Over a year ago, I was met with the news that BSURF would be canceled because of the pandemic. While many things have changed between then and now, I am grateful to have still been able to be a summer research fellow. In these past eight weeks, things have continued to change for me; I’ve grown, not only in my knowledge and research skills, but in my confidence, communication, and passion for science.
I remember my first day in Dr. Sherwood’s lab like it was yesterday: looking at flies under the microscope, learning about our project for the first time, and cringing every time I had to handle a fly vial. My first larval dissection took me about twenty minutes and looked horrendous. Now, I can do some in under five minutes and make them look better than my first. I also get embarrassingly excited when one of our crosses finally starts producing larvae, or when it’s finally time to look at my dissections under the microscope. Simply put, I have fully embraced the experience of being a fruit fly biologist. My time in the lab also came with the development of my personal skills – no longer being afraid to ask “dumb” questions, owning up to my mistakes without shame, and effectively communicating my science.
Despite not being able to get to every aspect of our project in these eight short weeks, my enthusiasm for our project is unchanging. I am beyond excited for what the future will hold for our lab, especially considering our results that remain to be analyzed. While research tends to be more failures than successes, the breadth of information I learned, and the valuable mentorship I received from Dr. Sherwood, are more than enough to compensate for the drawbacks of our research. I can’t wait to get back to BioSci in the fall and keep up the wonderful work we do!
Author: Shibani Mallik
Mentor: Nina Tang Sherwood, Ph.D.
Department of Biology
Spastin is a microtubule-severing protein important for microtubule degradation and growth. Spastin mutations in humans are known to cause Autosomal Dominant Hereditary Spastic Paraplegia (AD-HSP), a neurodegenerative disease of the motor system. Ubiquitous deletion of spastin in Drosophila also causes motor defects in adult flies, as well as defects in synaptic bouton morphology and function. While spastin is thought to be expressed in neurons, the true site of action of Spastin remains unknown. This study used the CRISPR-Cas9 genome-editing tool to generate spastin deletions in only neurons or glia using tissue-specific drivers, along with ubiquitous drivers, to discover Spastin’s site of action. This was accomplished through a series of genetic crosses, larval dissection, and immunofluorescence microscopy to visualize boutons at the neuromuscular junction. This study also evaluated the efficacy of using CRISPR-Cas9 as an editing tool in Drosophila. If Spastin is required only in specific tissues, then the larvae with certain tissue-specific drivers will display the mutant phenotype. A ubiquitous driver should also display the mutant phenotype, depending on the functionality of the CRISPR-Cas9 system. This study has important implications for future therapy development for AD-HSP and future projects on the spastin gene, among others implicated in microtubule development.
While my research occurs in a lab, it was very interesting to learn of our other peers’ research that happens in the field. I was particularly fascinated by Xitlali’s project and its intersection in the broader efforts of environmental nonprofits.
Xitlali’s project looks at the effects of urban development on the environment, by studying biodiversity in different areas of watersheds by their level of developed land. New Hope Creek’s watershed, located within Duke Forest, is barely developed, whereas Ellerbe Creek’s watershed is located in a highly developed area. The level of biodiversity between these two differing locations is thought to be affected by urban development, and its role in facilitating the drainage of storm surge. Rainwater is able to slowly drain into New Hope Creek following a storm surge because it is rich in soil, while Ellerbe Creek receives rainwater at high rates because of pipes and drainage infrastructure. The timing of these processes is thought to affect the biodiversity of the two creeks. The hypothesis is that New Hope Creek will likely have more biodiversity due to its soil-rich watershed, in comparison to Ellerbe Creek. This will be measured by looking at different species of aquatic insects in the watersheds. Insects are also thought to be better able to stay on rocks on New Hope Creek. However, there are thought to be more “resilient” species in Ellerbe Creek, to withstand harsher water upheaval.
It was great to witness the huge range of interests within BSURF, and Xitlali’s, in many ways, felt like a huge contrast to mine (minus the bugs). I could not imagine having to go out to rivers to do my work! Moreover, while many of our projects have clinical applications or contribute to tool-building, Xitlali’s has great implications for studying the effects of neighborhood and class divides, a topic I would normally study outside of my realm of biology courses. While I must admit ecology has never been a major interest of mine within biology, I found this chalk talk super interesting, and I can’t wait to see where this project goes!
While a day in the Sherwood lab looks a little different every day, there is always one constant to start (and end) my day: collections! The minute we get to the lab in the BioSci sub-basement, we bring out our fruit flies to pick out the ones we need. These could either be flies to continue adding to a cross or picking out the F1s of a cross important to our project. This process also unfortunately involves unceremoniously killing many flies that we don’t need by throwing them into our “fly morgue,” or a jar of ethanol.
Here’s where the day can take a turn. Sometimes, if our desired crosses have started producing larva in the third instar stage, or right when they’re about to pupate and metamorphosize, we pick out those larvae for dissection. While I’ve gotten much better at dissections since we started, I still find it to be somewhat of a painstaking process given how small they are. Once we’ve dissected them into flattened, stretched fillets (a word I still find hilarious), we go through a process of fixing and rinsing them to prepare for immunostaining. Once immunostaining is complete, which takes about 2-3 days, we can look at them under the fluorescence microscope and look at some really cool synapses (see below)! While it doesn’t seem like a lot in writing, these processes take up most of my time in the lab. Recently, we’ve been looking at fillets and counting boutons for our project.
However, if we are between stages of immunostaining, or simply do not have the larva we need, my days look a little different. Sometimes, we pore over our crosses to check for errors or try to work out the mysteries of getting a weird phenotype from a cross, and this involves a lot of time staring at a whiteboard, wondering what could have happened. Other days, we’re looking over readings. Some days, we might learn about something having to do with another project. For instance, we recently crushed up some flies that we’ll eventually PCR to check for recombination.
I end the day with evening collections, doing what we did in the morning, hopeful for some newborn F1 flies. No matter how our days might look, Jayden and I usually end around 6, walking out feeling satisfied with our work, but also tired after a long day of sitting at a microscope. Yet, I’ll wake up the next morning, excited to do it all over again.
Synapses! Photo by me 🙂
While I get to learn a little more about her every day, speaking in a more formal sense with my PI and mentor, Dr. Nina Tang Sherwood, was incredibly fulfilling and reassuring for an undergraduate like myself. I am of the lucky few whose PI is also their bench mentor, and I am even more lucky to have such an inspiring person like Dr. Sherwood by my side this summer.
Dr. Sherwood grew up in Hawaii around two parents who were also scientific researchers, to whom she accredits learning to love science and lab research. She fondly recalled going to her mom’s lab after school and getting to speak with the researchers in her lab, both experiences that fueled her passion to pursue research. For her undergraduate years, she went to UC San Diego, where she majored in biomedical engineering. It would be important to recall now that Dr. Sherwood’s lab is a neurobiology lab; as we’ve seen with other professors and PIs, not every career path is so straightforward. In fact, the first research lab she joined at UCSD was a physical chemistry lab!
However, in her junior year, Dr. Sherwood took a neurobiology course so captivating that it totally changed her path and set her up for her work today. This newfound interest also led to her decision to go to graduate school to continue studying neurobiology. She entered graduate school at Duke with the goal of understanding how synapses work, a goal she says has not changed to this day. Her current work on the spastin gene came about out of “total luck,” through a genetic screen that pinpointed a gene that just so happened to be heavily involved in synaptic development. In our conversation, she also reflected on her work specifically with fruit flies as a model system, describing the difficulty of genetics in mice (that she initially worked with) for the purpose of her work at the time she began her research. Her kind heart also made this somewhat difficult, as she found the prospect of hurting mice hard to justify. This lines up well with the fact that, had she not pursued research, she had actually thought about going to veterinary school for some time.
To round out our interview, we asked Dr. Sherwood if she had any advice for her younger self. She told us, ”Don’t be afraid to ask questions and don’t be self-conscious.” Seeing how far Dr. Sherwood has come, her words are of immense value to burgeoning researchers like me and (I’m sure) the rest of us in BSURF. We could all take her advice and start caring less about what others think of us, because at the end of the day, it is only our drive and determination that can get us where we want to be. I want to reiterate how grateful I am to be under the wing of a mentor who understands such fears and worries, and who inspires me to be more confident in myself as a student and scientist.
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). This picture shows the difference in synaptic morphology of the wildtype (figure 4a) and the complete spastin deletion (figure 4b).
When I first got to Duke, the word “research” intimidated me. Research implies thinking out of the box and asking big questions. For me, it implies sitting in a brightly lit lab somewhere on Science Drive. But scariest of all (in my opinion), it implies me becoming a “researcher.” Being a researcher comes with a multitude of responsibilities and expectations: that I am knowledgeable, dependable, inquisitive, among other traits we all know well by now. They say the best way to get over a fear is to face it head-on, and this is what this summer is offering me. So the question remains, what do I expect of my summer with BSURF?
First and foremost, I expect to be challenged. I expect to learn things I wouldn’t have learned in your average biology course. I expect to ask and be asked hard questions. I hope to become more confident in myself and my modes of thinking. I expect to fail over and over again, but to remain persistent and hopeful through it all. Yet, if there’s one thing I’ve always known about myself, it’s that I love a challenge. I’ve been a lover of the sciences for as long as I can remember, and to see that excitement and enthusiasm finally get channeled into research is an extremely gratifying feeling. With that in mind, I hope to learn much more about molecular genetics, a field I’ve been interested in since high school, and to finally get involved in exploring new questions and ideas about this exciting field. Being able to now take on a project in my lab allows me to delve deeper into different modes of exploration, specifically by exploring a new type of model system I haven’t been exposed to yet–fruit flies! I am very fortunate to be spending the summer in Dr. Nina Sherwood’s lab, where they use Drosophila as a model to study the spastin gene and its role in neural development. That being said, another goal of mine, although not quite important (but a little ironic), is to stop being so squeamish around bugs (spoiler: two days into my lab and I can say this is becoming much less of a problem). Although I’m just working with what are arguably some of the least intimidating of all bugs, I’m taking baby steps!
Now to address the first sentence of my post: “intimidated,” emphasis on the past tense. In talking not only with my PI but with other peers involved in research, as well as the pointers from Dr. Grunwald and Dr. Harrell, the anxiety I had around research has become adrenaline. However, that is not to say that I am 100% worry-free going into this experience. Nevertheless, I am beyond excited for my summer in the Sherwood lab, despite our short timeframe here at Duke. I can only hope to continue facing my fears head-on, and come out of this summer feeling fulfilled and proud of my work and my growth as a researcher.