672B Poster - 10. Cell biology: Cytoskeleton, organelles and trafficking
Friday April 08, 2:00 PM - 4:00 PM

Developing a Drosophila genetic screen for mutations that disrupt axonal ER organization


Authors:
Nishani Jeyapalan; Cahir O'Kane

Affiliation: Genetics Department, University of Cambridge

Keywords:
j. endoplasmic reticulum; v. cell biology of disease

Developing a Drosophila genetic screen for mutations that disrupt axonal ER organization
Nishani Jeyapalan, Cahir J O’Kane
Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, UK

Axonal endoplasmic reticulum (ER) consists of a continuous tubular membrane network. Its continuity and potential as a channel for long-distance communication have earned it the description "a neuron within a neuron". Mutations affecting ER-modeling proteins can cause the axon degeneration disease, hereditary spastic paraplegia (HSP), implying the importance of this network in axon maintenance. While the assembly, distribution, transport, and density of the network must be regulated to meet the needs of the neuron, we understand hardly anything of this regulation. To address this, our goal is to identify novel proteins involved in organizing and maintaining the ER network, using targeted CRISPR/CAS9 mutagenesis.

To allow easy visualization of axonal ER for screening, we identified Drosophila lines expressing GAL4 in small numbers of motor neurons innervating the adult leg, and used them to express fluorescent ER and plasma membrane markers, observed through undissected cuticle in adult legs. To allow tissue-specific CRISPR/CAS9 mutagenesis in the labelled motor neurons, we introduced a UAS-CAS9 construct into these flies, targeting CAS9 expression only to the labelled motor neurons utilising the Drosophila GAL4 system. Crossing this stock to any transgenic guide-RNA (gRNA)-expressing line allows tissue-specific somatic mutagenesis in the labelled neurons, and screening for axonal ER phenotypes as a consequence of tissue-specific gene disruption.

We have performed a pilot mutagenesis screen to observe axonal ER phenotypes in CAS9-mutated Drosophila motor neurons. We selected a small library of gRNAs, targeting a small selection of genes encoding ER-associated proteins, HSP protein orthologs, and motor proteins. We crossed these gRNA lines to the line expressing ER and PM markers and CAS9 in leg motor neurons, and tested the progeny for alterations in ER distribution in adult leg femoral motor axons.

Confocal screening showed a highly discontinuous axonal ER network as well as differences in ER and PM average intensities using several of these gRNA lines, in contrast to the more uniform continuous ER structure observed in control motor axons.

Having shown proof-of-principle for detecting axonal ER phenotypes caused by gRNAs in motor axon ER, we are now individually investigating gRNAs that cause axonal ER phenotypes via in vivo larval imaging, to ascertain whether axonal ER dynamics are also affected. Furthermore, we can now expand the pilot screen to encompass genes involved in several biological pathways, testing the roles of various gRNAs on axonal ER architecture.