First person – Emily McParland and Noah Gurley Free

Emily McParland

How would you explain the main findings of your paper in lay terms?

E.M. and N.G.: During animal development, formation of the body plan requires cells to move and change their shapes. One task of the cells is to maintain structural integrity while pushing and pulling on and moving around one another. The Peifer lab focuses on the proteins that maintain cellular adhesion, examining (1) how they link to one another and (2) how they are linked to the force-generating cytoskeleton within individual cells. The fruit fly protein Canoe (the focus of our work) and its mammalian counterpart afadin play a key role in maintaining this linkage. Canoe and afadin are regulated by the small GTPase Rap1, which binds and activates Canoe via two highly evolutionarily conserved regions of the protein (the RA domains). Interestingly, using biochemical and bioinformatic approaches, we found that both regions bind to Rap1 with similar affinity. To individually test the function of these two regions of Canoe, we generated fly mutants and analyzed their roles in development. Despite their similar biochemical properties, the two regions play strikingly different roles: while one of these regions (RA1) is crucial to normal development, the other region (RA2) appears to play a more supportive role. Remarkably, although RA2 is not necessary for normal body plan development, its deletion leads to defects in eye development, revealing deficits in cell junction support. Together, the findings presented in this work contribute to our understanding of cytoskeleton–cell linkage dynamics.

Were there any specific challenges associated with this project? If so, how did you overcome them?

N.G.: Many of the challenges associated with this project were technical in nature. For example, a key part of the project involved performing quantitative analysis of western blot data to compare expression levels of our four mutant proteins relative to endogenous Canoe protein. To do so, we initially planned to stain the membranes with two antibodies: one targeting the C-terminal region of Canoe (Cno-C) and another targeting the GFP tag on our mutant proteins. We presumed that the molecular weights of the wild-type and mutant Canoe proteins would be different enough such that the two bands would not overlap. This would have allowed us to compare fluorescence intensity of the GFP band relative to the Canoe band within the same biological sample. However, for two of our four mutants, the deleted domains were roughly the same molecular weight as the C-terminal GFP tag, thus causing our mutant proteins to exhibit similar migrations as wild-type Canoe protein. One way to overcome this issue would have been to lower the percentage of acrylamide in our gels, in order to increase the separation between proteins of similar molecular weights. However, this was not a practical solution: Canoe is a very large protein and the gel percentage necessary for sufficient separation would be so low that the subsequent transfer step would be made nearly impossible, due to the fragility of the gel. Thus, we had to pivot and come up with an alternative plan. After brainstorming some ideas with Emily and consulting with Mark, we ended up deciding to quantitatively compare across biological samples, measuring and comparing the intensity of the GFP band from the mutant embryo sample with the Cno-C band from the wild-type sample. Although throughout this project we ran into a number of frustrating roadblocks, these obstacles were rewarding as they challenged us to harness our creativity and pushed us through to completion.

When doing the research, did you have a particular result or ‘eureka’ moment that has stuck with you?

E.M.: Prior to starting my position in the Peifer lab, most of my research experience was working with mammalian jaw joint biomechanics – an entirely different field from cell biology. Much of that work was exploratory, and we had general expectations about what we would find. When I first started working with the mutant flies, I remember the excitement of seeing the results of the viability experiments. I had no idea which domains would be essential to fly viability and whether they would be able to replace one another. I distinctly recall graphing lethality for the four mutants and seeing the striking differences between domain functions. I am most fond of the excitement that came when we saw something that we had no expectations about.

Noah Gurley

N.G.: Throughout the course of this project, the question of ‘why have two RA domains’ was at the forefront of our minds. In trying to make sense of some of the data we were seeing, I scoured through the NCBI literature resources looking for publications involving proteins with tandemly positioned RA domains, and I eventually came across a few papers about phospholipase C epsilon (PLCε). One paper in particular (Rugema et al., 2020; doi:10.1038/s42003-020-01178-8) provided evidence to support a model in which one RA domain played a role in GTPase binding and membrane recruitment, while the other RA domain helped to facilitate interdomain interactions and stabilize basal activity. Coming across these data was incredibly exciting, as it helped expand my thinking of what could be happening with the RA domains of Canoe and gave me a deeper appreciation for the importance of reading existing literature and using it to inform working hypotheses.

Why did you choose Journal of Cell Science for your paper?

E.M. and N.G.: The central ideas of our work lie in questions about cell dynamics during development. Journal of Cell Science values careful experimentation on topics in cell biology, and we believe the questions that the Peifer lab asks fit these goals naturally. We have previously published in Journal of Cell Science and appreciate the overarching goals of the journal. We appreciate JCS and The Company of Biologists' commitment to a thorough and productive peer-review process that aided in clarifying the communication of our work.

Have you had any significant mentors who have helped you beyond supervision in the lab? How was their guidance special?

E.M.: Dr Nick Gidmark was my undergraduate mentor at Knox College (Galesburg, IL, USA). Early on in college, he encouraged me to pursue science and welcomed me into upper-level courses and projects in his lab. I had no prior experience and didn't know much about a career in science, but he went out of his way to introduce me to the joys (and hardships) of science. We have been working together since 2019, and we still collaborate and even teach a course together at Shoals Marine Lab. His mentorship and support thus far in my career have been essential to my growth as a scientist and person.

N.G.: Dr Mark Peifer played a pivotal role in shaping who I am today. First coming into my life as one of the two professors who changed the way I think about science, Mark would go on to serve as a mentor in my first research experience. Over the past five years, he has been one of my biggest supporters – believing in me during times when I doubted my abilities and seriously questioned whether I was the ‘right fit’ for science; he's taught me how to recognize my strengths and have confidence in myself. Perhaps most importantly, he showed me the importance of using your position as a leader to advocate for equity in science and research.

What motivated you to pursue a career in science, and what have been the most interesting moments on the path that led you to where you are now?

E.M.: My favorite part of pursuing a career in science is getting to ask and test questions that are interesting to me. Every day is a little bit different from the last, and I look forward to seeing where my research will lead me. I am grateful for my time in the Peifer lab. Although I have pivoted away from cell biology back to a PhD in functional morphology and comparative biomechanics, I still find myself gearing my thinking toward cell dynamics that I learned during my time in the Peifer lab. I enjoy thinking about animal movement through a series of scales, whether it's through cell movement, muscle biomechanics (the focus of my thesis) or skeletal movement. The combination of these skills has shaped me into the scientist I am today.

N.G.: During seventh grade, I developed a fascination for the remarkable complexity of cells, particularly in the context of animal development. I can vividly remember my astonishment when I learned that, by altering their molecular environments, individual cells can take on a wide array of distinct fates, which impart different biological and biochemical properties. When I first started my undergraduate education, medical school seemed like the perfect and only route to explore my biological curiosities. At the time, given that I was the first member of my family to pursue a degree in science, I was largely naive to the educational and career opportunities that would be available. Until that point, I had only ever met medical doctors, and I was unaware that I could pursue a career in science that was centered primarily around research and education. However, this changed as I studied under numerous professors who opened my eyes to the world of scientific research, especially Dr Mark Peifer, who gave me the opportunity to participate in research for the first time and helped shape me into the scientist I am today.

Who are your role models in science? Why?

E.M.: I am inspired by Dr Beth Brainerd and Dr Sharon Swartz, both professors in the Department of Ecology, Evolution and Organismal Biology at Brown University. They have made strong impacts on the advancement of knowledge and are leaders in their respective fields. I am most inspired by the excitement they exude when discussing their own and others' research questions. Both Dr Brainerd and Dr Swartz have welcomed me into the department over the past year, and I am lucky to have the chance to discuss my ideas with them, as they are both serving on my thesis committee. Importantly, they have challenged me to rethink my ideas about my work. I look forward to working with them throughout my PhD.

N.G.: I am particularly inspired by women in science, especially Katherine Johnson, Barbara McClintock, Jennifer Doudna and Emmanuelle Charpentier, who were/are pioneers in their respective fields despite facing many obstacles because of their gender. Both Johnson and McClintock were scientists at a time when women were frequently overlooked, underestimated, undervalued and openly discriminated against. Yet both women went on to make foundational contributions to the fields of astrophysics and genetics, respectively. Additionally, my own scientific endeavors in molecular biology (and the findings of this paper) would not have been possible without the unparalleled work of Doudna and Charpentier in discovering a role for CRISPR-Cas9 as a programmable tool for genome editing.

What's next for you?

E.M.: I am a second-year PhD student, so my most pressing next step is passing my qualification/candidacy exam! I look forward to developing plans for my thesis proposal and seeing projects come together that have been on my mind over the past year. My future goals are to teach at a small university that equally values teaching and research. I look forward to one day sharing my research interests with young students like myself, and I hope to inspire a next generation of scientists the way I have been inspired.

N.G.: I am currently a first-year PhD student in the Biochemistry, Cellular and Molecular Biology Graduate Program at Johns Hopkins University School of Medicine. As such, my next steps are to finish rotations, complete my coursework and find a thesis lab. I am really excited for the coming months, during which I will get the opportunity to explore different scientific disciplines, learn new techniques/model systems, find a new lab home and (possibly) make some novel discoveries. Following graduate school, I hope to pursue a career in academia.

Tell us something interesting about yourself that wouldn't be on your CV

E.M.: One of my favorite things is to identify species of plants and birds. My background is in neither of those fields, but with apps like iNaturalist and Merlin, I can learn so much about my surroundings with the click of a button.