Molecular mechanisms connecting endocytic and junctional complexes with epithelial pathology in C. elegans - The goal of my research program is to delineate the inner workings of membrane-associated protein complexes that are required to shape the form and function of tissues, and whose dysfunction leads to serious human diseases. We use a multi-disciplinary approach that combines deep genetic screens, cutting-edge transgenesis and live imaging in C. elegans, with biochemical assays of the homologous vertebrate complexes. We also collaborate with experts in structural biology and proteomics to reveal mechanistic details and binding partners of our purified complexes that we then further evaluate in vivo. Using this iterative approach, we have discovered two classes of proteins that act in opposition to regulate the predominant portal of entry into eukaryotic cells, clathrin-mediated endocytosis. The key player in this process, the clathrin adaptor complex AP2, is necessary for linking clathrin to the cell membrane to drive endocytosis. We discovered that NECAP family proteins recognize activated AP2 clathrin adaptor complexes that have been phosphorylated and return them to the inactive state, while endocytic initiators called muniscins/FCHo stimulate inactive AP2 complexes to adopt an intermediate conformation we have named ‘primed’. Together, these allosteric modulators of AP2 will enable us to investigate how cells select endocytic sites and cargo for internalization, while ensuring non-productive or inappropriate events are aborted. Recently, our studies to understand the physiological consequences of inactivating AP2-dependent endocytosis at the organismal level have opened new lines of research. In C. elegans, AP2 mutants exhibit a distinctive ‘cyst-like’ epidermal pathology. Through genetic screens, we have recently discovered mutant forms of the Inversin complex that mimic this phenotype. In humans, mutations in the Inversin complex cause lethal kidney cysts through unknown mechanisms, potentially linking what we see in our C. elegans model to the human disease. We have now isolated other mutations in the Inversin complex that reverse the cyst-like phenotype and will leverage these initial findings to determine the organization and output of this enigmatic complex. We find that ‘cyst’ formation also depends on the worm homolog of ASPP family proteins. Although poorly understood, mutations in ASPPs are clearly associated with heart, hair, and skin pathologies in mammals. Our biochemical and functional assays suggest ASPPs form membrane-associated arrays of protein phosphatase 1 (PP1). We will determine the molecular arrangement and activity state of this novel mechanism of phosphatase regulation using single-molecule imaging and optogenetic techniques. Both the ASPP and Inversin complexes localize to apical epidermal junctions; disrupting these complexes perturbs patterning of the apical extracellular matrix and underlying cytoskeleton. How these poorly characterized junctional complexes and membrane trafficking coordinate collagen-based and actomyosin networks remains an open question that we will address in the next five years by combining our established approaches with new ones, such as electron microscopy.
