THE ROLE OF CLIP-190, ORBIT/MAST, AND NEB IN MICROTUBULE ORIENTATION, NEURONAL MORPHOLOGY, AND AXONAL REGENRATION IN DROSOPHILA MELANOGASTER

Open Access
Author:
Skora, Joanna Magdalena
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Melissa Rolls, Thesis Supervisor
  • Craig Eugene Cameron, Honors Advisor
  • Scott Brian Selleck, Faculty Reader
Keywords:
  • microtubule dynamics
  • RNAi
  • da neurons
  • axon severing
  • Orbit/MAST
  • CLIP-190
  • neb
  • kinesin
  • +TIPs
Abstract:
Drosophila melanogaster dendritic arborization (da) neurons offer a model system in which to study microtubule orientation and the effects of disturbing microtubule dynamics. Microtubules serve a crucial function in the cell and are required for diverse processes ranging from cell division, cellular transport and attainment of proper cellular morphology. These cytoskeletal elements have an intrinsic polarity related to the polymerization of tubulin dimers during the dynamic process of their growth and shrinkage. The microtubule orientation in dendrites of Drosophila da neurons has been reported as 95% minus-end-out through EB1-GFP dynamics (Rolls et al., 2007). +TIPs are proteins that have been identified as binding or associating with the growing plus-ends of microtubules. Two +TIPs, CLIP-190 and Orbit/MAST, were investigated in this study to determine the effect of their knockdown by RNA interference on microtubule orientation, dendritic morphology, and the cellular response to axon severing. No significant effect was found on microtubule orientation or neuronal morphology in CLIP-190 knockdowns, while there was a significant increase in plus-end-out microtubules in Orbit/MAST knockdowns. These results indicate that Orbit/MAST may play a role in the establishment and maintenance of polarized microtubule arrays in Drosophila neurons, but this hypothesis requires further investigation. Both CLIP-190 and Orbit/MAST RNAi knockdown was found to compromise the ability of class I ddaE neurons to respecify one dendrite into an axon-like process following axon severing, indicating that both of these proteins may be required for cellular response to axonal injury. Another important aspect of microtubule function is directional transport of cargo in the cell. Kinesins are molecular motor proteins that use microtubules as tracks to transport cargo, and they are known as plus-end-directed motors. In this study, protein levels of the kinesin-like protein neb were reduced through RNAi and the effect of neb depletion was studied on microtubule orientation and neuronal morphology in da neurons. Knockdown of neb was found to significantly reduce branching complexity in dendrites of ddaE neurons. These studies shed light on the importance of microtubules and their associated proteins in proper neuronal structure and function.