A Question Years in the Making

In 2004, Dr. Nina Sherwood and her lab discovered the spastin gene in Drosophila in a gain-of-function screen. Spastin was found to diminish microtubules in the Drosophila larval neuromuscular junction (AKA, a synapse) when overexpressed, consistent with the fact that spastin is a microtubule-severing protein. So, with this logic, a loss-of-function mutation in spastin should prevent microtubule degradation, right? Wrong! The same results were discovered in the deletion of spastin, and this is because microtubule degradation by Spastin allows for the formation of free nucleation centers to allow for the growth of new microtubules. The Spastin loss-of-function mutation also showed smaller, bunched boutons at the presynaptic terminals (see picture below), unlike wild-type linear boutons, and behavioral changes like those observed in humans. Later findings by Dr. Emily Ozdowksi implicates the role of another gene, pak3, in the mutant phenotypes conferred by Spastin loss. Pak3 was recently found to act in the subperineurial glia. Loss of Pak3 alongside the loss of Spastin restored the wild-type phenotype, suggesting some interplay of these two genes. The question remains now, does spastin function in the glia, the neuron, or both?

This summer, I am working in Dr. Sherwood’s lab, along with my lab partner, Jayden, on identifying the site of action of spastin. What I am perhaps most excited about in this project is the use of the CRISPR-Cas9 system to determine spastin’s site of action. My interest in biology was sparked my freshman year of high school after doing a short paper on CRISPR’s role in genome editing, and to finally be involved in such research myself is something my 15-year-old self would have never seen coming. 

To accomplish such a task, we will be forming a lengthy series of crosses between different fly lines to both express the Cas9 protein in specific tissues using the UAS-Gal4 system (another method of inducing gene expression using “technology” adopted from yeast) via the use of tissue-specific promoters and fly stocks that express sgRNA (guide RNA, the instructions used by the Cas9 protein) to make partial or complete spastin deletions. Jayden and I formulated some crosses for two glial drivers, and we were just amazed at how quickly we were able to pick up all this new terminology and information in just a week. Once we achieve our desired phenotypes, we will perform larval dissections and immunostaining to observe the neuromuscular junction. While so far, larval dissections are proving to be extremely difficult to execute, I am very excited to see what this project will reveal after years of research on this gene!

Figure 4 from Sherwood et al. (2004)

Figure 4 from Sherwood et al. (2004). This picture shows the difference in synaptic morphology of the wildtype (figure 4a) and the complete spastin deletion (figure 4b).

 

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