PROJECT SUMMARY Cell polarity is the organization of the plasma membrane into discrete domains. In animal cells, polarity is particularly important because of their highly ordered, multicellular organization. In order to meet the demands that organized multicellularity places on polarity, animals have evolved specialized machinery, known as the Par complex, that controls polarization in a broad range of cell types. During development, stem cells must polarize before they divide in order to generate cellular diversity. It is the job of the Par complex to direct polarization in stem cells during mitosis. The Par complex consists of the adapter protein Par-6 and atypical Protein Kinase C (aPKC). It is the catalytic activity of aPKC which is responsible for the polarization of downstream components. In order to perform its catalytic duties at the start of mitosis, aPKC must get recruited to the cell membrane, where it polarizes membrane found fate determinants. This proposal is directed at elucidating how aPKC's interactions with lipids contribute to its proper localization and regulation of its catalytic activity. Within aPKC we have identified a lipid binding domain called the C1 domain. We hypothesize that regulation of the aPKC C1 domain governs the membrane recruitment of aPKC. We propose to test this hypothesis by characterizing the regulation of aPKC's C1 domain both at the molecular and cellular levels. Specific Aims: (1) Determine the intramolecular mechanism by which the C1 domain is autoinhibited. (2) Determine how the C1 domain is activated. Research design: To characterize the autoinhibition mechanism of aPKC's C1 domain, we will use mutagenesis strategies to dissect the intramolecular interactions that repress the C1 domain. C1 domain activity will be measured through aPKC's affinity for liposomes in vitro. Kinase activity assays will allow us to uncover how lipid binding affects the catalytic activity of aPKC. We will use Drosophila neuroblasts (neural stem cells) to track the localization of fluorescently-tagged aPKC in vivo. We will image aPKC at high temporal (every 20 seconds) and spatial resolution using live-cell confocal microscopy. From this data, we will compose 3- dimensional reconstructions to visualize the in vivo localization dynamics of aPKC. In addition to evaluating C1 repression, we will explore the modes of C1 activation. We will analyze how aPKC binding partners contribute to the activation of aPKC's C1 domain using liposome binding assays. Additionally, we will explore whether the C1 domain preferentially binds specific lipids by testing aPKC's affinity for liposomes with different lipid compositions. Finally, we will track the localization of phosphoinositide lipids in polarizing neuroblasts in order to address if aPKC localizes to sites of phosphoinositide enrichment in vivo. Learning how the regulation of the C1 domain controls aPKC's localization will provide the required context for addressing larger questions ...