Proceedings of The Physiological Society
King's College London (2011) Proc Physiol Soc 22, PC18
Trafficking of Roundabout by Commissureless in the Drosophila Central Nervous System
D. M. van den Brink1, G. Tear1
1. MRC Centre for Developmental Neurobiology, King's College London, London, United Kingdom.
Both longitudinal and commissural neurons in the Drosophila central nervous system are guided by a number of repellent and attractant cues. One of these is Slit, which is produced by cells at the midline. Slit signals through the Roundabout (Robo) receptor and acts as a negative signal to steer axons away from the midline. Commissural neurons express Robo but are insensitive to Slit prior to crossing the midline and become responsive to Slit after crossing. This differential sensitivity occurs through Commissureless (Comm), which regulates Robo protein distribution in the axon. Comm acts as an intracellular sorting protein that is able to sequester Robo within intracellular vesicles in commissural axons prior to crossing (Araújo and Tear 2003). To dissect the intracellular mechanism by which Robo is trafficked by Comm we have investigated the role of the Rab family of GTPases and additional components that regulate membrane protein transport. METHODS: Mutated forms of Rab proteins, acting as dominant negatives, were expressed in the Drosophila melanogaster CNS or cultured cells using the Gal4/UAS system (Brand and Perrimon 1993). Immunofluorescence techniques were used to analyze possible morphological changes in the CNS due to the overexpression of these proteins during embryonic development. RESULTS: Surprisingly, the growth of neither ipsi- nor contralateral projecting neurons was affected when dominant negative forms of Rabs were expressed in the CNS during development. We were able to show the efficacy of these dominant negative constructs, however, in a different developmental setting. The same dominant negative constructs expressed in a Drosophila cell line did show a role for one specific Rab protein in the trafficking of Robo alone (in the absence of Comm), while none had any effect on the trafficking of the Comm-Robo complex. To characterise the sorting process in more detail we are now studying the dynamics of both Robo and Comm in axons in vivo. Preliminary results show the process to be highly dynamic in living embryos. With the results of these ongoing studies we hope to gain insight into the precise regulatory mechanisms that direct the distribution of Robo and Comm within the extending commissural axons.
Where applicable, experiments conform with Society ethical requirements