Bio Patrika interviews Dr. Sridhar on his thoughts about “Understanding fundamental design principles of a cellular load-bearing machine”

Dr. Shreyas Sridhar’s interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Dr. Sridhar was born and bought up in namma Bengaluru. It is here at the Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR) that he completed his MS-PhD dual degree course under the supervision of dynamic mentor, Prof. Kaustuv Sanyal. His research question was to understand the organization and evolutionary adaptation of the kinetochore in the human fungal pathogen Cryptococcus neoformans, an organism that causes fungal meningitis and pneumonia. He obtained his Ph. D. degree in 2020. He is currently a post-doctoral researcher in the laboratory of Prof. Tatsuo Fukagawa at Osaka University, Japan. Here he works to understand how the kinetochore-centromere interface, a key interaction network hub driving accurate chromosome segregation, is organized in vertebrates using a multidisciplinary approach. During his time away from work, he enjoys travelling on two wheels not only to explore places but also to admire nature and unique culture associated with each place. Here, Shreyas talks about his PhD work titled “Bridgin connects the outer kinetochore to centromeric chromatin” published as a first author in Nature Communications (2021).

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

Billions of cells are born each day in an adult human body to replace the dead cells. When a cell divides, in a process called mitosis, the generation of daughter cells with identical genetic information is vital. A complex network of molecular events coordinates to achieve this equal segregation of chromosomes. Dysregulation of these pathways results in newly formed daughter cells with abnormal chromosome content: a hallmark of genetic disorders, cancers, and a leading cause of drug resistance amongst human fungal pathogens. Hence, a detailed understanding of these molecular events is important to find a means of therapeutic interventions.

Chromosomes duplicate at the synthetic (S) phase of the mitotic cell cycle. Duplicated chromosomes are segregate equally by the chromosome segregation machinery at the mitotic (M) phase of the cell cycle. The chromosome segregation is driven by a dynamic spindle shaped structure made up of microtubules that capture one copy of duplicated chromosomes to pull into each daughter cells. Microtubules interact with a chromosome at the centromere DNA-kinetochore protein complex1.

Figure 1. The kinetochore is a load-bearing machine essential for accurate chromosome segregation.

Although the kinetochore structure is integral to propagate spindle forces required to separate duplicated chromosomes, one each towards the opposite spindle poles, many key kinetochore protein subunits seem to have lost across species during evolution. These loss events often involve components of the inner kinetochore that interact with centromere DNA and play critical roles in recruiting the microtubule-binding outer kinetochore. So how does a kinetochore adapt to these lost components of the chromosome segregation machine? Could an understanding of this adaptation aid in identifying the core design principles of this load-bearing machine?

To address this very question, we focused on the highly diverse fungal kingdom. In our recently published study in Nature Communications, we have shed light on the adaptive evolution of the multi-protein kinetochore complex using the medically relevant human fungal pathogen Cryptococcus neoformans2. We describe the recurrent loss of key inner kinetochore proteins amongst the fungi of the phylum Basidiomycota, which encompass economically important mushroom-forming fungi as well as several human and plant pathogens. C. neoformans is one such human fungal pathogen causing the often fatal cryptococcal meningitis and pneumonia, resulting in ~200,000 deaths yearly worldwide3.

Utilizing a biochemical screen in C. neoformans, we identified several novel components of the chromosome segregation machinery, including a previously unknown kinetochore protein that we named “bridgin”. Our work suggests that bridgin is recruited to the outer kinetochore and its recruitment helps to connect the outer kinetochore to the underlying centromeric DNA. This connecting function of bridgin was found to be critical for accurate chromosome segregation in C. neoformans, which lost several evolutionarily conserved key inner kinetochore proteins present in organisms ranging from yeast to humans.

Further to our surprise, we identified bridgin as ancestrally related to the human tumor cell proliferating marker Ki67. Thus, in the event of functional divergence, possibly driven by the loss of kinetochore proteins, bridgin may have gained a kinetochore function. Critical components, such as bridgin and other identified proteins that facilitate accurate chromosome segregation, may be utilized as attractive therapeutic targets for anti-fungal development.

Figure 2. Cell-cycle dynamics of bridgin (cyan) during C. neoformans mitosis. Histone H4 (magenta), a chromatin-associated protein, marks the segregating genetic material.

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

Centromere DNA sequences as well as the subunit composition of kinetochores that form on it are rapidly evolving across species. Are the rapid changes in centromere DNA sequence force kinetochore subunits to evolve? How a kinetochore adapts and how it is driven to these changes are key unanswered questions. In our study, through the identification of a novel kinetochore protein, bridgin, we reveal an unconventional mechanism connecting the outer kinetochore to centromeric DNA. This bridge may reinforce the outer kinetochore resulting in effective force propagation and facilitating accurate chromosome segregation. Our findings shed significant light on the functional adaptation of the kinetochore through evolution and predict factors that may help drive these innovations. This work identifies bridgin, that shares limited homology with its putative human relative and can be used as a molecular target to develop antifungals.

“This work identifies bridgin, that shares limited homology with its putative human relative and can be used as a molecular target to develop antifungals”

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

As a researcher, discovering a novel player and mechanism within a critical cellular pathway is what I aspired to achieve during my Ph. D. This study has enabled me to realize a lot of that. There are two key highlights that I can describe as my eureka moments.

The first was the microscopic visualization of bridgin at the kinetochore. We labelled many candidate proteins, obtained after our biochemical screen, within cells with a green fluorescent protein (GFP). After screening several candidates that did not localize exclusively to the kinetochore, I observed bridgin to localize exclusively to the kinetochore in a cell cycle phase-dependent manner. It was a thrilling moment, and I knew we had a good lead. The second event was the finding that bridgin, an outer kinetochore protein, can interact with the distant centromeric DNA. After our initial discovery of bridgin as a protein recruited to the outer kinetochore, we attempted to address its function towards mediating accurate chromosome segregation. Being an outer kinetochore protein, located proximal to spindle microtubules than centromeric DNA, we expected a corresponding role in spindle interaction. But to my surprise, during a visit to our collaborator’s laboratory in Japan, I found that a basic stretch within the protein interacted with DNA. This observation in light of the loss of critical DNA interacting inner kinetochore proteins in C. neoformans helps shape our hypothesis for bridgin function.

What do you hope to do next?

My interest in further evaluating how the kinetochore structure is organized and assembled and understanding the consequences of its evolutionary alterations have brought me to the lab of Prof. Tatsuo Fukagawa at Osaka University. Using in vivo vertebrate cell culture systems and in vitro biochemical and structural studies, we plan to address some of these questions. Since I just began with my post-doctoral studies here, I am hopeful of conducting exciting science, looking forward to interesting results, and continue learning

Where do you seek scientific inspiration?

I was fascinated with the world around me ever since I was young, be it marine biology, archaeology, etc. Back then, scientists on television were a significant source of inspiration. Currently, I take inspiration from peers and mentors, reading general scientific articles and literature or listening to talks. I am often amazed by fascinating scientific discoveries and their elegant approaches taken to tackle challenging issues. This usually motivates me to look at my questions and ask how I can better address them or contribute to my field’s understanding. It’s amazing how complicated a rather simple-looking cell is!

How do you intend to help Indian science improve?

I’m happy I chose the career path of a researcher and would be delighted to assist other aspirants with any aspect they may have doubts about. I sincerely hope that the science we have performed excites and motivates the talented student community in India. With science taking great strides in India, I would be thrilled to return to India at some point in the future. I am hopeful to have an opportunity to work with the research community back home and be grateful if I can share some of the experiences/ skills learned during my post-doctoral training towards aiding Indian scientific ambitions.

References

  1. Sridhar, S. et al. Centromere and Kinetochore: Essential Components for Chromosome Segregation. Gene Regul. Epigenetics Horm. Signal. 259–288 (2017). doi:10.1002/9783527697274.ch9
  2. Sridhar, S., Hori, T., Nakagawa, R., Fukagawa, T. & Sanyal, K. Bridgin connects the outer kinetochore to centromeric chromatin. Nat. Commun. 12, 146 (2021).
  3. Rajasingham, R. et al. Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis. Lancet Infect. Dis. 17, 873–881 (2017).

Email: shreyas.sridhar5@gmail.com

Learn more about lab of Prof. Kaustuv here https://molecularmycologylab.wixsite.com/kaustuv

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