Thomas Pucadyil, faculty from IISER Pune, is the only Indian scientist to be awarded the HHMI International Research Scholarship this year. Out of 1400 applicants, including Pucadyil, 41 scientists from 16 countries won this prestigious award. His lab studies how cells produce vesicles.
Q. Why is it important we know about how cells produce vesicles?
This process impinges on practically every aspect of cell physiology. The intricate distribution of organelles you see displayed in Cell Biology textbooks — is maintained that way by the dynamic production and consumption of vesicles.
Anytime you starve cells of essential nutrients, these organelles either change their shape or disappear entirely. To maintain organelle shape and identity, cells need to produce a continuous stream of vesicles. For all the progress we have made in this field, we still don’t know enough about where or how the vesicles for forming organelles arise.
Q. Could you give us a summary of research in the field — what is known, and what are some of the open questions?
The simplest forms of life that developed on early-Earth, invariably ended up encased in a lipid bilayer. In high school and college Cell Biology courses, students learn about the many features of the bilayer - that it is selectively permeable, very flexible — easy to bend and twist, but extremely difficult to rupture. The same reason that makes it choice substance for containing life forms, also makes it difficult to ferry membrane proteins from one part of cell to another. The vesicular transport system is believed to have evolved because membrane proteins can’t be transported like soluble proteins. To move them, part of a membrane has to bud-out as a vesicle which ultimately fuses with another (target membrane).
The field knows a lot about possible fission catalysts. It’s kind of funny in that practically everything we know about fission has come from studying dynamin, a protein involved in budding vesicles from plasma membrane.
The counterpart of mammalian dynamin protein in fruit fly is called shibire. The name translates to “limbs gone to sleep”; for a remarkable limb paralysis seen in Shibire mutants. AT 27 deg, this fly looks just like any other; but when taken to 34 deg, it collapses with sudden paralysis. What’s even more remarkable is that when you bring it back to 27 deg, the fly returns back to normal- flying about as if nothing happened! It is very rare to get such a ‘full circle’ phenotype. We know now that the reason this phenotype is seen is because the mutation prevents the formation of neurotransmitter vesicles. And since that process is so rapid, you can see the phenotype in such a short period of time.
Ever since these reports, dynamin has been the gold standard for other proteins involved in fission. This protein has a GTPase domain. If you scan the genome for other proteins that also have the [GTPase] domain, you could find other candidate proteins involved in membrane fission. This is what’s called a “candidate-based approach”; you are only likely to find something like dynamin. That’s one of the reasons we decided to not take that approach. Rather we devised an assay where we score for fission activity, in our hope to expand the repertoire of molecules involved.
We want to figure out a “parts-list”, which are the molecules involved in budding vesicles out of membranes; with the hope that if you figure out enough of them, you could draw generalisations on how the process works. We start with highly simplified model systems where we figure out the process of budding of vesicles.
Q. How does your lab study formation of vesicles?
In my lab, we have devised a new method to study membrane fission. The traditional method of studying membrane fission is very laborious. It uses polystyrene beads trapped inside an optical trap. If you touch the bead to the surface of a large vesicle and draw it back, you pull out the membrane in the form of a tube. When I started, this was all there was available, in the form of technique. I did not want to go down this route because it is not trivial at all. It requires a lot of time and effort, and the best-case scenario is you end up with one tube. We wanted an assay system that gives us an array of [membrane] tubes; something that’s robust; doesn’t require elaborate technical expertise and is amenable to downstream fractionation approaches. So these were our motivations behind developing a new assay.
Our method to make membrane tubes requires a small amount of lipid and is relatively easier. We call these supported membrane tubes (SMrTs). To start, you coat a glass coverslip with polyethylene glycol or PEG, the reason being that membranes stick to glass very well and that would cause a membrane tube to collapse. On this coated coverslip, we spread some lipids. Once dry, the coverslip is placed inside a flow cell. When exposed to buffer inside the flow cell, lipids self assemble to form a membrane that takes the shape of very large vesicles. We then flow buffer to extrude these vesicles into long and narrow membrane tubes. The outcome is that you see hundreds of these tubes arranged parallel to the flow of buffer. Proteins of interest are then passed onto these tubes and fission is observed as tubes getting cut. This high-throughput system can be assembled in a matter of minutes and is highly reproducible. (Watch a video of membrane fission on Pucadyil Lab website).
Q. How was the idea of supported membrane tubes conceived?
The bulk of my PhD (at CCMB Hyderabad) was spent studying membrane proteins. Close to finishing, I was thinking about getting into reconstitution biology - make your own membrane, add your protein of interest and see if it shows anything interesting. My first foray into reconstitution biology started while I was still at CCMB. By then I knew how to play around with model membrane systems. With that technical expertise I went to The Scripps Research Institute for postdoctoral training, where I studied membrane fission. The first model membrane system to study fission came out of my work there. In my lab at IISER, we built upon this work.
Q. Could you tell our readers the process of selection for HHMI International Research Scholarship?
The grant is for five years. The entire application period was 6-9 months (till interview). The application itself was very straightforward; does not emphasize on an elaborate proposal. They fund very diverse types of applications, which are reviewed by an international panel.
The interview took about 30-45 minutes during which they asked about experimental details of the proposed research; where you see yourself in 5 years, etc. There were also questions about support system at your institute - ambience and facilities; as well as internal process of evaluating faculty.
Q. In a previous interview, you attributed your desire to pursue a research career in Biology to a summer rotation experience. What would you say, can be done to kindle this enthusiasm for research in students?
A formal set-up for education is necessary. But for most students, their exposure to science is predominantly reading textbooks and research articles, about what discoveries others have made. Sadly through this process, we end up deifying laureates, but we don’t tell enough about doing science. As a result, the process of doing science can seem very distant.
It was no different for me, until I did the summer rotation at Amitabha Chattopadhyay’s lab at CCMB (where I would end up doing PhD). It was during this time that doing science seemed within reach.
I think it is crucial for students to have some independence in studying science, much before PhD. This is critical not just for doing research, should they so choose in their future. Education in science should give them tools for any kind of future they desire.