Bio Patrika interviews Mr. Kesarwani on his thoughts about “live-cell tyrosination sensor”

Mr. Shubham Kesarwani’s interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Mr. Kesarwani is currently a PhD student in the lab of Dr. Minhajuddin Sirajuddin at Institute for Stem Cell Research and Regenerative Medicine (inStem), NCBS, Bangalore. He published a paper titled “Genetically encoded live-cell sensor for tyrosinated microtubules” as a first author in Journal of Cell Biology (2020).

How would you explain your paper’s key results to the non-scientific community?

Our recent publication in the Journal of Cell Biology, features a technical advancement to study tyrosinated microtubules with a live cell sensor. Microtubules are cellular cytoskeletal elements which are assembled from the polymerisation of alpha and beta tubulin proteins. These are hollow tube-like structures and forms a dense network of widespread filaments inside the cell. Microtubules are dynamic polymer which undergoes spontaneous polymerisation and depolymerisation inside the cell. They play a pivotal role in various cellular activities, which include (but not limited to) maintaining cell shape, intracellular transport of molecules/proteins, cell division, and cellular movements. Several chemical alterations to the tubulins in the form of addition or removal of certain reactive chemical groups or amino acids can regulate microtubule functions inside the cell and are broadly known as posttranslational modifications (PTMs) of microtubules. Detyrosination and tyrosination of tubulin are well known reversible microtubule PTM. The terminal tyrosine of the alpha tubulin can be enzymatically removed to generate detyrosinated microtubules and this process can also be reversed by a separate enzyme which restore tyrosine back at the terminal of alpha tubulin. Generation of both tyrosinated and detyrosinated microtubules is highly regulated process inside the cell which results in unique interaction of certain microtubule-associated proteins to the microtubule to control cellular functions.

Figure 1. Microtubule are polymerised by alpha and beta tubulin dimers. The terminal tyrosine (Y) of alpha tubulin is the site for reversible posttranslational modification (PTM). The terminal tyrosine can be removed by an enzyme to generate detyrosinated microtubule. This process can also be reversed to restore the tyrosine back on alpha tubulin.

Abnormal regulation of tyrosination or detyrosination process has resulted in various disease pathologies of brain, heart and several forms of cancers. The standard way of studying these modifications inside a cell is based on labelling these microtubule populations with PTM specific antibodies in the fixed cell. Although this method has helped us understand the organization of modified microtubules inside different cell types but we still don’t understand when these modifications occur and how they regulate microtubule functions inside a cell. To overcome this challenge, we have for the the first time generated a live cell sensor against tyrosinated microtubules. This sensor when fused with a suitable fluorescent protein, can be used to specifically detect and follow the real-time changes of tyrosinated microtubule inside the cell. We further validated the sensor’s robustness for live-cell studies and found that the binding of this sensor to the microtubules doesn’t perturb any microtubule or cellular functions. We have successfully utilized this sensor to track real-time microtubule dynamics in a live cell and studied the effect of several anti-cancer drugs (nocodazole, colchicine and vincristine) on microtubule. We show that each of this drug has a distinct mechanism by which they depolymerise microtubule and prevent the cell from dividing.

Figure 2. The represented image marks the cellular microtubule with the tyrosination sensor.


“[…] sensor is superior to all the existing microtubule markers for live cell study […]”

What are the possible consequences of these findings for your research area?

The tyrosination sensor has a huge potential to study the posttranslational modifications of microtubules and its associated enzymes in live cells. Currently, the tyrosination sensor can be used to understand the real-time mechanism of any new anti-cancer therapeutic drug which targets microtubules. Since most of the microtubules in dividing cells are tyrosinated, therefore, the tyrosination sensor can be used to study organization and dynamics of microtubule in these cells. This sensor is superior to all the existing microtubule markers for live cell study as they all perturb microtubule dynamics and functions, which is not the case with our tyrosination sensor.

What was the exciting moment (eureka moment) during your research?

There were many exciting moments during my research, but I would say the eureka moment in my research came when I could detect microtubules in live cells using my tyrosination sensor. It took me some time before I could figure out the suitable fluorescent proteins such as the blue fluorescent protein (TagBFP) or the red fluorescent protein (TagRFP-T) which when fused with tyrosination sensor can be used to detect tyrosinated microtubule inside a cell. This was an important moment as most of the commonly used fluorescent proteins (GFP, mCherry) were not ideal for live cell imaging of tyrosinated microtubules. Finally, I could see the microtubules under a microscope with my tyrosination sensor using TagBFP and TagRFP-T fluorescent protein fusions.

What do you hope to do next?

Our next goal is to extend this methodology to isolate live-cell sensors for the other microtubule PTMs such as detyrosination, glutamylation and acetylation modifications. These sensors will aid to study the onset and role of these PTMs in regulating microtubule functions during different cellular processes.

Where do you seek scientific inspiration?

My main scientific inspiration comes from my research goals and from my efforts in achieving them. My perseverance and patience have always paid in good scientific output, which has always inspired me to take a step forward towards new challenges to address my research hypothesis. I also seek motivation from my peers and mentors who guide me through all the odds in my career and their constructive feedback on my research has always improved my logical thinking and made me a better scientist.

How do you intend to help Indian science improve?

In my view, India is a potential country where diverse research is being carried out. My future efforts will contribute to the progress of my country by bringing new technologies and innovations in science. As a research scholar, my focus is always to generate a reproducible scientific output that anyone can replicate in the future. With my current work, I have initiated a new approach to the study of microtubule PTM by contributing with a live cell sensor for tyrosinated microtubule study. I want to take this method forward for my future research endeavors to generate many more microtubule PTM sensors and also inhibitors for the clinically relevant protein targets. A large part of our research is supported by the Government funds with very little industrial involvement. We often face financial problems to afford the essential resources to carry out an ambitious research project. This could be resolved by building a strong collaboration with the experts in the field from both academia and industry. Therefore, our next challenge would be to focus on such research, which not only generates good scientific outputs for publications but also attracts industrial funding to support commercialization and serve as a source for revenue generation.

Reference

Kesarwani S, Lama P, Chandra A, Reddy P P, Jijumon A S, Bodakuntla S, Rao B M, Janke C, Das R, and Sirajuddin M. Genetically Encoded Live-Cell Sensor for Tyrosinated Microtubules. J. Cell Biol, 2020 , 219.

Learn more about Dr. Minhaj Sirajuddin’s lab research interests http://cytoskeleton-lab.org/.

Author introduction and research interests

I was born in Lucknow, Uttar Pradesh. I have obtained my primary education in Lucknow, followed by that I have moved to Delhi for under-graduate degree and then to Varanasi for my post-graduate studies. Currently, I am pursuing Ph.D. under Dr. Minhajuddin Sirajuddin at inStem, Bengaluru. My Ph.D. project aim to understand the post-translational modification mediated regulation of microtubule assembly and functions. My broad research interest is to understand the biomechanics of cells regulated by various cytoskeleton proteins.

Email: shubhamk@instem.res.in

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