Commissureless (Comm) and Roundabout (Robo) are key regulators of axon guidance at the Drosophila CNS midline. Robo encodes a receptor for the midline repellent Slit and prevents ipsilaterally projecting axons from crossing the midline. Comm encodes a novel transmembrane protein that is required for commissural axons to cross the midline. Genetic studies suggest the function of Comm is to regulate Robo receptor activity. The broad goal of this work is to understand how Comm functions. First, how does Comm regulate Robo? Second, what are the functions of Comm family members? Third, how does Comm function in neuromuscular synaptogenesis?
Using a cell culture assay, we demonstrate that Comm forms a complex with Robo and facilitates clearance of Robo from the cell surface. This activity requires a defined region of the Comm cytoplasmic domain for clearance activity and the transmembrane domain region for complex formation. Similarly, the Robo transmembrane and juxtamembrane regions are required for complex formation and Comm-mediated clearance. The identification of lysine residues in the juxtamembrane region of Robo as important for Comm-mediated clearance suggests that ubiquitination of Robo may be required for internalization of the Robo-Comm complex.
We have identified two additional Comm family members (Comm2, Comm3) and tested their ability to regulate different Robo family members. We find that Comm can clear all three DRobos (DRobo, DRobo2, DRobo3), while Comm2 only clears DRobo efficiently, and Comm3 does not efficiently clear any of the Robo family members. As a first step in our genetic analysis of comm2, we isolated two P-element insertions in the 5'-UTR of comm2. Molecular genetic studies indicate that these insertions in comm2 influence the transcription of comm, despite residing approximately 25kb 3' of comm. This is consistent with other data that suggests Comm cis-regulatory regions are large and complex.
Comm is expressed by all muscles in the periphery and is required for initiation of neuromuscular synaptogenesis. Our findings suggest that the conserved tyrosine residues identified as important for endocytosis of Comm and for Comm-mediated Robo clearance in S2 cells are not absolutely essential for Comm function in muscles.
|Commitee:||Beattie, Christine, Vaessin, Harald|
|School:||The Ohio State University|
|School Location:||United States -- Ohio|
|Source:||DAI-B 79/09(E), Dissertation Abstracts International|
|Keywords:||Axon guidance, Comm2, Comm3, Commissureless, Roundabout, Synaptogenesis|
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