Open Access Dissertation
Date of Award
Mark F. VanBerkum
Guidance receptors such as Frazzled affect cell shape and motility by directly or indirectly modulating the cytoskeleton. Fra is particularly needed for the formation of the posterior commissures in a developing Drosophila embryo. The cytoplasmic tyrosine kinase, Abelson Kinase (Abl) enhances the loss of commissures observed in fra mutant. Abl physically interacts with Frazzled to help guide commissural axons across the midline. Furthermore, the loss of commissural axons is only seen when the actin dynamics are perturbed. Abl is also known to regulate actin-dependent processes underlying formation of filopodia, microspikes and membrane ruffles. So, we established a Drosophila S2 cell culture paradigm to study how Frazzled and Abelson may co-regulate actin-dependent morphology. Ectopic expression of Frazzled in S2 cells produces long filopodia-like extensions in over 70% of the cells when plated on concanavalin A coated cover slips. These phalloidin stained filopodia are reduced when either the P1 or P3 cytoplasmic motif is removed. Frazzled is localized to the tips of these filopodia, and is associated with an increase in phosphotyrosine levels as well as alterations in localization of proteins modulating actin dynamics and also known to interact with Abelson. Loss of Abl by RNA interference causes S2 cells to form peripheral microspikes as well as a decrease in phosphotyrosine staining at the edge of the cell. When Abl is knocked down in Fra expressing cells, they are unable to extend filopodia but microspikes persist. Moreover, overexpression studies reveal that while Abl deltaF may be still functional but the kinase activity of Abl is particularly critical for Fra dependent filopodia. Together, these data indicate that Fra may be regulating Abl to control the actin dynamics underlying the formation of the commissures.
Varughese, Bridget Elsa, "Frazzled And Abelson Interact To Regulate The Actin Cytoskeleton In Drosophila" (2011). Wayne State University Dissertations. 223.