Parsing molecular determinants of asymmetry in planar cell polarity signaling - PROJECT SUMMARY/ABSTRACT Conotruncal heart malformations are the most common congenital heart disease. Genes associated with planar cell polarity(PCP) signaling are implicated in the etiologies of conotruncal heart malformations, as PCP signaling is necessary for the coordinated cell movements needed for proper outflow tract development. The broad objective of this proposal is to determine the basic molecular mechanisms of this key developmental signaling pathway, which are currently poorly understood. Establishment of PCP involves sorting PCP-associated proteins into complexes with distinct compositions that are distributed to mark a vector of polarity used for downstream readouts, such as coordinated cell movement. These complexes physically interact between adjacent cells to facilitate alignment of intracellular polarity vectors across whole tissues, aligning with broader axes, exemplified by the proximal-distal axis in the Drosophila wing. Complexes are linked by trans intercellular homodimers of the PCP protein Flamingo (Fmi), with PCP proteins Frizzled(Fz) recruited to one side of the cell-cell junction and Van Gogh (Vang) recruited to the other. Previous work suggests that Fz interacts laterally with Fmi and changes Fmi homodimer affinity to preferentially bind Fmi:Vang complexes. Additional work suggests a novel, adhesion- independent role for Fmi in modulating Fz binding to its downstream signaling partner Dishevelled (Dsh). Fz- Dsh binding is known to be sensitive in a dose-dependent manner to PI(4,5)P2. Thus, this work investigates how interactions between Fmi and Fz drive two key aspects of early establishment of planar cell polarity. I hypothesize that lateral interactions between Fmi and Fz I. promote interactions favoring the formation of intercellular trans interactions with Fmi:Vang complexes, and II. amplify PCP signaling through an adhesion-independent, PI(4,5)P2-mediated feedback loop that recruits cytosolic signaling partners. Aim 1 will characterize the asymmetric interactions between Fz:Fmi and Fmi:Vang complexes by reconstituting the proteins into a lipid- nanodisc system, using total internal reflection fluorescence (TIRF) microscopy to measure asymmetric binding affinity, and using cryogenic electron microscopy to identify structural determinants of asymmetry. Aim 2 will characterize a potential PI(4,5)P2-mediated feedback loop by characterizing PI(4,5)P2 availability, determining the Fmi-dependence of PI(4,5)P2 levels near PCP complexes, examining whether recruitment of Skittles, a lipid kinase that produces PI(4,5)P2 can rescue Dsh binding in a Fmi null phenotype, and investigating whether Fmi can directly bind Skittles. This project thus sets the stage for the development of a detailed mechanistic understanding of PCP, which is essential for understanding normal heart development as well as the origins of conotruncal heart disorders. The applicant will receive additional training from expert mentors in membrane protein biochemistry and cryogenic-electron microscopy, in single-molecule biophysics, and training in Drosophila genetics, building an excellent foundation for an independent research career working on the molecular mechanisms that underpin human development and physiology.
