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Spatial and Temporal Dynamics of the Notch Ligand Delta During Development in Drosophila Melagonaster

Final Report Summary - DELTA (Spatial and Temporal Dynamics of the Notch Ligand Delta During Development in Drosophila Melagonaster)

This project was aimed at characterizing the intracellular movement of Delta, one of the two so called "DSL" ligands for the transmembrane receptor Notch in Drosophila. Notch signalling is one of a handful of molecular pathways that mediate animal cell-cell communication. These signalling pathways arose as early as the earliest multicellular animals and are crucial to maintaining homeostasis and fine-tuning tissue development. As a result, malfunction of the Notch pathway in humans has been implicated in a multitude of diseases including a number of cancers. Even though animal forms, sizes and behaviours have diversified enormously, these central molecular pathways have been exquisitely conserved. Thanks to this conservation, we can take advantage of simple model organisms, like the fruitfly Drosophila, in order to study the mechanism of action of central players in this pathway, like Delta. As has been repeatedly demonstrated, the conclusions extracted from these easily tractable systems can then be applied to human models and exploited for the design of drug screens.

DSL-Notch is a contact mediated communication pathway, since both ligand and receptor are embedded in the cell membrane. Previous work from our lab and many others has established that DSL proteins must be modified by ubiquitin and then endocytosed in order to signal. This is quite surprising, as clearance of the ligand from the cell surface would be expected to reduce (not to stimulate) signalling. A solution to this paradox came with the realization that these special proteins send their signal as they become internalized by exerting a mechanical force on the Notch receptor in the adjacent cell. However, a possibility that has not been explored yet is whether Delta can continue influencing cell behaviour after its initial internalization. For this reason, we set out in this project to characterize the trafficking parameters of Delta inside a Drosophila cell and to see whether the route and the motility of Delta in the signalling cell can affect its ability to signal. For this we used transgenic technology to express a version of Delta fused with a fluorescent protein, called Tomato, so that we can detect Delta in live cells and tissues. We also combined this transgene with a number of other genetic tools, such as fluorescent markers for specific intracellular membrane compartments (endosomes) and modifying enzymes that target Delta and affect its endocytosis and trafficking. The latter refer to two enzymes of the E3 ubiquitin ligase family, called Neuralized and Mindbomb1. These add ubiquitin moieties to Delta intracellular domain (Dl ICD) and this modification triggers Delta internalization. By coexpressing Dl-Tomato with Neur, we were able to increase the amount of internalized Dl-Tomato.

After ensuring that the Tomato fusion had not impaired the functionality of Delta, we concentrated our efforts in obtaining time lapse sequences of Delta-Tomato expressed in larval epithelial tissues. This was very challenging and required the development of culturing and imaging methods never used before in our lab. We managed to resolve many of the problems and were able to visualize moving particles of Delta in live tissue. In parallel we used more conventional fixed tissue microscopy to identify the endosomal compartment that the Delta particles represent. Our data point to the fact that it is an early endosomal compartment, based on its molecular characteristics. Indeed when we assayed the activity of Delta after compromising the assembly of a later endocytic compartment, we did not find any defects, suggesting that later steps of membrane trafficking in the cell are not important for Delta function.

These initial results have already spurred a series of followup experiments that are currently actively pursued in our group. We hope to have a more complete picture of Delta intracellular movement in the near future, which will undoubtedly contribute to the mechanistic unravelling of this intensely studied and medically relevant signalling pathway. We expect that clinical researchers will extrapolate this mechanistic information to gain a deeper understanding of Notch-related pathologies. For example they could use the subcellular distribution of the human Delta proteins (Dl1, 3 or 4) to discern and classify different types of pathologies.