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Title: Visualizing roles of spastic paraplegia proteins in organizing axonal ER in live Drosophila
Author: Sohail, Anood
ISNI:       0000 0004 7651 4489
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Axons possess a continuous network of smooth tubular endoplasmic reticulum (ER), extending from the nuclear envelope throughout the neuron to synapses. Mutations affecting proteins with intramembrane hairpin domains that model tubular ER membrane can lead to the axon degenerative disease, hereditary spastic paraplegia (HSP). However, the extent and mechanisms by which HSP proteins contribute to axonal ER organization and dynamics are unclear. To understand these mechanisms, there is a need to visualize axonal ER in wild-type and mutant live axons. I have therefore aimed to develop these tools in Drosophila larvae and adults, and use them to visualize mutant phenotypes. Firstly, I developed a system to visualize fluorescently marked ER in individual axons in adult fly legs, and tested how this can be used to investigate the effects of loss of intramembrane hairpin HSP proteins on ER in adult legs. Secondly, known mutations affecting HSP hairpin proteins reduce the axonal ER network but not severely; I hypothesized that additional HSP ER membrane proteins might contribute to residual tubule formation; these include Arl6IP, also reported to promote ER tubule formation. I generated transgenic flies to overexpress a fluorescently tagged eGFP::Arl6IP1, and found that this fusion protein localizes within axonal ER. To study whether loss of Arl6IP1 function affects axonal ER, I tested the effects of knockdown on this compartment, but found no consistent effects. To achieve stronger loss of function, I also generated a mutant stock that lacked one of the transmembrane domains and showed a slight developmental delay in homozygous Drosophila larvae. Like mutations in a number of other HSP hairpin proteins, this lesion is homozygous viable, and further characterization of its phenotype will help elucidate how Arl6IP1 contributes to modeling the axonal ER network. In conclusion, my work shows the utility of GFP markers of axonal ER, it can facilitate faster screening for other genes that potentially regulate ER structure and for ageing phenotypes that are not apparent in larval stages, and suggests Arl6IP1 as another HSP protein with a role in axonal ER organization.
Supervisor: O'Kane, Cahir Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Axonal ER ; neurodegeneration ; drosophila