Analysis of developmental regulation and disease modeling of cargo trafficking in zebrafish

Abstract

Cargo trafficking is the process by which proteins and lipid carriers are transported between intracellular compartments. Mutations in components of cargo trafficking machinery have been associated with a diverse collection of diseases that affect select tissues, but the mechanisms leading to cell-type specific deficits are largely unknown. Here, I use forward and reverse genetics approaches in zebrafish to uncover the developmental and physiological functions of several cargo trafficking components.

First, using a positional cloning strategy, I discovered an essential function of erc1 in regulating chondrocyte cell shape changes and cell survival during early cartilage development. I show that in chondrocytes Erc1 localizes to the cell cortex and regulates microtubule polarity and post-Golgi cargo trafficking. By genetic mosaic analysis and live cell imaging I established that Erc1 functions cell autonomously to maintain cell shape during chondrocyte growth. Furthermore, Rab8a and Kinesin-1 act in the same cargo trafficking pathway as Erc1 to regulate chondrocyte cell shape. My data indicate that Erc1-dependent regulation of microtubule polarity spatially directs extracellular matrix cargo delivery at the plasma membrane to shape cell growth.

In a second project, I used a gene knockdown strategy to study the function of sar1b during dietary lipid absorption. Using a pulse-chase dietary lipid feeding assay, I show that Sar1b is required for clearance of lipid droplets from enterocytes, and I discover an unexpected function of Sar1b in dietary cholesterol uptake from the intestinal lumen. Genetic replacement experiments with sar1b and its close paralog sar1a show that while sar1 paralogs have redundant functions in growth and morphogenesis, sar1b is specifically required for dietary lipid absorption. My study offers the first animal model of Anderson disease, a lipid malabsorption syndrome caused by mutations in SAR1B.

Finally, in a collaborative project, I helped uncover structure-function relationships of the Sec23 paralogs using genetic replacement experiments in sec23a-/- chondrocytes. We show that an 18 amino acid divergent peptide sequence in Sec23a confers cargo specificity for collagen trafficking. Our data provide the first experimental evidence that Sec23a influences COPII cargo selection.