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Breaker of Boundaries: Interview with Dr. Thomas Pucadyil

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Thomas Pucadyil2Dr. Thomas Pucadyil runs the Reconstitution Biology lab in IISER Pune. His research is based on a novel approach to address questions in membrane biology that he pioneered during his postdoctoral work. His techniques have found great success, as evidenced by the many accolades he has received, the most recent of which is the Shanti Swarup Bhatnagar award last year. He elaborates on his research and his perspectives on doing science in a brief discussion with BS MS student Rahul Iyer. Can you briefly explain the focus of your research? Every single living cell is contained by a lipid bilayer that forms a membrane. Basically, if you throw in a bunch of amphipathic molecules they spontaneously assemble into a membrane. Their self-assembly is dictated by non-covalent forces, no genetic code or template is required for their formation, and they form very resilient compartments in water. Lipids have thus evolved to be the perfect compartmentalizing agent to contain the chemical reactions necessary for life. Membranes are also embedded with membrane proteins, which are affected by and in turn affect, membrane properties. For a lot of cellular processes such as budding of vesicles, division, or even existing in sub-compartments as most eukaryotes do, membrane proteins are required to work against the resilience of membranes. Membrane fission is one such process, which I basically try to understand by identifying the essential players and their mechanistic function. Membrane composition is critical for all these processes, so I asked whether I can stitch membranes together from scratch and mimic events that occur in the mechanism of the process I’m looking at. This is essentially the reconstitution approach. I wanted to mimic clathrin-mediated endocytosis, which people have been working on since the ‘50s. If I’m successful, then I’ve understood the salient features of the process. Even if I fail, I can explore what stalls the process and revise what we know about it. A similar approach had been tried for cell division. Bacterial division appears to be managed by very critical and highly conserved proteins. So if you get these four proteins inside a vesicle and have these bind the membrane, do you see division? As it turns out you don’t, and while no one has yet managed to mimic the process in vitro, the approach has highlighted other proteins’ functions necessary for the process. What led you to work in membrane biology? My interest in membrane biology started when I was working with Amitabha Chattopadhyay at CCMB. My PhD thesis was based on understanding how lipids in the membrane contribute to function of the serotonin receptor in the brain. This necessitated a deeper understanding of how cell membranes function. As I read more about this complex entity, my interest shifted from neurobiology to membrane biology. A fantastic paper that came out in 1996 from Pietro de Camilli’s lab in Yale who looks at endocytosis, got me hooked. For context, this paper described epsin as a critical protein for endocytosis that functions to bend membranes to extreme degrees for endocytosis. To get the same degrees of curvature with vesicles, you need to bombard them with sound waves of high energy. This paper, cleverly titled ‘Curvature to the ENTH degree’ (ENTH being the membrane-bending domain in epsin, geddit?), shows how exactly epsin manages to counter the hydrophobic effect for an essential physiological processes like vesicle budding. This got me thinking - can other proteins perform similar functions? Can you use this to make a minimal living system from scratch which can grow, bud and reproduce? The one that thing I realized by this point was that you cannot understand life unless and until you understand membranes. At this time, we also began to see a lot of disciplines interested in this area, even aside from cell and molecular biologists. Physicists were trying to understand the energetics of membrane structure based on membrane components. Ecologists found lipid composition plays a role in thermal tolerance, specifically in migrating fishes. Neurobiologists looked at neurotransmitters and their respective membrane receptors. In fact, the effects of anaesthetics and alcohol were elucidated as more was uncovered about membrane behaviour. Once the genome got sequenced, we found that one-third of all proteins are membrane proteins and about 50% of these belong to the class of seven-transmembrane domain G-coupled receptors, which includes the serotonin receptor. The fact that every third drug passed over the counter acts on one of these G-coupled receptors got pharmacologists involved as well. It was a hot topic, but also a challenging field. While a lot was known about proteins involved in different cellular processes, how they carried out their function was not known in most cases. So it was an exciting area, and coming in with a different approach added to the challenge. So with this vision, you were applying for a postdoctoral position... For my post doc, I intentionally wrote to cell biologists focusing on membranes because inputs from cell biologists are needed to know the direction this should take. You need both reductionist and non-reductionist perspectives. Most of the people I applied to responded positively to the idea but could not fund this or did not want to invest in this unconventional direction. I eventually got accepted by Sandy Schmid from The Scripps Research Institute, who said ‘This idea is perfect because I have no clue what you’re talking about but you apparently do, so let’s do this!’ I started working on dynamin, a protein known to be involved in membrane fission in order to form synaptic vesicles. So I stitched together a membrane following a recipe of common phospholipids, formed a neck typically seen in clathrin-mediated fission, and introduced dynamin to this assay. The question was - given that dynamin is necessary for fission and release of this vesicle, is dynamin sufficient?  We successfully devised our first fission assay which told us that dynamin is indeed sufficient. We could also see the regulation of these reactions in a very modular sense: an early module for formation of the bud/neck, and then a late distinct module where dynamin acts at the neck. Now you can ask how it knows when to come in and how does it exactly execute this function? What makes it more interesting is that the yeast can carry out fission with only clathrin, whereas the worm or the fly requires dynamin as well. Evolutionarily, an investment in making dynamin critical for fission resulted in better regulation of this process. However, I still could not demonstrate this process in real time, which is what I had set out to do. After your post doc, you joined IISER Pune and set up the Reconstitution Biology Lab. How has your research focus evolved since? While setting up my lab here, the question in my mind simply was - what are the possible ways one can separate a given membrane compartment into two? The phenomenon and its extent were documented very well, but little was known about how it takes place. This was a big question to address, so I had to break this into smaller parts. I had to set up assays that would allow me to understand the sorts of proteins that manage separation of compartments. For my post doc I had devised an assay with a recipe to mimic a vesicle to study dynamin, but could I now tune this to mimic fission in organelles? For this we devised another assay: membranes with a long neck which has a thin radius. This is a wonderful system to monitor multiple independent events taking place at the neck in one shot. We called it the Supported Membrane Tubes (SMrT). Using this assay, we found that the action of dynamin is to constrict the membrane until its breaking point. However, we found that the rates of constriction varied, despite the conditions of the assay remaining constant. This was baffling because we are working with reductionist systems for the purpose of controlling these reactions. Changing the assay system led us to conclude that this is due to stochasticity in energy states at the molecular level in dynamin. We have also asked whether dynamin is the only molecule that can release vesicles. When we added brain lysates to membrane tubes, fission took place so we initially concluded that lysates had dynamin. It did have dynamin, but a closer look revealed multiple dynamin independent pathways, each having different intermediates. Based on this, we are currently looking in the following directions. One: can we build a complete repertoire of molecules that can catalyse fission? Two: where and how do they function in a cell? Three: how similar and different are the pathways to fission in case of each of these proteins? Four: what are the determinants of each protein being used in their respective contexts? For example, for dynamin we have found the determinant to be the curvature of membrane, but when it comes to mitochondria, dynamin will sense it to be flat in the same way as we sense the earth. We managed to show that a distinct mitochondrial dynamin belonging to the dynamin protein family is responsible for fission in mitochondria. As we increased the size of membrane tubes, the regular dynamin could not catalyse fission after a point whereas the mitochondrial dynamin would. Currently, we are working on what brings about regulation of mitochondrial fission. The nice bit in using all these assays is that we aren’t losing sight of cell biology - for all our findings of the determinants in vitro, we check if that holds true in real cells. In fact what we also try to do as we improve our assays is to revisit previous experiments. Since we now have improved readouts, we just might find something new. We have actually reported three new fission catalysts so far doing exactly this, which could not have been identified by the crude readouts of earlier fission assays. There are still fundamental questions in the field we have yet to answer, like how membraneless organelles like lipid droplets organize and localize themselves? Or how is cell shape and size determined mechanistically when this information is not genetically encoded? What determines the fixed number of organelles for a particular cell? In my opinion, the way forward is to focus on devising proper assays for the questions being asked, especially when it comes to signals and sensors in the regulation of any process. What are your views about life at IISER Pune and your many roles in this institute? It has been quite an experience. Compared to my peers who have now found positions in established institutions, I find I can induce more change but with more responsibility. Each of us in the department has ideas about how to run a graduate program. There is no perfect recipe to run a lab, or to mentor students. Collectively speaking, because there is diversity in viewpoints in the kinds of questions being asked and in the expertise we cover, we fare better. Between disciplines, however, the common ground has unfortunately been reducing in the past few years. Truly speaking, it is the students who are keeping it together, especially the BS-MS students because they are the catalysts who cross-fertilize each discipline. Looking back now, it has been kind of difficult to go about setting your lab, have teaching to do, work on getting funding, make sure students graduate with a decent PhD thesis, and to help them secure a position. As a guide, my first priority is to ensure that each student who works in the lab on these assay systems leaves knowing that addressing questions in contemporary research is within reach. For short term rotation periods, they start working with how to make these model membrane assay systems and see membrane fission with their own eyes. Then I leave it to them to decide the direction they want to take. Even if it is not extraordinary, it should be original. There is a lot of emphasis on hypothesis-based research, and while this is important, I put more emphasis on curiosity-driven science. This is what reinstates your faith in science. Then you can decide which direction to take your research. In my opinion, the current process of predetermining objectives and title of a project constrains the whole rotation experience. The one thing I require is keeping tabs on current developments in biochemistry, material science, cell biology, and organismal biology, which we partly do in our journal club sessions. Having students from different backgrounds also helps greatly. I was made chair of the screening committee that screens faculty applicants to the biology department a while ago. My constant endeavour and effort goes into increasing diversity. In terms of their field of expertise, in terms of the biological systems they are working on, more diversity is what makes the department stronger as a whole. Taking a membrane analogy, a pure lipidic membrane system can go from very solid to very fluid within a change of 1 or 2 degrees Celsius. What truly makes a cell membrane functional is all kinds of lipids and proteins, such that even if there is a dramatic shift in temperature, it still stays fluid and keeps functioning. That is possibly the most important thing for us to strive towards. That also comes from getting students from diverse disciplines, diverse viewpoints, and totally doing away with departments within biology. I honestly think departments have no place in contemporary biology. What do you do in your downtime? Currently, mostly doodling. I have been interested for a while now in depicting my research as cartoons, because everything we do can be reduced to an intuitive concept. I think a lot about the communication of my work. Professionally, I have always been excited by the storytelling aspect of putting together a paper. Using data to create a sequence of schematics and results, just the presentation of information in a manuscript is an art form, in my opinion. Otherwise, interests like music, the arts, they come and go in phases. I keep up with current science fiction from time to time, although nothing of late feels inspired. Most of today’s science fiction is in movies, and a majority of the focus is in special effects. Those are great, but when everyone’s doing it, well, no one really stands out. Maybe it’s me being cynical, but I haven’t found anything path breaking like Bladerunner, or even box office hits like Star Trek, for a long time now. - Interview by Rahul Iyer