First person – Olivera Mitevska

Olivera Mitevska

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

During mitosis, microtubules attach to structures on chromosomes called kinetochores; this process enables accurate chromosome segregation. When microtubules are unattached, the outermost kinetochore region, called the corona, expands and forms crescents that can encircle the entire chromosome pair; however, once accurate microtubule attachments form, the corona is stripped down along microtubules in part by the molecular motor dynein. This enables the cell to progress through mitosis by silencing a signal (checkpoint) that prevents premature anaphase entry. Moreover, the protein Lis1 is a conserved dynein regulator that localizes to the corona. In our paper, we find that cells depleted of Lis1 cannot disassemble the corona properly and fail to exit metaphase in time due to persistent checkpoint activation. Moreover, we also find that cells depleted of Lis1 have more than twice the number of errors in anaphase. We demonstrate that an interaction between Lis1 and dynein is required to localize both molecules to the kinetochore and that kinetochore-bound Lis1 can limit erroneous microtubule attachment. However, cytoplasmic Lis1 (which has severely diminished dynein-binding capabilities) can drive proper corona disassembly.

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

The project built upon my supervisor Dr Auckland's postdoctoral work, which found that dynein-driven stripping is antagonized by the corona components CENP-F and Nde1, which form part of the CENP-F–Nde1–Ndel1–Lis1 (FNNL) complex. Thus, given the emerging evidence of Lis1 as a dynein activator and its association with the FNNL complex, when we first looked at Lis1 kinetochore localization, we initially performed experiments on siNdel1 and siNde1-treated cells; however, the data from these experiments proved too complex and beyond the scope of our project. Indeed, because of the multitude of events happening at the kinetochore and the large number of functionally distinct molecules involved, attributing certain processes to specific protein–protein interactions can be challenging. Moreover, because our proteins of interest have broad roles within the cell, we had to think creatively to design experiments with proper controls that considered alternative explanations. For example, one of our control measures was to first test whether Lis1 depletion could perturb the formation and/or maturation of end-on microtubule attachments before exploring other possibilities. Another challenge came with the interpretation of our data, as there were often multiple explanations for the same phenomena. For instance, the processes by which dynein promotes the formation and stabilization of mature bi-oriented microtubule attachments could be disrupted by our separation-of-function mutant Lis1-5A in several ways; however, based on our data we've come to favour some possibilities more than others. We also had to factor in a finding from a publication that came out during the preparation of our manuscript – that human Lis1 has additional dynein-binding sites on the stalk. Beyond that, the commercial RNAi primers that were tested did not work, so we used primers designed by Dr Auckland. Finally, a personal challenge of mine was to get up to date with the latest discoveries in the kinetochore field, as it was my first time working on this topic!

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

I remember going into the lab to find Phil, my supervisor, and Lydia, one of the co-authors, talking about how our mutant Lis1-5A had entirely cytoplasmic localization. We expected that the Lis1–dynein interaction would be important for localizing dynein to the kinetochore but didn't think it would go both ways! I was both confused and quite excited by this. I experienced similar feelings when we observed that both tagRFP-Lis1- and tagRFP-Lis1-5A-transfected cells had restored the removal of corona proteins. We had to pause and rethink our line of reasoning – did Lis1 contribute to corona disassembly independently of dynein? I think this goes to show the importance of keeping an open mind when approaching new experiments and always thinking critically. It was also very interesting to see how Lis1 depletion can more than double the rate of anaphase errors, and further interpreting this result was truly engaging. As far as eureka moments go, I would have to say that for me, interpreting the results has been less spontaneous and more gradual; each time I looked at the data, I understood it a little bit better and came that much closer to a logical explanation.

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

In the Auckland lab, we strive to keep updated on developments in the kinetochore field and the wider mitosis community. Journal of Cell Science stands out in its ability to curate science that is both well executed and highly relevant to the lab's work. In fact, JCS is one of the journals that we routinely check for new publications! Moreover, Phil (my project supervisor) had previously published in JCS, so we were already acquainted with the journal's quick and orderly submission and peer-review process. I feel incredibly lucky that my first paper was accepted for publication in JCS and I'm looking forward to reading the other exciting papers that JCS has to offer.

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

I am very grateful that I had the opportunity to work in the Auckland lab because Dr Auckland is an outstanding mentor. Throughout the project, I was always encouraged to have my own ideas and to work at a level of independence that I think is rare for undergraduate fellowships. He has also helped me tremendously with my science communication skills and has given me great career advice. I would also like to thank Prof. Eggert Ulrike and Prof. Jody Rosenblatt (Randall Centre, King's College London, UK), Prof. Andrew McAinsh (University of Warrick, UK) and Prof. Geert Kops (Hubrecht Institute, Netherlands) for sharing resources and/or guidance in the preparation of this manuscript. Furthermore, I'd like to thank Prof. Gordana Dimeska and Dr Slobodan Tofiloski (Faculty of Natural Sciences and Mathematics, North Macedonia), Dino Atanasov (Genlight LLC, North Macedonia), and my mentors at Petnica Science Centre, Serbia, for giving me the resources and guidance to explore science as a high school student and develop the skills for my fellowship at the Auckland lab.

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?

Throughout high school, I had the privilege of attending four seminars at the Petnica Science Centre, Serbia. These experiences introduced me to the basics of scientific reasoning and gave me the opportunity to learn in a practical lab setting. I later decided to look for opportunities in my home country, North Macedonia, and was lucky to be hosted in Dr Gordana Dimeska's genetics lab at the Ss. Cyril and Methodius University in Skopje, where I conducted a simple project to measure the genotoxicity of the largest river in the country. Around this time, I also started reading books about philosophy of science and was instantly intrigued. It has always been my dream to help people, and through immersing myself in the ways of scientific thinking I became convinced that the best way for me to do that is to spend my life trying to push the boundaries of scientific understanding. I became involved in this project through an undergraduate research fellowship offered by my university King's College London, where I'm currently in my third year on the MSci Pharmacology with Professional Placement Year course. To me, science represents the ultimate amalgamation of disciplines – from logic and maths to art and sociology – and for that I find it incredibly fascinating.

Who are your role models in science? Why?

Growing up, I was in awe of Jane Goodall and Marie Curie, who made astonishing discoveries despite facing numerous obstacles. More contemporary role models for me would be Dr Jennifer Doudna and Dr George Church, whose work in genetics and limitless mentality I find remarkable. Their stories motivated me to explore the possibility of pursuing a career in science. Moreover, my mentors have in many ways been my role models, as I have been very fortunate to have good mentors! My greatest role model in science, however, is my mother, who has been working in science for the past 30 years.

What's next for you?

Over the next year, I will be researching circadian biology in the central nervous system as part of a professional placement at the Cader lab, Nuffield Department of Clinical Neurosciences, University of Oxford. The circadian rhythm regulates physiological processes with a rhythmic pattern of ∼24 h and wields vast influence over health and disease. Here, I will be focusing on elements of the molecular clock, their expression pattern and functionality in several brain regions of interest. Following that, I will return to London to finalize my MSci degree. I'm not entirely sure what will happen afterwards, but I know that in the long-term I want to build a career in biomedical research. I would particularly like to work on translational research, helping develop treatments all the way from bench to bedside.

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

I love cooking and can spend hours perfecting a recipe! I also like to spend time in nature, go to gigs/concerts, visit museums and exhibitions, and going thrifting.