James H. Keen, PhD
James H. Keen, PhD
Expertise and Research Interests
The long-term goal of my lab has been an understanding of the structure of clathrin-coated membranes and their role in membrane transport processes within mammalian cells. It is well established that plasma membrane coated pits mediate endocytosis, and that clathrin coated buds and tubules in the TGN are involved in targeting and recycling of newly synthesized proteins. But clathrin coats are also present on vacuolar and tubular domains of endosomes where they are involved in sorting to late endosomes, or to retrograde or recycling pathways, respectively, through mechanisms that are only partially understood. As the early/sorting endosome is a nexus for virtually all extracellular materials entering cells as well as many intracellular trafficking cargoes, understanding sorting mechanisms and structures in the endosomal region remains a key challenge with many implications for human health and disease. In this context, we have identified and characterized distinct clathrin coated structures in the endosomal region that exhibit extremely rapid but localized movement. Accordingly we termed these structures 'Gyrating'- or 'G-clathrin', and they appear to be clathrin coated buds along or on the ends of membrane tubules. We have shown that G-clathrin accumulates internalized cargo destined to be recycled to the plasma membrane, including transferrin and c-Met receptor, and that they function in rapid recycling from the endosome which remains a largely uncharacterized pathway. In recent work, we have shown that the dynamics of G-clathrin requires completion of the Arf6 guanine nucleotide cycle but that Arf1 can also support its formation. Similarly, both BFA-sensitive and -resistant guanine nucleotide exchange (GEF) proteins modulate G-clathrin levels, together indicating that overlapping mechanisms are operative in G-clathrin regulation. The complexity of regulation appears to parallel the multiplicity of endosomal sorting pathways.
Current efforts utilize two major approaches. First, we are dissecting the regulation and function of G-clathrin in rapid recycling pathways and identifying the cargos transported, with a focus on the role of GGA proteins and other potential regulators and adaptors. Second, we are interested in better visualizing the structure and function of G-clathrin in intact cells, using correlative light-electron microscopy (CLEM) in collaborative efforts with the lab of Graça Raposo (Institut Curie, Paris) at the ultrastructural level, and interactions and interconversions of G-clathrin with other endocytic and membrane compartments using high-speed live cell imaging.
Our methodological approaches utilize widefield, TIRF and spinning disk confocal fluorescence microscopy and associated photobleaching/photoactivation and image analysis procedures, the development and application of novel "molecular traps" to probe functions in intact cells in collaboration with John Williams (City of Hope, Duarte CA), and conventional cell and molecular biology techniques such as siRNA-mediated protein depletion and exogenous expression, and others.
- A unique role for clathrin light chain A in cell spreading and migration
- Exosome-mediated transfer of αvβ3 integrin from tumorigenic to nontumorigenic cells promotes a migratory phenotype
- Integrins influence the size and dynamics of signaling microclusters in a Pyk2-dependent manner
- Clathrin light chains are required for the gyrating-clathrin recycling pathway and thereby promote cell migration
- Patched-1 proapoptotic activity is downregulated by modification of K1413 by the E3 ubiquitin-protein ligase Itchy homolog