Tag Archives: Titin

My Summer Experience Observing Changes to Sarcomeres with Titin Regulation

Muscles are comprised of repeating units called sarcomeres, which can be seen as bands in myofibrils. Prior research has shown that the consistency of the lengths of these bands correlates to muscle strength – the more consistent the bands, the stronger the muscle. My project focuses on the relationship between the consistency of these sarcomeres and titin, the longest known protein which is proposed to maintain sarcomere length. Our lab developed an inducible skeletal muscle specific Bmal1 knockout mouse that demonstrates a to a longer titin isoform. By altering titin and comparing the variance of lengths of these sarcomeres to a control group, I hope to demonstrate a correlation between sarcomere length and the expression of titin. My project takes a histological approach to study changes in sarcomere length and A-band centrality in the skeletal muscle by staining for alpha actinin and myosin. These images are overlaid and peak fluorescence is measured. Knowledge from this experiment would contribute to knowledge about titin and could eventually lead to more targeted drugs or therapies. Results of my experiment thus far indicate that there is a trend toward a longer sarcomere length and no significant change in variance, but there are still other stains and trials I would like to do to strengthen my findings. If my hypothesis is supported then my project would support the assertion that titin has a structural role in the sarcomere.

The myosin stain allowed me to see if there were any differences in the location in the A-band in relation to the Z-line.

The alpha actinin stain labels the z-lines, allowing the sarcomere to be visualized and measured.

Realities of Research

Research in Dr. Esser’s lab was very different from what I had anticipated. Generally, people think of scientific research as solitary, but what I have found is there is a lot of collaboration in academic research. When we encountered an error in the RNA sequencing program of the model, and we reached out to other people at the university in order to help understand and correct this error. Additionally, we borrowed equipment such as the cryostat (a machine used to cut muscle at cold temperatures) and microscopes from other labs. Lab meetings were also a useful part of my experience because they allowed for brainstorming when I faced challenges. In addition to the RNA sequencing obstacle, there was also a problem with a control antibody stain because there was fluorescence in an unexpected region. After discussing the problem, we came up with the hypothesis that the secondary stain for one of the regions was attaching to the conjugated antibody of another region, which was why we were seeing the unexpected fluorescence. To correct this, I applied the antibodies in two steps instead of combining them. All of these obstacles were easier to overcome because I had people around to assist me, an invaluable asset that I did not expect.

Day-to-Day of a Scientist

I look forward to coming in to the lab because I like the challenge of overcoming the unforeseen obstacles. Designing ways to solve problems is definitely my favorite part of working in a lab. The production of ideas about how an experiment or procedure should work and testing the methods is exciting. While these obstacles can be a nice challenge, they can also become tedious. The problem with the RNA sequencing was especially frustrating because it was out of our hands. Despite these complications, my research was overall very enjoyable. I am very appreciative of the APS for allowing me to pursue my interests this summer, and would encourage others to do a program because the experience was very rewarding.

Joseph Mijares, also known as Robby, is a junior at the University of Florida in Gainesville, FL. He is grateful to be a 2017 Undergraduate Summer Research Fellow in Dr. Karyn Esser’s Lab at the University of Florida.  Robby plans on applying to several MD and MD-PhD programs after his junior year, with hopes of attending after graduation.
Atomic Force Microscopy on Titin, the Mega Protein

This summer in Flagstaff, Arizona at Northern Arizona University (NAU) I worked with Dr. Samrat Dutta in the Nishikawa Lab for biomechanics performing atomic force microscopy (AFM) on the N2A region of titin molecules at different pulling speeds in both the presence and absence of calcium. Titin is found in vertebrate muscles and is the largest known protein molecule (Nishikawa et. al, 2011). Currently, we know that titin functions in passive muscle movement. However, it may provide an important addition to our current understanding of both active and passive muscle function (Nishikawa et. al, 2011). Understanding titin isn’t just revolutionary for muscle theory, Nishikawa’s lab is applying this new information to improve prosthetics. AFM is a non-conventional type of microscope (shown below) that allows us to record the stiffness and stability of biomolecules such as titin by pulling on its spring-like domains. The titin is chemically attached to a surface and the AFM traverses that surface and records changes in its topography using a laser. This experiment allows us to predict the behavior of titin and its contribution to muscle force under different conditions.

Working with AFM has been a steep learning curve for me. AFM wasn’t a process that I was at all familiar with before this summer. With guidance from Dr. Dutta over the course of these 10 weeks I’ve learned about the chemistry, function, and potential of AFM. Unfortunately, we received low usable data yields and this may have been a result of the protein unfolding before the experiment began. As a result, there was a lot of tweaking of our methods to gather a larger set of more accurate data. The analysis of our data afforded me an opportunity to learn physics and chemistry beyond the scope of my university classes. However, we have not yet completed the analysis of our data. I look forward to seeing the outcome of our experiment and contribute to the ever-growing data on titin. Hopefully, my research will answer how much force titin can contribute and in comparison to previous works, does the N2A region of titin react differently than other regions.

What was it like working in the lab?

Dr. Kiisa Nishikawa’s lab group is filled with scientists doing various projects in all different disciplines of the muscle physiology field. Throughout my time at NAU, I had the opportunity to network and learn from all different kinds of people such as postdocs, graduate students, and full professors. They all guided me through my new environment at NAU and supplied me with both professional and scientific knowledge from their different disciplines. Working in a collaborative group meant having support during disappointing moments and always having someone to run ideas by. When the experiment wasn’t producing the amount of data we expected, a team of graduate students and postdocs helped my mentor and I brainstorm possible causes and solutions. This brainstorming session was how we determined that dialysis may be a useful alternative to our previous protein purification method. Working with a large team also means that you must share resources and space which, can make things more difficult. Overall, this summer was an invaluable experience in many aspects and it wouldn’t have been possible without the American Physiological Society (APS). I want to thank APS for allowing me and so many other undergraduates the opportunity to contribute to different fields of research.


  1. Nishikawa, K. C., et al. Is Titin a ‘Winding Filament’? A New Twist on Muscle Contraction. Proceedings of the Royal Society B: Biological Sciences 279(1730), 981–90, 2011.
Blair Thompson is studying biology at Scripps College in Claremont, California. She is a 2017 fellowship recipient of Integrative Organismal Systems Physiology (IOSP) funded by the APS and a grant from the National Science Foundation Integrative Organismal Systems (IOS). She worked with Dr. Samrat Dutta and Dr. Jenna Monroy at Northern Arizona University this summer. After graduation, she plans to attend medical school and become a physician.
Understanding Muscle Force During Cyclic Movements: Does Titin Play a Role?

During cyclic every day movements, such as running, jumping, and walking, our muscles go through cycles of shortening and stretching. While there has been extensive research on muscle function for the last 50 years, there is no current muscle model that can accurately predict natural movements. For example, when active muscle is stretched, it produces more force than expected based on current theories of muscle contraction. Likewise, when active muscle shortens, it produces less force than predicted by current theories. For years, scientists have been measuring properties of muscles under highly controlled conditions. The classic force-velocity relationship shows that force generated by a muscle is inversely related to the velocity of the shortening. However, this relationship changes during natural, more life-like movements. Recent work suggests that for a given velocity, muscle force is higher during cyclic contractions than the traditional force-velocity relationship. My research investigates the role of the elastic protein titin in the force-velocity relationship measured under different conditions. Using a mouse model with a mutation in titin, I conducted in vitro muscle experiments to compare the force-velocity relationship in cyclic and controlled (isotonic) conditions. Hopefully, my results will shed light on titin’s role as a spring in active muscle. If titin truly does store energy like a spring, this could account for the extra force and lack of force in the stretch-shortening cycles. This research will allow us to better understand movement on a whole organism scale, which can prove quite useful in prosthetic design and bioengineering, for example.

Much like the active muscle, doing research in a lab goes through cycles, except instead of stretch-shortening cycles, it is periods of challenge and reward. Some days, you go into lab, collect great data, and leave feeling utterly fulfilled. However, other days, you go into lab and it seems as though you spent your entire day trouble-shooting. Mainly though, our experiments worked and we were able to collect useable data. We have yet to fully analyze our results, but preliminary results seem to support our expectations.

In general, I have found my lab group experience to be very similar to my experience with playing college soccer. Both activities involve a group of people working toward a common goal. While in soccer, your team is working together to win, in the lab, there are many scientists working together to uncover a truth. Collecting and analyzing data is a collaborative effort and, to me, that was the best part of summer research. Working as part of a lab team allows you the opportunity to constantly learn and build off of others. It teaches you to adapt, be open to new ideas, and to use your time efficiently. The worst part of day-to-day life in the lab, is that sometimes data collection does not go as planned and you need to figure out what went wrong.  However, this aspect doesn’t seem so bad when you have your lab team to help brainstorm.

Overall, my time in the lab has been an incredible experience. It has helped me grow as both an individual and as a scientist and has stimulated my interest in future research opportunities. It is an experience I would highly recommend to other undergraduate students!

Lindsay Piwinski attends Pitzer College in Claremont, CA. She is a 2017 Undergraduate Summer Research Fellow (UGSRF) doing research with Drs. Jenna Monroy (Pitzer College) and Kiisa Nishikawa (Northern Arizona University, Flagstaff, AZ). She hopes to attend graduate school in the future and continue pursuing research.