Author Archives: Lauren Sar

BSURF was a wonderful experience. When first hearing about and applying to the program, I had no idea what to expect from a sumer research experience. I knew that I wanted to try something with biological and virological research in my time at Duke but had no idea how to get involved or how to find the right lab and mentor to help me get a good experience. In hearing about BSURF, I learned that this program would help to navigate this confusing process and give more experiences on how research works and how to be a good researcher. I was very excited for this type of opportunity even though I did not know what to actually expect in the research itself.
Now at the end of BSURF, I can happily say that BSURF was everything it promised and more. I was able to join a lab in virology that helped me to better understand what research in virology and microbiology is through the help of wonderful mentors. I was able to get good advice from both my mentors in my lab, Vanessa and Dr. Horner, and the leaders of the BSURF program, Austin, Dr. Harrell, & Dr. Grunwald. I was able to make great friends with other students interested in biological research and spend lots of time discussing research and other interesting topics with programmed events. I am very glad that I was able to be a part of this experience!

Last Tuesday, one of Duke’s Nobel Prize winners came to speak to BSURF, Dr. Lefkowitz. To my surprise, Dr. Lefkowitz spoke very briefly about the focus of his lab work. However, his talk very quickly became one of my favorites because of his focus on determination in changing careers, applicable goals for success in science, and charismatic anecdotes.

Dr. Lefkowitz obtained a B.S. and MD from Columbia and was quickly met with a difficult problem after graduation. All doctors were required to enlist for the Vietnam war. In order to circumvent going to Vietnam, he joined a group of doctors at the NIH nicknamed the yellow berets, getting his first research experience there. He spoke of the great struggle he had in the first months there, as he never intended to do any research in his career, and his eventual fondness for research because of this initial struggle in the program. He then described his eventual return to research after some time as a clinician, his opening of his lab at Duke, and the work in his lab that led to his Nobel Prize. What I enjoyed most about his talk was his tips for success that he learned throughout this journey. He told us to focus, build this focus around interesting questions and not techniques, do lots of experiments, don’t talk yourself out of experiments, be bold, take risks, and fail, but don’t be afraid, learn to tell a good story, be ambitious, be persistent, and prepare, among other things.

What stuck with me about this section of his talk was not the exact tips themselves, but rather the anecdotes he told along with each one to better engage with the audience and explain what he meant by each one. With this, he additionally mused on the necessity of jokes (so long as you’re a funny person), his love for Duke basketball and his friendship with Coach K, and the absolute necessity of chocolate in order to become a Nobel Laureate. It was these stories that drew me most to his talk and were used excellently to better understand and characterize himself and his pathway in research. Dr. Lefkowitz wonderfully shared his life and his research with us this week with some excellent advice. I will certainly be eating more chocolate after hearing from him!

Lauren Sar

Mentors: Vanessa Gutierrez, PhD, Stacy Horner, PhD

The antiviral innate immune system is a vertebrate’s first line of defense against disease, sensing non-self patterns such as dsRNA and activating a larger immune response mediated by interferon. The two proteins RIG-I and 14-3-3ε are known to play a central role in this system. RIG-I is the molecule that senses dsRNA. RIG-I then binds to 14-3-3ε, which moves RIG-I to signal sites for a signal transduction cascade. This cascade leads to the production of interferon, beginning a wider antiviral response. The molecular mechanism by which these proteins interact is unknown. It is known that 14-3-3ε binds somewhere in RIG-I’s 2-CARD domains and that 14-3-3ε traditionally binds to post-translational modifications (PTMs). However, it does not bind to any PTMs in the 2-CARD. Interestingly, 14-3-3ε has a PTM, ufmylation, which is required for its’ interaction with RIG-I. The 2-CARD of RIG-I contains 2 LC3-interacting regions (LIRs), motifs known to interact with ufmylation. Here we investigate whether these LIR motifs mediate the binding of RIG-I and 14-3-3ε. Utilizing the expression of mutant plasmids with deletions of these LIRS, we show that both LIRS impact the interaction of RIG-I with 14-3-3ε.

In our program this week, all BSURFers gave 8-minute ‘Chalk Talk’ presentations in which we outlined background details and the main goals of our project, almost like a drawn-out form of a paper abstract. I thoroughly enjoyed hearing about the various projects that everyone was working on. It reminded me of how widespread the interesting fascinations of biology are- from ecology and bugs in Rena’s project to biochemical stabilization of protein complexes in Kyle’s project.  

One talk that I found quite engaging happened to come from my lab’s next-door neighbor, the Ko lab. Sam works in the Ko lab, studying a type of bacteria called Yersinia pestis, better known as the plague. Sam did an excellent job drawing a connection between his individual project and the importance of research on the plague, citing its current high fatality rate when infected and the previous plights caused by the disease, killing nearly 1/3 of the world population at its peak. After catching everyone’s attention with the importance of his project and research on the plague, Sam dove into the details of his research. 

He explained how his lab more widely was investigating a singular nucleotide polymorphism on the gene for an immunoreceptor that is associated with a change of phenotypes when infected with plague. He and his mentor are looking at the protein made by this gene, the immunoreceptor, and trying to understand the interaction between this immunoreceptor and the Y. pestis bacteria. They found previously that a particular portion of this protein on the extracellular membrane of the cell, the IG-like domains 1, 2, & 3 are interacting with the bacteria. Thus for his project, Sam is going to design mutant plasmids of this gene that remove different combinations of these IGs from the protein and overexpress this to see how this affects the interaction between the bacteria and the protein through a method called flow cytometry.  

I greatly enjoyed the overall set-up of Sam’s presentation as well as the content of his research. I thought that he did a very good job of explaining the substance of his research by starting with the larger context and then explaining the more specific details of his project. I am excited to see where this project goes in the future! 

Each day in lab can easily become very hectic and I often find myself learning new procedures, having interesting conversations on topics I’ve never heard of, and meeting new people every single day. However, a pattern has begun to emerge in my weekly schedule as I have begun to perform similar experiments multiple times in the past few weeks. For instance, I use a particular line of ‘immortal’ human cells called 293T cells daily for my experiments. To prep these cells for experiments, I must go through a 3 day process of plating these cells to grow, transfecting them with a desired plasmid, infecting them with a virus, and harvesting them to be tested in various ways. I usually perform this cycle two times a week and find my harvesting days, often the days that I also perform experimental tests, to be the most exciting. My most recent harvest day, Friday, was yet again a fairly exciting day: 

On Friday, I arrived at lab a bit after 9 and placed my things at my desk, talking to the two postdocs with desks surrounding mine as I settled into lab for the day. Then I went to the tissue culture room in our lab to harvest my cells that I had been preparing over the past two days. This procedure takes about an hour in total, involving checking the cells to confirm they’re living, removing the cells from the plate, dividing the cells into smaller test tubes for my two different experiments for them, a luciferase assay and a western blot, pelleting these cells so they can be stored, and plating new cells to be used in my next round of experiments. 

After this, I met with the principal investigator in my lab, Stacy Horner, to talk about my project and help prepare for my chalk talk in the following week. She was able to guide me on the next steps in my project, as well as give me advice on teaching on the lab’s research and presenting in general for my chalk talk. After this meeting, I went back to my lab bench to conduct experiments on the cells I had harvested previously. I began with the luciferase assay, a procedure that measures interferon induction in the cell through tagging with a fluorescent substance, luciferase. This procedure usually takes around 1-1 ½ hours to set up. After this assay was finished, I constructed graphs on my computer to display the data and average it with other replicates of the same experiment. Then I used the rest of my harvested cells to perform a western blot, which helps to visualize whether or not a particular protein is being expressed in the harvested cells. This procedure takes a few hours, requiring quantification of the concetration of protein, separation of protein by size on a gel, and identification of the protein with antibodies. All throughout these experiments, I was talking to my labmates about various subjects in lab as well as other fun and less lab related topics. When this experiment was completed, I ended my day and headed out of lab around 5. 

The principal investigator in the Horner lab, Dr. Stacy Horner, is the co-director of the Duke Center for RNA Biology and an associate professor of medicine and of molecular genetics and microbiology at Duke, having opened her lab at the university in the 2013-2014 academic year. I have been working in her lab for about a month now, with multiple opportunities to get to know her better and learn more about her journey into the world of microbiology and host-virus interactions, as well as how she got to her current position. Recently I was able to meet with her to talk about this journey to where she is now.  

She grew up in Minnesota, where she first found a general interest in biology and chemistry in school but knew little of the research opportunities in these fields. As she searched for colleges that would enable her to explore her interests in biology and chemistry, she ended up attending Gustavus Adolphus College. She said she wanted to choose this school because it was a small liberal arts college in Minnesota that enabled her to explore both biology and chemistry with a biochemistry major. Going into college, she said that she knew very little about the world of research. She was able to dive into research through her undergraduate classes but did not consider going to graduate school in a PhD program until later in her undergraduate career as she learned that unlike other degrees, many PhD programs in the sciences do not require one to pay tuition and instead pay the students. Through this knowledge and encouragement in undergrad, she began to pursue graduate school for a PhD program in molecular biology. 

She joined the lab of Dr. Daniel DiMaio at Yale University, where she began to research human papillomaviruses. Throughout her time in graduate school, she did not believe that she would become a professor or principal investigator in a lab in the future. Dr. Horner said that she thought that she might not be creative or capable enough to constantly come up with the questions and ideas for a lab. It was when she was talking with mentors about her plans for the future that her mind was changed. When she told her mentors that she would not pursue postdoctoral studies and a professorship because of these reasons, they said that they thought she did have the skills to pursue the path to professorship. It was with their encouragement that she decided to pursue postdoctoral work. 

In her postdoctoral work, Dr. Horner studied the regulation of the innate immune system concerning Hepatitis C in the lab of Dr. Michael Gale at the University of Washington. She then opened her lab in the Duke school of medicine where she currently pursues research in RNA virus host interactions, often concerning the innate immune system and Hepatitis C. Stacy has been incredibly welcoming, knowledgeable, and enthusiastic as I have gotten to know her in joining her lab. I am very excited to talk more and learn more with her in lab! 

For this summer, I am working in the Horner lab, a lab in the the molecular genetics and microbiology department of the Duke school of medicine. The Horner lab is widely investigating RNA virus-host interactions with the immune system, specifically focusing on positive sense RNA viruses such as Zika, Dengue, Sendai, and Hepatitis C viruses interacting with the innate immune system. My mentor Dr. Gutierrez, a postdoc in the lab, is currently investigating ufmylation, a post translational modification, and its potential substrates in the innate immune system pathway. My specific project is attempting to characterize the protein-protein binding site of RIG-I and 14-3-3e, two proteins at the start of the innate immune system.
We set to determine whether the LIR motifs in the 2-CARD domain of the RIG-I protein mediate binding between RIG-I and 14-3-3e. The decision to investigate this particular motif came about by the lab’s investigation into previous studies of these proteins. It was previously found that 14-3-3e often binds in a phosphorylation binding pocket. However, it is known that 14-3-3e also binds in other non-canonical methods not involving phosphorylation (Pennington et al, 2018). RIG-I is one of the proteins known to interact without phosphorylation and thus must use a different binding method (Snider et al, 2021). It is currently being explored whether 14-3-3e may also bind at the site of another post translational modification, ufmylation, as this modification has been shown to be present on 14-3-3e by the Horner lab.
It was previously found that RIG-I binds to 14-3-3e through the 2-CARD domain of the RIG-I protein (Liu et al, 2012). It is not known what part within this domain binds to 14-3-3e. RIG-I contains two LIR motifs in the 2-CARD domain. The lab suspected that RIG-I may bind through one or both of these LC3 Interacting Regions, also known as LIR motifs, because these regions have been shown to mediate binding at post translational modification sites for other protein-protein interactions.
With this background, I was given the project to determine if RIG-I binds to 14-3-3e through the LIR motifs in the 2-CARD domain of RIG-I. To test this, we will be utilizing a RIG-I KO cell line and inserting plasmids encoding for RIG-I with mutated LIR motifs, disabling the function of these motifs. These lines will be tested to ensure expression of the RIG-I protein with a western blot, to determine function of the innate immune pathway with the mutated protein through a luciferase assay for IFNb (a major product of the pathway), and for binding of RIG-I to 14-3-3e through co-immunoprecipitation. If a LIR motif is not integral to the binding of 14-3-3e and RIG-I, IFNb should still be produced and the two proteins should precipitate together while RIG-I is expressed with a mutant on that motif. If a LIR motif is integral to the binding of 14-3-3e and RIG-I, IFNb should not be produced and the two proteins should not precipitate together while RIG-I is expressed with a mutant on that motif.
Upon completion of this experiment, this project has two likely paths forward. If the LIR motifs are not likely to be necessary for RIG-I and 14-3-3e binding, more potential suspected binding sites on RIG-I for this interaction may be investigated. If the LIR motifs are likely to be necessary for RIG-I and 14-3-3e binding, the binding site for this interaction on 14-3-3e may be investigated.

Pennington, K., Chan, T., Torres, M. et al. The dynamic and stress-adaptive signaling hub of 14-3-3: emerging mechanisms of regulation and context-dependent protein–protein interactions. Oncogene 37, 5587–5604 (2018). https://doi.org/10.1038/s41388-018-0348-3

Snider, D. L., Park, M., Murphy, K. A., Beachboard, D. C., & Horner, S. M. (2021). Signaling from the RNA sensor rig-I is regulated by ufmylation. PNAS. https://doi.org/10.1101/2021.10.26.465929

Liu, H. M., Loo, Y.-M., Horner, S. M., Zornetzer, G. A., Katze, M. G., & Gale, M. (2012). The mitochondrial targeting chaperone 14-3-3ε regulates a rig-I translocon that mediates membrane association and innate antiviral immunity. Cell Host & Microbe, 11(5), 528–537. https://doi.org/10.1016/j.chom.2012.04.006