Bio Patrika interviews Dr. Gadadhar on his thoughts about “finding novel mechanism behind male infertility”

Dr. Sudarshan Gadadhar’s interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Dr. Gadadhar is currently Senior postdoctoral research fellow in the lab of Prof. Carsten Janke at Institut Curie, PSL Research University, Department of Genome integrity, RNA and Cancer CNRS, France. Sudharshan published a paper titled “Tubulin glycylation controls axonemal dynein activity, flagellar beat and male fertility” as a first author in Science journal (2021). Article featured on Science journal cover page.

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

One of the most essential structural and functional components of every cell in our body is the cytoskeleton. It contains 3 major components, among which are the microtubules (MTs), tiny tubes made up of tubulin proteins. The microtubules once formed, undergo modifications, wherein enzymes add additional amino acids on the surface of the microtubules, a process called posttranslational modification (PTM). MTs have various functions in our cells, among which they play a key role in the function of tiny hair-like structures on the cells of our body, called cilia and flagella, which are rich in MTs. A major example of a flagellum is the sperm tail, which is important for the sperm to swim towards the egg, required for male fertility and hence, sexual reproduction. The flagellum has to beat in an exact and coordinated manner to allow straight-line swimming of sperm, failing which can lead to male infertility.

Figure 1: Tubulin glycylation regulates sperm motility, thus fertility. (A) Sperm tails are rich in proteins, microtubules, which have two key modifications, glutamylation and glycylation. (B) In the absence of glycylation, the sperm cells swim predominantly in circles, as compared to normal sperm cells, which swim in a straight line as they have glycylation. Copyright: Gadadhar S. et al, 2021. Science.

MTs in the flagella contain a specific tubulin modification called glycylation (Fig 1A). Despite being discovered in the early 1990s, very little was known about the function of this modification. In this collaborative study, we took a closer look at the role of this modification in mammalian cilia and flagella function. We established a system where we removed the proteins responsible for bringing about glycylation of microtubules by deleting their DNA sequence. We found that in the absence of glycylation, the sperm tail showed defects in the rhythmic beat pattern. Moreover, very interestingly, we saw that the sperm were swimming mostly in circles (Fig 1B). This caused a reduction in fertility in male mice. To find out why lack of glycylation caused perturbed sperm motility and male subfertility, we imaged the sperm at high resolution using cryo-electron microscopy. We visualized the molecular structure of the flagellum and its molecular motors. The tens of thousands of tiny molecular motors, called dyneins, are responsible for rhythmically bending the microtubules to produce the waves required for sperm movement and steering. Our analysis of the mutant flagella showed that though the flagella was correctly built, the loss of glycylation affected the coordinated activity of the dynein motors. This explains why the sperm had defects in the flagella beat, and thus spinning sperm.

Abnormal sperm cells swim predominantly in circles, as compared to normal sperm cells. Copyright: Gadadhar S. et al, 2021. Science.

For the first time, our study establishes the role of glycylation in controlling sperm movement at the molecular level.

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

For the first time, our study establishes the role of glycylation in controlling sperm movement at the molecular level. Moreover, it is one of the prime examples of how microtubule modifications directly control the function of other proteins in cells. It also provides a novel mechanism that could be responsible for male infertility. Moreover, sperm is just one of the many cells with motile cilia in the body. This shows that tubulin modifications could play vital roles in regulating functions of other motile cilia, like respiratory cilia in the trachea, the motile cilia in the brain ventricle required to flow cerebrospinal fluid and so on. Thus, our work opens the door to a deeper understanding of diseases, such as developmental disorders, cancer, kidney disorders or respiratory and vision disorders.

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

Our lab had established earlier that glycylation might have a role in stabilizing the structure of cilia and flagella (Bosch Grau et al., 2013; Gadadhar et al., 2017). However, we found cells with cilia and flagella intact in our mice with no glycylation. It was also difficult to find stark physiological defects in these mice. All tissues analysed looked okay. But when I was imaging the sperm swimming, along with Luis Alvarez, we saw that the way the flagella were beating was different. Immediately, we felt something was not okay, and it was super interesting for us as we felt we had hit something exciting here. This was confirmed when we saw the sperm swimming in circles. I had goosebumps, as this showed us what happens when glycylation is lost in the sperm.

What do you hope to do next?

A key observation is that glutamylation increases in the absence of glycylation. So, the immediate question is to see if the mice with high levels of glutamylation show similar spinning sperm. Also, we still do not know what happens when glutamylation goes down. Does glycylation increase? And if so, how does this affect cilia and flagella function. And finally, many cells have cilia that do not move but have roles in cellular function and tissue homeostasis. How do tubulin modifications play a role in these functions? Thus, the work has opened various new paradigms to address the roles of tubulin modifications in cilia and flagella function.

Where do you seek scientific inspiration?

From my undergraduate days, I was always fascinated at seeing minute cellular structures under the microscope. I have always been fascinated by how, with the advent of newer technologies, we get more in-depth knowledge of the fascinating molecular processes that happen within our bodies. This drives me to understand these finely regulated processes more intricately. I am also inspired by my great mentors, both my PhD supervisor and my current supervisor, as well as the wonderful collaborators who are highly motivating and push me to find that little extra. Scientific discussions with them always bring out a new angle into the problem and make me look at things differently.

How do you intend to help Indian science improve?

I want to contribute to Indian science by bringing my scientific research to the public, interacting with school students. Apart from this, I would like to train school and college students to perform simple biological experiments. It is essential to inculcate the research mindset at an early age. I would like to come back to India and continue my research as the research facilities in India are well-equipped to carry out cutting edge research. I will establish new methods to study cilia and flagella, from my expertise as a postdoc.

References

Gadadhar, S., Alvarez Viar, G., Hansen, J.N., Gong, A., Kostarev, A., Ialy-Radio, C., Leboucher, S., Whitfield, M., Ziyyat, A., Touré, A., Alvarez, L., Pigino, G., and Janke, C. 2021. Tubulin glycylation controls axonemal dynein activity, flagellar beat and male fertility. Science. 371:eabd4914.

Bosch Grau, M., Gonzalez Curto, G., Rocha, C., Magiera, M.M., Marques Sousa, P., Giordano, T., Spassky, N., and Janke, C. 2013. Tubulin glycylases and glutamylases have distinct functions in stabilization and motility of ependymal cilia. J Cell Biol. 202:441-451.

Gadadhar, S., Dadi, H., Bodakuntla, S., Schnitzler, A., Bieche, I., Rusconi, F., and Janke, C. 2017. Tubulin glycylation controls primary cilia length. J Cell Biol. 216:2701-2713.

Author introduction and research interests

I have done my PhD with Prof. Anjali Karande at the Department of Biochemistry, Indian Institute of Science (IISc) Bangalore. In my PhD, I constructed immunotoxins for targeted cancer therapy using plant toxins. I looked at the efficacy of the immunotoxins, and also established their intracellular trafficking pathway within cells. In my postdoctoral tenure, I have worked on understanding the role of tubulin posttranslational modifications (PTMs) in regulating mammalian cilia function, where I have established that, like the motile cilia in mammals, primary cilia also undergo tubulin glycylation, necessary to stabilize the primary cilia. I have also revealed the first molecular mechanism of how tubulin glycylation regulates sperm flagellar beat, swim patterns and thus, male fertility (Gadadhar S. et al., 2021. Science). In future, I would like to understand the molecular fine-tuning of complex regulatory circuits that are integral to proper cellular functioning and organ development. More importantly, to reveal how disruption of such fine-tuning can lead to deleterious diseases.

Webpage: shorturl.at/boN29

Email: Sudarshan.Gadadhar@curie.fr

Prof. Anjali Karande lab at IISc Bangalore http://biochem.iisc.ernet.in/AAK%20files/anjali%20lab.htm

Prof. Carsten Janke lab at CNRS https://bit.ly/2MWgI5r

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