First person – Prashun Acharya Free

Prashun Acharya

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

Our ears contain tiny hair-like structures called stereocilia, which detect sound vibrations and allow us to hear. These structures rely on a special motor protein known as Myo7A, which is essential for keeping them organized and functioning properly. Mutations in Myo7A can lead to various forms of hearing loss, including a dominant form known as autosomal dominant DFNA11 deafness. Interestingly, some DFNA11 variants cause Myo7A to become overly active instead of impairing its function. This excessive activity results in the mistargeting of Myo7A to incorrect locations within the hair cells of the inner ear. Normally, Myo7A functions like a car with brakes – moving only when necessary. These DFNA11 mutations remove the brakes, causing the protein to move continuously even when it shouldn't, which disrupts its normal function. Using a kidney epithelial cell culture model, we studied Myo7A and discovered that the fifth IQ motif, part of the lever arm of the protein, regulates its activity. We found that mutations in the lever arm can significantly alter Myo7A function. Moreover, the resulting overactivity of Myo7A is not limited to one specific part of the protein – mutations in various areas, including the motor domain, can all lead to excessive activity. This finding helps explain why DFNA11 is considered a dominant genetic disorder; even when an individual has one normal copy of the gene and one variant copy, the overactive Myo7A produced from the variant gene can still cause hearing loss. Overall, these insights provide scientists with a better understanding of how these variants lead to hearing loss and could help in developing treatments in the future.

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

Yes, we definitely encountered a few hurdles along the way. One of the main challenges was the lack of a suitable cell culture model for studying Myo7A function inside cells. We initially used CACO-2BBE enterocytes, but this model had some significant limitations, including that they're slow to grow and properly organize mature microvilli, which are crucial for our research. We needed something faster and more efficient. So, we decided to switch to LLC-PK1-CL4 proximal tubule kidney epithelial cells. These CL4 cells were a game-changer because they polarize much faster – within just 3 to 4 days – and they form well-developed apical microvilli. They also provide a ‘surrogate’ actin track that is comparable to the actin core found in inner ear stereocilia. Additionally, they naturally express CALML4, a Myo7A myosin light chain our lab previously discovered, which is only expressed in epithelial cells. Myo7A is also natively expressed in the proximal tubule cells of kidney tissue, making the LLC-PK1-CL4 cells an excellent model to investigate this motor. Another big challenge was figuring out which mutations could activate Myo7A targeting in epithelial cells. We knew we needed to find mutations that activated Myo7A targeting, but we didn't know exactly where to look. To tackle this, we took a systematic approach. We created libraries of mutations in the lever arm, and then screened them to find the ones that significantly enhanced microvillar targeting. It was a trial-and-error process, but it allowed us to zero in on the mutations that had the most impact. It was time-consuming but ultimately rewarding. By methodically testing each mutation, we were able to identify the ones that made the biggest difference. This approach really helped us move forward and get the results we were looking for.

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

Absolutely! There was one moment that really changed everything. For years, people had tried to express active Myo7A in cultured cells using HeLa and COS cells, but they kept hitting roadblocks. It seemed like these commonly used cells just didn't have the right environment for it. The regulatory mechanisms of myosin are complex, and those cell lines were likely missing key components, such as the right binding partners and actin tracks. The big moment came when we reconstituted the R853C mutation – a known DFNA11 autosomal dominant deafness variant – into Myo7A and expressed it in cultured kidney epithelial cells. That's when things got really interesting. We finally saw Myo7A become active and target to microvilli tips using its motor activity. Myo7A R853C was not just active inside these cells – it was super active. We repeated the experiments multiple times and after seeing the same result, it became clear that we had stumbled upon something really significant, a huge clue that this mutation actually makes Myo7A overly active rather than just disrupting its function. Up until that point, we assumed the DFNA11 deafness was associated with a loss of function, but this finding flipped the story. Instead of a broken protein, we realized the problem was actually an overactive one, meaning the key to treatment might not be restoring function but rather finding a way to slow it down to restore normal regulation. It was one of those moments that reminds you why you do research in the first place.

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

We chose Journal of Cell Science for our paper because it is a leading journal that focuses on significant advancements in cell biology, aligning perfectly with the core themes of our research. JCS is a well-respected journal in the field, ensuring both credibility and visibility. We believe that JCS will provide the right context and audience for our research, facilitating meaningful discussions and advancements in the field.

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

As an international student, adjusting to a new country was quite challenging. I was shell shocked and filled with doubts. First of all, I would like to thank my PI, Dr Crawley, for being a supportive advisor. I would also like to acknowledge our former lab members, Maura Graves and Samaneh Matoo, for their invaluable support and guidance. Initially, I had little-to-no exposure to research skills, but I worked hard to learn from the aforementioned lab members and from the skills taught by Dr Crawley. With their mentorship, I have developed my skills and experienced personal growth.

Beyond the lab, they have provided insights into career paths, networking opportunities and the nuances of navigating academia and industry. I would also like to give a special mention to my friend Apurba Bhattarai, who has provided immense friendship and support throughout my PhD journey.

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?

During my undergraduate years, I was really fascinated by biochemistry and molecular biology and would get excited doing experiments in this area during my undergrad lab work. Although the lab resources available during my Biotechnology undergrad program were quite limited, I was really driven by the applications of biotechnology and the potential to contribute to advancements that improve human health and cure diseases. With this in mind, I pursued my PhD in Cell and Molecular Biology in Dr Crawley's lab. The research skills that I learned in the lab and the discovery that we made about how Myo7A is regulated and how it causes DFNA11 deafness has been another motivation to continue research to find potential treatment for individuals experiencing hearing loss.

Who are your role models in science? Why?

During my PhD research, I considered my PI, Dr Crawley, to be a significant role model. His passion for science is pure and unmatched. His ability to approach problems from multiple angles and maintain curiosity, even in the face of experimental failures, has taught me what it means to be a true scientist. He emphasizes that science is not only about obtaining results but also about asking the right questions. His commitment to curing deafness and to the journey to find potential treatments for individuals with hearing loss serve as powerful motivations for my continued pursuit of science and research.

What's next for you?

I am seeking postdoctoral researcher positions.

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

I love traveling and exploring new places, and I also enjoy playing sports. If I weren't a researcher, I would have pursued a career in sports. I am still open to the possibility of becoming a sports scientist.