Modulation of Cell Motility by EGF-like Repeats in Dictyostelium discoideum

Dictyostelium discoideum is a social amoebozoan that is used a model system for studying a variety of cell and developmental processes, especially cell motility and chemotaxis. Genome analyses suggest that this model organism possesses a higher percentage of Epidermal Growth Factor (EGF)-like (EGFL) repeats than any other sequenced eukaryote, including humans. EGFL repeats share strong sequence similarity with EGF. In mammals, EGF binds to an EGF receptor (EGFR) to initiate intracellular signalling that regulates a diversity of cellular processes including cell motility and chemotaxis. Some EGFL repeats, like EGF, have also been shown to increase the rate of cell motility by binding to the EGFR and activating EGFR-dependent signalling. Despite their abundance in Dictyostelium, a function for EGFL repeats in this model eukaryote had not previously been studied. This thesis presents a collection of studies that investigated the function of a specific EGFL repeat from the extracellular, cysteine-rich, calmodulin (CaM)-binding protein CyrA. A synthetic peptide (DdEGFL1), equivalent in sequence to the first 18 amino acids of the first EGFL repeat (EGFL1) of CyrA, was shown to increase random cell motility and cAMP-mediated chemotaxis via a novel signalling pathway that did not require either of the two cAMP receptors that are active during early development of Dictyostelium. Several intracellular signalling components were identified and then incorporated into a model detailing the signal transduction regulating EGFL repeat-enhanced cell movement in Dictyostelium. Finally the expression, secretion, and localization of CyrA are presented to couple the findings from studies on DdEGFL1 function with those for the full-length protein. In mammals, a protein that localizes to the extracellular matrix (ECM) and modulates cellular processes by binding to a cell surface receptor and initiating intracellular signalling is termed a ‘matricellular’ protein. The research presented in this thesis suggests that CyrA is the first matricellular protein identified in Dictyostelium.Ph