The dynamics of clathrin-mediated endocytosis in developing embryos

Clathrin-mediated endocytosis (CME) is a pathway that remodels the plasma membrane in order to internalize vesicles that contain extracellular cargo such as iron-bound transferrin. Studies of CME have benefited from live-cell imaging where copies of endogenous proteins are visualized by fluorescent protein fusions that are introduced via genome-editing to preserve native protein stoichiometries. Markers of CME such as AP2, DNM2, and ARPC3 can combine in complex and heterogeneous ways, complicating the analysis of data. Here, I describe the development and validation of computational tools that organize and classify different kinds of events marked by the presence of one or more proteins participating in CME. These tools rely on dimensionality reduction to take high-dimensional information (i.e., >100k CME events with highly variable motion profiles and abundance of markers) and compress it into human-interpretable results that describe the kinetics of the markers in their respective groups (Chapter 1). These tools were used to study the dynamics of branched actin network assembly, a force-generating mechanism inside cells, and revealed that sites of stalled CME assemble branched actin networks at one side of the budding CME pit to assist in vesicle internalization (Chapter 2)./nChapter 3 describes the generation of a collection of fluorescently-labeled, genome-edited zebrafish in which diverse complexes and proteins have been tagged: AP2 adaptor complex, Arp2/3 complex, clathrin triskelia, caveolae, and beta-catenin. First, I outline the strategy we used to rapidly generate reagents required for genome-editing. Next, I show that these tagged proteins localize in a living organism in both expected and novel ways by imaging embryos with high-resolution Airyscan microscopy. I first highlight the ability to detect caveolar structures in developing skin and notochord. Next, I show that we can detect canonical WNT signaling marked by beta-catenin accumulation in the nuclei of several cell types for the first time in a living embryo. The beta-catenin line also revealed potentially novel structures found in neurons and skin. The Arp2/3 complex showed diverse labeling in the form of pegs and ridges during skin wrinkling, including a potentially overlooked apical circumferential ring; punctate labeling in neurons, circulating blood, vascular endothelium, and the swelling endoskeletal disc in the pectoral fin; dense, unresolvable meshes during cloacae morphogenesis; and dense, uniform-appearing accumulation in crawling immune cells. I also observed co-localization of the Arp2/3 complex and AP2 in the pectoral fin, documenting the first evidence for branched actin network assembly use during CME in cells not grown in the artificial environment of a glass surface./nBy following the localization of microinjected, labeled transferrin, a key cargo in blood development, I made a surprising observation: transferrin accumulation via CME occurs most abundantly in the vascular endothelium, not in the circulating blood. By providing labeled transferrin at the earliest stages of blood development, I found that emerging red blood cells only consume transferrin much earlier than the onset of circulation. Furthermore, by visualizing the presence of endogenous AP2 and clathrin, I found that emerging blood stem cells have upregulated CME that peaks in frequency above the nearly non-existent levels found in the cells that precede them, the dorsal aorta endothelium./nMy zebrafish data demonstrated the utility of studying endogenous markers of cellular pathways that highlight the known use of these proteins as well as producing new insights into their use.

Advisor: Drubin, David
Swinburne, Ian