A Genome-Wide RNAi Screen Identifies Regulators of Cholesterol-Modified Hedgehog Secretion in Drosophila

Hedgehog (Hh) proteins are secreted molecules that function as organizers in animal development. In addition to being palmitoylated, Hh is the only metazoan protein known to possess a covalently-linked cholesterol moiety. The absence of either modification severely disrupts the organization of numerous tissues during development. It is currently not known how lipid-modified Hh is secreted and released from producing cells. We have performed a genome-wide RNAi screen in Drosophila melanogaster cells to identify regulators of Hh secretion. We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI) but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway. We provide evidence that both proteolysis and cholesterol modification are necessary for the efficient trafficking of Hh through the ER and Golgi. Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg) morphogen secretion in vivo. These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion

Plasmodesmata: Channels for Viruses on the Move

The symplastic communication network established by plasmodesmata (PD) and connected phloem provides an essential pathway for spatiotemporal intercellular signaling in plant development but is also exploited by viruses for moving their genomes between cells in order to infect plants systemically. Virus movement depends on virus-encoded movement proteins (MPs) that target PD and therefore represent important keys to the cellular mechanisms underlying the intercellular trafficking of viruses and other macromolecules. Viruses and their MPs have evolved different mechanisms for intracellular transport and interaction with PD. Some viruses move from cell to cell by interacting with cellular mechanisms that control the size exclusion limit of PD whereas other viruses alter the PD architecture through assembly of specialized transport structures within the channel. Some viruses move between cells in the form of assembled virus particles whereas other viruses may interact with nucleic acid transport mechanisms to move their genomes in a non-encapsidated form. Moreover, whereas several viruses rely on the secretory pathway to target PD, other viruses interact with the cortical endoplasmic reticulum and associated cytoskeleton to spread infection. This chapter provides an introduction into viruses and their role in studying the diverse cellular mechanisms involved in intercellular PD-mediated macromolecular trafficking