First person – Md Hashim Reza Free

Md Hashim Reza

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

Budding yeasts are difficult to visualise, owing to their small size, and resolving the subcellular structures within these cells is further limited by the diffraction limit of conventional microscopy techniques. To overcome this, various advanced microscopy techniques (super-resolution microscopy) have been developed to improve resolution. However, these techniques are complex and expensive, and therefore restricted to a few users. Ultrastructure expansion microscopy (U-ExM) provides an alternative, complementary and inexpensive approach, which utilises physical expansion of samples. Once expanded, samples can be studies with an improved resolution, enabling the visualisation of more subcellular structures than with conventional microscopy. Here, we optimise fungal cell wall digestion and demonstrate isotropic four-fold expansion of Candida albicans, a human fungal pathogen. Via snapshots, we study the organellar segregation dynamics during cell division. Using U-ExM, we resolve the distribution of the spindle pole body (SPB) protein Tub4, present between two different layers of SPBs that nucleate microtubules. U-ExM also helps in studying the differences in protein levels at these structures. Furthermore, we show how U-ExM can be utilised to study subcellular organisation in several non-model pathogenic yeasts.

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

We struggled with tubulin staining in Candida albicans. We wanted to validate the SPB-like structures stained with a pan-labelling dye for tubulin staining. We tried various anti-tubulin antibodies, dilutions and incubation times but with little success. Later, we created a strain in which one copy of Tub2 was tagged with GFP at the native locus while the other untagged wild-type copy was left undisturbed. Finally, after a few attempts, one of the anti-GFP antibodies worked, and we could see both nuclear and cytoplasmic microtubules in the expanded C. albicans cells. From optimising U-ExM in C. albicans with help from Hiral Shah, a postdoctoral fellow in Dey's lab, to teaching this technique to some of my colleagues in India, I was fortunate to receive support from all my co-authors – Srijana, Rohit and Hiral – for this work.

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

The first time I saw expanded cells of C. albicans, together with the isotropic expansion of the bud-neck, will always stick with me. I remember sharing some of the first images with my mentor and colleagues working on C. albicans, and one person replied ‘These images look like EM images’. I loved that! Later, looking at multiple images stained with a pan-labelling dye made us realise that SPBs were present in a side-by-side arrangement in C. albicans, with a shorter metaphase spindle. This unique arrangement of SPBs before anaphase onset seems to be a conserved feature in some yeasts.

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

Journal of Cell Science has a broad readership focused on fundamental cell biology and our work on examining subcellular structures across pathogenic fungi seemed perfectly suited to the special issue on Imaging Cell Architecture and Dynamics.

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

I have been blessed to have many people, mentors (my PhD supervisors, the late Dr J. Manjrekar and the late Prof. B. B. Chattoo, and my postdoc supervisors, Prof. K. Sanyal and Dr G. Dey), family and friends, who have shaped my scientific career. Dr Manjrekar, a kind and noble person, was a remarkable scientist who always emphasized work–life balance, and his words “Good science is good, but good people are better” will stick with me forever. I observed the balancing act of doing science and administration from Prof. Chattoo who had a remarkable ability to foresee the upcoming challenges and advancement in the field. Prof. Sanyal's passion for science, attention to tiny details and openness to collaborations pushed one to do better. Gautam Dey has been instrumental in this work, supporting my fellowship applications, welcoming me into his lab, giving me the freedom to try multiple approaches for this project, and involving me in other projects as well. I would also like to acknowledge Felix and Jana from Dey's lab for all the discussions during my stay at the European Molecular Biology Laboratory (EMBL), Heidelberg.

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?

I give all the credit to my teachers, Ms Sangeeta, Dr N. Narayanaswamy and Dr P. H. Reddy who all made biology so beautiful, fun and interesting. I must thank Dr Y. Singh at the Institute of Genomics and Integrative Biology (IGIB, New Delhi), with whom I did my summer internship during my bachelor's degree. Working in his lab gave me the experience I needed and confirmed that I wanted to be in science doing research. The moment that defined my passion and love for cell biology came right after my PhD thesis submission when I tagged one of the vacuolar transporters, Mnr2, with a fluorophore in the rice blast fungus Magnaporthe oryzae. I was amazed by the clustering–declustering dynamics of Mnr2 at different stages of development under a microscope.

Who are your role models in science? Why?

My mentors, whom I have watched closely and learnt so much from, inspire me to be in science. If I must name one person, it would be Prof. Maya Schuldiner from the Weizmann Institute of Science. Her work on inter-organellar crosstalk at the interface of membrane contact sites closely aligns with mine, and I would love to study these processes in relation to the cell cycle and how they have evolved in pathogenic yeasts. I hope to meet her one day.

What's next for you?

I am looking for a faculty position to start my lab. Fungal species display remarkable plasticity in spindle positioning, which is central to the equal distribution of duplicated DNA during asymmetric cell division. Yet, we observe diversity in spindle positioning at the pre-anaphase stage. Understanding the factors that govern these differences and why different organisms have adapted to different ways of spindle positioning are some of the questions I am interested in.

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

I love cooking, playing badminton and football.

Is there anything instrumental related to this work?

The exchange of ideas and collaboration is an integral part of the progression of science. I had the opportunity to visit Dr Dey's lab at EMBL, Heidelberg thanks to the support from the EMBL, Corporate Partnership Programme (CPP) Fellowship and the European Molecular Biology Organization (EMBO) Scientific Exchange Grant. At EMBL, we optimised U-ExM in C. albicans and used multiple dyes to study subcellular structures; later, we completed a few experiments in India. With the knowledge gained from this visit, I shared it not only with some of my colleagues in the lab but also with others from my institute.