A major challenge in developmental biology is to understand how quantitative information is properly relayed between cells or tissues. Transforming growth factor- beta superfamily members (TGF-betas) are critical, evolutionarily conserved messengers for cellular communication. We are using the genetic tools available in Caenorhabditis elegans to investigate several outstanding questions in intercellular TGF-beta gradient formation. The long-term objective of this study is to understand the molecular and cellular basis for the trafficking of TGF-2 molecules between cells using C. elegans. Our central hypothesis is that transport of TGF- beta between cells is mediated by a network of interactions among TGF-betas, specific extracellular proteins including proteoglycans, endocytosis machinery in the receiving cells, and other signaling pathways. To test this hypothesis, we will answer the following questions: 1) How does glypican protein sequester TGF-beta ligand? Glypicans are proteoglycans that are conserved extracellular TGF-beta regulators. Alteration of the glypican protein core is associated with developmental defects and cancers, and we have shown that glypican core protein restricts TGF-beta activity. We hypothesize that a specific region within the glypican core protein sequesters TGF-2 at a specific site to prevent TGF-2 from activating cell surface receptors. We will first identify the site(s) within the glypican protein that affect TGF-beta activity by mutating regions of the glypican and asking if the altered glypican retains function in our in vivo bioassays. We will then determine if an altered site that is required for glypican activity is sufficient by itself to inhibit TGF-beta activity. In addition, we will confirm that glypican mutant function is correlated with its ability to bind TGF-beta. Last, we will identify the region of TGF-beta that interacts with the glypican core protein. 2) How is TGF-beta signaling affected by other extracellular TGF-beta regulators? We hypothesize that other proteoglycans, proteoglycan-modifying enzymes, proteases, extracellular matrix constituents, directed endocytosis, and other signaling pathways define TGF-2 longevity and signaling range. We will characterize the roles of novel regulators we have identified in a sensitized screen, including extracellular proteins, endocytosis machinery, and another cell signaling pathway receptor. We will also 3) Identify new regulators of TGF-2 localization using a sensitized RNAi screen. Our studies to answer these questions employ a combination of transgenic and biochemical methods. The approach is innovative because we can directly observe effects of regulators on the localization of a fluorescently tagged TGF-beta we generated, and we have used this tool to screen a genomic RNAi library and identify candidates. The proposed research is significant because it is expected to produce a deeper and substantially expanded understanding of how TGF-beta ligands are regulated as they travel between cells. This contribution will have a positive impact on the cell signaling field because it provides a broad foundation from which to understand how graded growth factor distribution is achieved during development and is altered in disease states including cancers.
Misregulated cell-to-cell communication underlies a number of human developmental disorders and cancers. This work will characterize the functions of known and novel molecules and fundamental mechanisms that regulate the transport of messenger proteins between cells; which will clarify not only normal developmental processes; but also related pathological states including birth defects; developmental disorders; and cancers. Furthermore; this work will identify potential therapeutic targets to combat these diseases.
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