First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Ryan Hart is first author on ‘ Myosin II tension sensors visualize force generation within the actin cytoskeleton in living cells’, published in JCS. Ryan conducted the research described in this article while a Research Technician in Indra Chandrasekar's lab at Sanford Research, Sioux Falls, USA. He is now a PhD student in the lab of Mark Huising at University of California, Davis (UC Davis), USA. He is interested in mechanisms of actin cytoskeletal rearrangement in response to inhibitory GPCR activation.

Ryan Hart

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

Much like how our bodies use muscles to move, individual cells require a much smaller ‘muscle’ referred to as non-muscle myosin II B (NMIIB) to facilitate cellular movement and maintain their shape when stimulated. When a cell moves, NMIIB bound to its actin cell skeleton (called a cytoskeleton) contracts and stretches, exerting force. To measure these forces, we inserted a DNA sequence encoding the ‘Tension Sensing Module’ (TSMod) into a specific region of the DNA sequence for NMIIB. This results in an NMIIB protein containing the TSMod within its tail region, where NMIIB experiences significant force during contraction. The TSMod, originally developed in Dr Martin Schwartz's lab, functions by tethering two fluorescent proteins between a spider silk protein ‘spring’. As these fluorescent proteins move while the spring expands and retracts, changes in force can be tracked be viewing the fluorescent signal under a microscope. We were thrilled to see that the NMIIB TSMod functioned properly, even in cells where the native NMIIB protein had been removed and replaced by the new NMIIB TSMod construct. To further validate this tool, we observed changes in the measured forces when either NMIIB or the TSMod function was independently disrupted. This confirmed that the changes in force were in fact a result of the NMIIB TSMod protein behaving in a similar manner to NMIIB in normal healthy cells. Collectively, this paper describes how we generated and developed a new tool to measure a process that has been very difficult to quantify – the forces generated along the actin cytoskeleton of a living cell.

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

At the beginning of this project, the most difficult part was identifying a region of the NMIIB sequence suitable for inserting the TSMod sequence. We overcame this challenge by reviewing seminal work on NMIIB structure and function by Robert Adelstein and Jim Sellers, which helped us identify a region of the tail domain that would not interfere with ATPase activity, actin binding or bipolar filament formation. Once the target region was identified, introducing the TSMod presented another challenge due to the close similarity between the sequences of the two fluorescent proteins. Ultimately, I was able to complete the construct, and this experience in cloning tricky plasmids and sequences will help me immensely throughout the rest of my scientific career. The final challenge was completing the manuscript after I had moved on to my graduate work at UC Davis. This was resolved through consistent communication and teamwork across numerous investigators, with my current Principal Investigator allowing me to use my current lab's imaging set up to complete the revision experiments.

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

The eureka moment for this project occurred over the span of a week. After struggling with the construct generation, we finally received sequencing results confirming that we had successfully generated the NMIIB TSMod as designed in silico. I quickly moved to transfect this positive clone into HEK293 cells, and we were very excited to see that the structure of the fluorescent protein was indistinguishable from an NMIIB overexpression vector (there was a lot of high fiving going around). To this day, seeing the construct in the cells for the first time remains one of my favorite moments as a researcher. The excitement only grew a month later when we tested the construct using a fluorescence-lifetime imaging microscopy (FLIM) setup and observed changes in the Förster resonance energy transfer (FRET) ratio, confirming that the sensor was working as designed.

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

Journal of Cell Science is a high-quality, well-established journal with an excellent reputation in the field of cell biology. When searching for a home for this paper, we were naturally drawn to Journal of Cell Science, and I was very happy with the rigorous yet fair review process and timeline path to publication. I am thrilled to have my work published in Journal of Cell Science.

A HEK293 cell expressing the fluorescent sensor NMIIB TSMod. This was one of the original images taken of the construct in living cells in May 2016.

A HEK293 cell expressing the fluorescent sensor NMIIB TSMod. This was one of the original images taken of the construct in living cells in May 2016.

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

I have been incredibly lucky in my scientific career to have mentors whose guidance extends well beyond the bench. When completing my master's degree, I met my research mentor, Dr Neal Chamberlain. Dr Chamberlain went out of his way not only to discuss our science but also to model how to balance both professional and personal responsibilities. It was during this time that my love for research and the pursuit of making sense of observations was truly ignited. I became enamored with research and the pursuit of the unknown, and I have worked in academic research labs ever since, with the goal of completing my doctorate later this year.

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?

My work during my master's degree solidified my love of research. Since then, I have sought out positions in numerous academic research labs across the USA while supporting my partner through their medical training and work. This opportunity to do what I love, combined with the unique experiences and learning opportunities at each stop, has continually motivated me to become a better researcher. In terms of interesting moments, beyond observing the NMIIB TSMod functioning as expected for the first time, many of the observations I have made during my graduate work stand out as some of my favorites. I joined another imaging intensive laboratory, and observing real-time changes in the actin cytoskeleton in primary tissues has consistently been one of the most interesting aspects of my research.

Who are your role models in science? Why?

I have had four important mentors at each stage of my journey: Dr Chamberlain at A.T. Still University, Dr F. Anthony Willyerd at Phoenix Children's Hospital, Dr Indra Chandrasekar at Sanford Research and Dr Mark Huising at UC Davis. Each of these mentors has taught me that research, while a serious and often intense endeavor, can also be fun. They have all provided valuable insights into balancing research responsibilities with family life and how to prioritize to find satisfaction on both fronts. I consider each of my previous mentors to be my main role models in science, and I hope to one day emulate the same strengths they demonstrated daily.

What's next for you?

I am currently wrapping up my doctoral research and beginning to draft the manuscripts describing my thesis research. After graduation next spring, I hope to pursue a post-doctoral position that will further improve my scientific skill set while also preparing me for a career as an independent investigator at an academic institution.

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

I have an amazing family – my spouse is an incredible pediatrician, and our two young children provide new (and exciting) challenges every day. I cannot say which role in my life is the most difficult but balancing all these responsibilities together is very fulfilling.

Ryan Hart’s contact details: UC Davis, One Shields Avenue, Briggs Hall 196, Davis, CA 95616, USA.

E-mail: [email protected]

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et al. 
(
2024
).
Myosin II tension sensors visualize force generation within the actin cytoskeleton in living cells
.
J. Cell Sci.
137
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jcs262281
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