor signalling could disrupt cadherin-activated cell signalling. The integrity of cadherin-based contacts and the activity of Src at the cell-cell contacts were both disrupted in the presence of high levels of EGF. Analysis of E-cadherin and RPTPα immunoprecipitates suggested that activation of cadherin-bound EGFR might disrupt Src activation by displacing E-cadherin-RPTPα binding. Finally, analysing the subcellular distribution of these proteins revealed that, in response to high levels of EGF, E-cadherin, β-catenin, EGFR and pEGFR are internalised together in phospho-cortactin-rich endosomal-like structures. Therefore I propose that E-cadherin adhesion activates a cell-signalling pathway involving c-Src that functions to dynamically regulate the actin cytoskeleton and to maintain the adhesive strength of cell-cell adhesions. Perturbation of cadherin-activated Src signalling downregulates cadherin function and promotes the disassembly of cell-cell adhesive contacts. The concept of a cadherin-activated Src signalling pathway provides a new way to think about cadherin biology. Instead of merely functioning as passive glue holding two cells together, E-cadherin functions as an adhesion-activated signalling receptor. Dysregulation of E-cadherin-activated Src signalling and downregulation of cell-cell adhesions could be a mechanism promoting the invasion and metastasis of epithelial tumours.

Thesis (Ph.D.)--McMaster University, 2005. / Supervisor: John L. Brash. Includes bibliographical references. Also available online.

Tez (Tıpta Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, Nöroşirurji Anabilim Dalı, 2006. / Bibliyografya var.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 3 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.