Defining interaction interfaces important for the establishment of cell polarity in Drosophila neuroblasts (948.8)

Asymmetric cell division leads to the differentiation of daughter cells by aligning the mitotic spindle to polarized fate determinant cortical domains during mitosis. In Drosophila neuroblasts, cell polarity is established through the Par complex protein Bazooka (Baz aka Par‐3) binding to Inscuteable (Insc), while proper mitotic spindle orientation is established through Partner of Inscuteable (Pins) and the G protein, Gai. Loss of any component leads to loss of polarity and subsequent lethality in the developing fly. Although these interactions are fundamentally important, the physical nature of Insc’s interactions with its binding partners is poorly understood. To explore this idea further, we asked: What are the interaction interfaces between Insc and its binding partners? We identified and characterized the minimal interaction interface between Insc, Baz, Pins, and Gαi by MBP affinity chromatography using different constructs of Insc and assaying for binding of the partners. Our study of Insc has revealed that Pins/Insc binding is conserved with their mammalian orthologs using a motif within the ankyrin repeat 1 ‐ 2 region. Baz/Insc binding requires folded Insc ankyrin repeats 2 ‐ 3 plus one adjacent ankyrin repeat. Gαi binds to Insc in a GoLoco‐like manner in the ankyrin 3 ‐ 4 linker. We find that Insc competes with GoLocos for Gai binding. We also find Insc/Gai binding to be nucleotide specific. Using this data we will generate point mutants, which disrupt the individual pathways and look for phenotypes in the developing fly. Grant Funding Source : Supported by NSF REU Site Program in Molecular Biosciences ‐ BIO/DBI 1063144