Cabernard, ClemensDelgado, Melissa2023-08-142023-08-142023Delgado_washington_0250E_25303.pdfhttp://hdl.handle.net/1773/50244Thesis (Ph.D.)--University of Washington, 2023Asymmetric cell division (ACD) is a highly conserved developmental process utilized to create novel cell populations. ACD can be molecular, physical or a combination of the two. In molecular asymmetry, sibling cells inherit distinct sets of RNA or proteins while in physical asymmetry the sibling cells differ in their size. Although much is known about molecular asymmetry in regard to mechanisms and functions, physical asymmetry remains poorly understood. Thus, I sought to gain a better understanding of the establishment and role of physical asymmetry in Drosophila neuroblasts. Utilizing biochemical and fly genetic approaches to molecularly characterize the mechanism by which Protein Kinase N (Pkn) induces an apical to basal Myosin flow, over 50 potential binding partners of Pkn were identified. Initial characterization of a few selected top candidates including Lethal Giant Larvae, Shaggy, and Twins showed a similar phenotype to pkn mutants. Thus, these candidates warrant additional analysis in order to determine whether they directly interact with Pkn.To assess the functional consequences of altered physical asymmetry as well as molecular asymmetry, a nanobody approach was employed. Altering physical ACD by trapping Myosin to the apical cortex of neuroblasts resulted in symmetric as well as inverted asymmetric sibling cells. The changes to sibling cell size were found to alter cell fate via changes in cell cycle timing as well as the number and nuclear size of cells postulated to have a neuroblast fate. Thus, cell size alone can alter cell fate. Based on these results, I wondered whether inverting molecular asymmetry could also result in altered cell fate. Thus, I chose to trap the scaffolding protein Miranda, which shuttles the basal determinants Prospero and Brat to the basal cortex, to the apical cortex of neuroblasts utilizing a nanobody approach. In addition to Miranda’s mislocalization, a portion of Prospero was found to be trapped to the apical cortex. Alterations to molecular asymmetry resulted in changes in cell cycle timing as well as the number and nuclear size of cells postulated to have a neuroblast fate. Although Miranda and a subset of Prospero was trapped apically, I found polarity to be maintained. Taken together, this research has demonstrated alterations in physical as well as molecular asymmetry can affect cell fate within Drosophila neuroblasts. Thus, I have begun to elucidate the mechanisms and functions behind physical asymmetry.application/pdfen-USnoneAsymmetric cell divisionMolecular asymmetryNeuroblastsPhysical asymmetryProtein Kinase NBiologyDevelopmental biologyCellular biologyBiologyThesis