Design-driven, multi-use research agendas to enable applied synthetic biology for global health

Many of the synthetic biological devices, pathways and systems that can be engineered are multi-use, in the sense that they could be used both for commercially-important applications and to help meet global health needs. The on-going development of models and simulation tools for assembling component parts into functionally-complex devices and systems will enable successful engineering with much less trial-and-error experimentation and laboratory infrastructure. As illustrations, I draw upon recent examples from my own work and the broader Keasling research group at the University of California Berkeley and the Joint BioEnergy Institute, of which I was formerly a part. By combining multi-use synthetic biology research agendas with advanced computer-aided design tool creation, it may be possible to more rapidly engineer safe and effective synthetic biology technologies that help address a wide range of global health problems.     

Teaming up: from motors to people

When I reflect on how I became a cell biologist and why I love being one today, one thing that comes to mind is the many terrific collaborations I have had. The science I am most proud of from my graduate and postdoctoral training would not have been possible without working in teams with other scientists. Now, in my own group, much of our best work is being done collaboratively, both within the lab and with other labs. In this essay, I will highlight my experiences working in teams as a trainee, the role teamwork has played in my own research group, and how important I think collaborative science is for the future of biological research.     

The discovery of polyribosomes

By the early 1960s, evidence had accumulated that proteins were synthesized from special RNA copies of genes, named "messenger RNAs" (mRNAs), not directly from the stable RNAs found in the ribosomes of the cytoplasm. Yet, precisely how the protein chains were assembled along the RNA and, in particular, the relationship between the mRNAs and the ribosomes during protein synthesis, was obscure. In this account, I discuss how my laboratory found that multiple ribosomes traverse each mRNA, yielding the structures known as polysomes. This work led on to the first physical determination of the coding ratio, new insights into how protein chains are initiated, and an early suggestion that chloroplasts and mitochondria in eukaryotic cells might ultimately have been derived from symbiotic bacteria.     

In search of evolutionary developmental mechanisms: the 30-year gap between 1944 and 1974

The approach I have elected in this retrospective of how I became a student of evo-devo is both biographical and historical, a case study along the lines of Waddington's The Evolution of an Evolutionist ('75), although in my case it is the Evolution of an Evo-devoist. What were the major events that brought me to developmental biology and from there to evo-devo? They were, of course, specific to my generation, to the state of knowledge at the time, and to my own particular circumstances. Although exposed to evolution and embryology as an undergraduate in the 1960s, my PhD and post-PhD research programme lay within developmental biology until the early 1970s. An important formative influence on my studies as an undergraduate was the work of Conrad Hal Waddington (1905-1975), whose writings made me aware of genetic assimilation and gave me an epigenetic approach to my developmental studies. The switch to evo-devo (and my discovery of the existence of the neural crest), I owe to an ASZ (now SICB) symposium held in 1973.     

(M,R)-systems, (P,M,C)-nets, hierarchical decay, and biological aging: reminiscences of Robert Rosen

I have the pleasure to present a number of personal experiences that I had with Robert Rosen, both as his student and as a research colleague, and I will describe how this affected my academic career over the past decades. As a matter of fact, Rosen's work with (M,R)-systems as well as his continuing mentorship guided me into my own research in gerontology and geriatrics. Amazingly, this still continues to affect my work in complexity theory after 30 years.     

Using a course-long theme for inquiry-based laboratories in a comparative physiology course

I developed an inquiry-based laboratory model that uses a central theme throughout the semester to develop in undergraduate biology majors the skills required for conducting science while introducing them to modern and classical physiological techniques. The physiology laboratory uses a goal-oriented approach, with students working cooperatively in small groups to answer basic biological questions. The student teams work to develop skills associated with experimental design, data analysis, written and oral communication, science literacy, and critical thinking. The laboratory curriculum is a research-based model that offers the advantage of students asking open-ended questions by use of a variety of techniques. For the students and instructor alike, this presents an exciting and challenging approach for learning physiology and basic biological principles. Another advantage of this laboratory model is that it is flexible and adaptable; the central theme can be any that the instructor chooses, and the goals and techniques developed are based on student and instructor needs and interests. Students who have completed this model at Loyola College in Maryland have become equipped with the skills essential for any area of the biological sciences and, most importantly, showed elevated excitement and commitment to learning.     

Prediction and design of macromolecular structures and interactions

In this article, I summarize recent work from my group directed towards developing an improved model of intra and intermolecular interactions and applying this improved model to the prediction and design of macromolecular structures and interactions. Prediction and design applications can be of great biological interest in their own right, and also provide very stringent and objective tests which drive the improvement of the model and increases in fundamental understanding. I emphasize the results from the prediction and design tests that suggest progress is being made in high-resolution modelling, and that there is hope for reliably and accurately computing structural biology.     

Seven transmembrane receptors—A brief personal retrospective

Receptors have fascinated biologists for more than a century and they have fascinated me for the entirety of my own research career. The seven transmembrane receptors, also known as G protein coupled receptors, represent the largest of the several families of plasma membrane receptors, comprising more than a thousand genes and regulating virtually all known physiological processes in mammals. Moreover, they represent one of the commonest targets of currently used drugs. I have spent the entirety of my research career working on these receptors. Here I set down some personal reflections on the evolution of the field during the past 35 years, hanging the thread of the story on some of the work from my own laboratory.     

Balancing teaching and research experiences in doctoral training programs: lessons for the future educator

While a variety of alternative careers has emerged for Ph.D. life scientists in industry, business, law, and education in the past two decades, the structure of doctoral training programs in many cases does not provide the flexibility necessary to pursue career experiences not directly related to a research emphasis. Here I describe my efforts to supplement my traditional doctoral research training with independent teaching experiences that have allowed me to prepare myself for a career that combines both into a combined educational program. I describe the issues I have come across in finding and taking part in these endeavors, how these issues have affected my work in pursuing my Ph.D., and how my experiences translate into my hopes for a future education-based career in molecular and cell biology.     

Gaia Pigino: Inside the cell

Gaia Pigino studies the molecular mechanisms and principles of self-organization in cilia using 3D cryo-EM.

Gaia Pigino was only 3 yr old when she became fascinated with nature in the beautiful countryside of Siena, Italy, where she grew up. The neighbor’s daughter showed her a hen in the chicken coop, and they caught it in the act of laying an egg. Gaia remembers, “This was for me almost a shock, as my experience about eggs was that they come directly out of paper boxes!” Her father was also an important part of awakening Gaia’s curiosity for the amazing things in nature. He used to bring home the award-winning magazine Airone, the Italian equivalent of National Geographic. Gaia never missed an issue; even before learning to read, she could spend hours looking at the captivating photos of the wildlife. She wanted to understand what she was seeing, and maybe because of that, she was determined to do science.Gaia Pigino. Photo courtesy of Human Technopole.Gaia took her first “scientific” steps with Professor Fabio Bernini and Professor Claudio Leonzio at the University of Siena, where she studied bioindicators of soil contamination and detoxification strategies of soil arthropods as part of her PhD project. But it was later, when she joined the laboratory of Professor Pietro Lupetti and met Professor Joel Rosenbaum, a pioneer of cilia research, that Gaia discovered the world of 3D EM and felt her place was “inside a single cell.” She solidified her interest in the structure of protein complexes of cilia and flagella and boosted her passion for cryo-electron tomography (ET) in the laboratory of Professor Takashi Ishikawa, first at the ETH Zurich and then at the Paul Scherrer Institut in Switzerland. In 2012, Gaia started her own laboratory at the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, Germany, with the vision of creating a truly interdisciplinary laboratory. Her team combines techniques from different fields such as biophysics, cell biology, and structural biology to answer open questions in the cilia field. Gaia recently moved countries again—this time to take over the position of Associate Head of the Structural Biology Research Centre, at the Human Technopole, Milan, Italy.We reached out to Gaia to learn more about her scientific journey and future research directions.What interested you about cilia?The first thing that attracted me toward cilia and flagella were some EM micrographs, by Professor Romano Dallai in Siena, that showed the beautiful geometrical microtubular structures of sperm flagella. I was intrigued by the apparent perfection of these organelles that clearly showed me that a cell is a coordinated system of complex molecular machines, the mechanism of many of which we do not understand. Soon after, Professor Joel Rosenbaum introduced me to the bidirectional transport of components inside cilia, which, he explained to me, is required for both assembly and function of virtually all cilia and flagella, from the motile cilia in our lungs to the primary cilium in our kidneys. He called it intraflagellar transport (IFT) and compared it to a Paternoster elevator, where the individual cabins were what we now call IFT trains. I was completely fascinated by the IFT system, the structure, the function, the dynamics, and the mechanism of which were still largely unknown. Quickly, I realized that in addition to IFT, cilia represent a virtually infinite source of open biological questions waiting to be solved, from the mechanics and regulation of the beating to the sensory function of primary cilia, and their importance for human health.What are some of the scientific questions currently of interest in your laboratory?In the past few years, we have made substantial contributions to the current understanding of the structure and the mechanism of the IFT (1, 2, 3). Currently, we are investigating how the structure of IFT trains relates to their functions by looking, in cryo-electron tomography, at how anterograde trains transform into retrograde trains and at how different ciliary cargoes are loaded on the trains. Beside this more classical line of research, we are exploring other approaches to study IFT, for instance we have developed a method to reactivate IFT trains in vitro on reconstituted microtubules. We want to use this approach to investigate the behavior of IFT trains, and their motors, in experimentally controllable conditions, e.g., in the presence of only certain tubulin posttranslational modifications. We have also made interesting discoveries about the distribution of tubulin posttranslational modifications on the microtubule doublets of the axoneme and how this spatially defined tubulin code affects the function of different ciliary components. We hope we will be able to share these new “stories” with the structural and cell biology community very soon!What kind of approach do you bring to your work?I believe that the main reason for why science became an integral, and dominant, part of my life is because it provides infinite riddles and continuous challenges. I have always been curious about how things work in nature, but I quickly realized that learning from books didn’t satisfy me. My desire was to be at the frontline, to be among the ones that see things happening in front of their eyes, at the microscope, for the first time. I wanted to be among the ones that make the discoveries that students read about in textbooks. Thus, what I bring to my work is an endless desire of solving biological riddles, curiosity, creativity, determination, and energy, with which I hope to inspire the members of my team. My laboratory uses an interdisciplinary approach; we use whatever method, technique or technology is needed to reach our goal, from the most basic tool to the most sophisticated cryo-electron microscope. And if the method we need does not yet exist, we try to invent it.A young Gaia Pigino (3 yr old) the day she discovered how eggs are made. Photo courtesy of Giancarlo Pigino.Could you tell us a bit about the Structural Biology Research Centre at the Human Technopole (HT)?At the HT Structural Biology Centre, we are working to create a vibrant and interdisciplinary scientific environment that will attract molecular, structural, cell, and computational biologists from all over the world. We are creating fantastic facilities, including one of the most well equipped and advanced electron microscopy facilities in Europe—and likely the world—headed by Paolo Swuec. My team, together with the teams of my colleague Alessandro Vannini and the research group leaders Ana Casañal, Francesca Coscia, and Philipp Erdmann, already cover a vast range of competences and know-how from classical molecular and structural biology approaches, such as crystallography and protein biophysics, to cryo-CLEM, cryo-FIB SEM and cryo-ET, all of which allow us to address questions in cell biology. Our goal is to create a scientific infrastructure and culture that will enable biologists to obtain a continuum of structural and functional information across scales.What did you learn during your PhD and postdoc that helped prepare you for being a group leader? What were you unprepared for?I learned that everyday research is mostly made of failures, but that with the right amount of obsession, persistence, curiosity, and creativity, it is always possible to succeed and discover new things. Being given the freedom to develop your own ideas and your own project very early in your career is a treat; science is not only about having good ideas! One needs to follow up on these ideas with intense work and troubleshooting to make them reality. In addition, I realized that being fearless and attempting what is considered too difficult by others, despite challenges, can turn into a worthy learning experience. Also, how you present your work to the scientific community matters for swinging the odds of success in your favor. Different places might work in very different ways, and conducting good science does not only depend on you, but also on the possibilities given to you by your environment.What was I unprepared for?—I guess several things, but one comes immediately to mind: I underestimated how much being responsible not only for my own life and career, but also the career of students, postdocs, and others in the laboratory, would affect me personally.Structure of the 96-nm axonemal repeat reconstructed by cryo-ET and subtomogram averaging. Image courtesy of Gonzalo Alvarez Viar, Pigino Lab.What has been the biggest accomplishment in your career so far?This is a tricky question for me... I tend to look into the future more than celebrating the past. I fight to succeed in something, but as soon as I conquer it, I find it less of an achievement than the thing I could conquer next. Nevertheless, I am happy about the discoveries and the papers published together with my students and postdocs (1, 2, 3, 4, 5). I am extremely excited about the fact that after many years of work I am now leading an interdisciplinary laboratory, where we combine techniques from different fields. I am also happy that three times my husband and I were able to move from one world class academic institution to the another to start exciting and fitting jobs and could still live together in the same place. We worked hard for this, but we also got lucky.What has been the biggest challenge in your career so far?I studied French in school; I had almost no exposure to spoken English until the end of my PhD. To avoid having to show my English insufficiencies, I did hide beside the board of my poster at the first international conference I attended in 2004! It took me a while to overcome this barrier and feel confident to express my thoughts and ideas in English.What do you think you would be if you were not a scientist?I had been a good fencer during my youth. I was a member of the Italian National Team between ages 14 and 19 and saw quite a bit of the world, which was cool! When my sporting career failed, due to diabetes, I was torn between art and science. I guess that in a parallel universe, I am a wildlife photographer and a potter specialized in wood kiln firing. [Gaia confesses that she misses “the amazing and addictive adrenaline rush of a good fencing match!”]Any tips for a successful research career?Do not compare your performances to the ones of the people at your career stage; compare yourself with people that are already successful one level higher than you currently are at. For example, if you are a PhD student, ask yourself what in your current performance separates you from being a good postdoc—once a postdoc, what is missing to be a good PI.     

Biologists behaving badly: vitalism and the language of language

A comparison is made between Biologos, the "language of language" that predominates in current infocentric biology, and Logos, the classic bringer of form to chaos. The immaterial information on which Biologos is based is seen to bear intriguing similarities to just the sort of disembodied formative powers that an aggressively materialist biology has long derided. I address these issues by meeting a (perhaps only hypothetical) charge that my own work is in some sense vitalist, first with the usual flat denial, then with a countercharge. My third move is a nontraditional one, meant not as capitulation or acquiescence, but as an acknowledgement that the terms of this debate, never clear, continue to be remarkably ill-defined. The question of how best to think about development, or epigenesis--the process whereby organisms come into being--remains a legitimately contested and difficult one.     

Quantifying your body: A how-to guide from a systems biology perspective

During the coming decade we will see an accelerated digital transformation of healthcare. Leading this change within the institutional medical community are both the move to digital medical records and the use of digital biomedical measurement devices. In addition to this institutional evolution, there is a non-institutional, bottom-up, unorganized, highly idiosyncratic movement by early adopters to "quantify" their own bodies. In this article, I share my decade-long personal experience of tracking many blood and stool biomarkers, which provide insight into the health or disease of major subsystems of my body. These results are interpreted in the context of the genetics of my human DNA and that of the microbes in my gut. Even though I am a computer scientist and not a medical professional, by using commercially available tests and a systems biology integrative approach, I have become an early example of Leroy Hood's vision of the emergence of predictive, preventive, personalized, and participatory (P4) medicine. It is an individual's story illustrating how each of us can contribute to realizing this paradigm shift.     

Evolutionary convergences and parallelisms: &;their theoretical differences and the difficulty &;of discriminating them in a practical &;phylogenetic context

The importance of evolutionary parallelisms and their differences from evolutionary convergences have been historically underappreciated, as recently noticed in Gould's last book `The structure of evolutionary history'. In that book, Gould make an effort to distinguish and to reinterpret these concepts in the light of the new discoveries of the last decades on developmental biology and genetics, presenting the elegant metaphor of `Pharaonic bricks versus Corinthian columns'. In this paper I will briefly discuss these concepts, and will argue that, despite the advances that have been made to define them in theory, it is rather hard to differentiate them in a practical phylogenetic context. In order to do so, I will provide some few examples from my own empirical studies on the last years of one of the most morphologically and taxonomically diverse groups of Vertebrates, the catfishes.     

Crystallography, evolution, and the structure of viruses

My undergraduate education in mathematics and physics was a good grounding for graduate studies in crystallographic studies of small organic molecules. As a postdoctoral fellow in Minnesota, I learned how to program an early electronic computer for crystallographic calculations. I then joined Max Perutz, excited to use my skills in the determination of the first protein structures. The results were even more fascinating than the development of techniques and provided inspiration for starting my own laboratory at Purdue University. My first studies on dehydrogenases established the conservation of nucleotide-binding structures. Having thus established myself as an independent scientist, I could start on my most cherished ambition of studying the structure of viruses. About a decade later, my laboratory had produced the structure of a small RNA plant virus and then, in another six years, the first structure of a human common cold virus. Many more virus structures followed, but soon it became essential to supplement crystallography with electron microscopy to investigate viral assembly, viral infection of cells, and neutralization of viruses by antibodies. A major guide in all these studies was the discovery of evolution at the molecular level. The conservation of three-dimensional structure has been a recurring theme, from my experiences with Max Perutz in the study of hemoglobin to the recognition of the conserved nucleotide-binding fold and to the recognition of the jelly roll fold in the capsid protein of a large variety of viruses.     

Ye Tian: Surveilling stress to live longer

Ye Tian investigates how mitochondrial stress signaling pathways regulate longevity using C. elegans as a model system.

An avid reader, Ye Tian used to save up her child allowance with the sole purpose of buying science fiction books. Reading and solving mathematical problems were her favorite hobbies; indeed, she liked mathematics so much that she was about to enroll herself as an architecture major but finally chose biotechnology. Ye moved from her hometown in the Northwest of China, Baoji—famous for housing the Zhou dynasty’s bronzeware and being close to the Terracotta Army—to Beijing for her college and graduate studies.Ye is proud of being among the earliest researchers working on Caenorhabditis elegans in her country; for her PhD studies, she joined the lab of Hong Zhang, who at that time has just established the first C. elegans lab in China at the National Institute of Biological Sciences in Beijing. Ye identified epg-2 as an adaptor for cargo recognition during autophagy. In 2010, she crossed the Pacific toward the U.S. West Coast for her postdoctoral training in the aging field with Andrew Dillin, first at the Salk Institute in San Diego and then at the University of California, Berkeley. There, she discovered that mild mitochondrial stress during development in worms rewires their chromatin landscape to establish specific gene expression patterns throughout the lifespan and promote longevity.Ye Tian. Photo courtesy of Ye Tian.Ye came back to China at the end of 2016 to start her own lab at the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences. Her research team studies mitochondrial stress signaling pathways and their interplay with aging. We chatted with her to learn more about her next scientific plans.What interested you about the interplay between mitochondria and aging?I became interested in mitochondrial biology during my postdoc in Andrew Dillin’s lab. Since the origin of eukaryotic cells, mitochondria have been a driving force of evolution. During reproduction, mitochondria are passed from the mother to the offspring through egg cells and they exhibit a unique inheritance pattern. As essential hubs that dictate cellular metabolism, it is clear now that mitochondria and the nucleus maintain a bidirectional communication. Early life “stressed” mitochondria communicate with the nucleus to induce gene expression changes that are beneficial on longevity and persist throughout the lifespan. The fact that mitochondrial function is crucial to aging fascinated me; I wanted to continue exploring that topic further, and that’s why I established my lab around the question of how mitochondrial surveillance mechanisms regulate the aging process.What are you currently working on? What is up next for you?My research team focuses on the interplay between mitochondrial stress signaling pathways and aging. The first work that my lab published was a project that I started during my postdoc. The Dillin lab reported a phenomenon in which perturbations of mitochondria in neurons induced a mitochondrial stress response in the peripheral tissues and hypothesized that a secreted signal molecule, named after mitokine, is required for the cell non-autonomous regulation (1). The identity of this molecular signal remained elusive for almost ten years until we found that a secreted Wnt ligand, EGL-20, functions as the mitokine to coordinate mitochondrial stress signaling across tissues and promote longevity of the organism (2). We are also interested in how the crosstalk between mitochondria and the nucleus influences lifespan. We found that mitochondrial perturbations alter the nuclear epigenome to induce longevity via the histone deacetylation complex NuRD in response to cellular acetyl-CoA levels, the key metabolite at the entry point of the Krebs cycle (3).Lab group picture; current lab members (2021). Photo courtesy of Ye Tian.Our latest work stemmed from a serendipitous observation that neuronal mitochondrial stress is sensed by and transmitted through the mitochondria in the germline. Intergenerational, maternal inheritance of elevated levels of mitochondrial DNA via the mitokine Wnt/EGL-20, which causes the activation of the mitochondrial unfolded protein response (UPR^mt), provides descendants with a greater tolerance to environmental stress. This makes the offspring live longer (4).Among our short-term scientific plans, we’re determining how mitochondria functions during the aging process at both the genetic and biochemical levels and searching for ways to apply our findings from C. elegans to neurodegenerative disease models in mammals.What kind of approach do you bring to your work?The curiosity about how things work drives me; what I enjoy the most is when I see things happening in front of my eyes and when I figure out why they occur that way. That enthusiasm is what I try to spread to my team every day. In the lab, we rely on C. elegans as our model system and on genetics to dissect complex biological processes like aging. We have also adapted modern biochemical and imaging techniques as well as bioinformatics to complement our genetic studies. I’m a geneticist at heart, and I like to initiate a project with a well-designed genetic screen. The best part is that the screen often leads me to answers I was not expecting, and that’s genuinely inspiring!What did you learn during your PhD and postdoc that helped prepare you for being a group leader? What were you unprepared for?Like most scientists, my research career has gone through ups and downs. I had to change my research project in the last year of my graduate school; that was nerve-racking, but I eventually managed to redirect my thesis and get exciting results under time pressure, thanks in large to the support of my parents, mentors, and lab mates. That helped me prepare to become a principal investigator; I gained confidence in problem solving, and since I’ve experienced the stress of dealing with last-minute scope changes firsthand, I connect better with my students.I guess, as many other non-native English speakers, I wasn’t prepared for writing grants and papers fluently in English. This issue wasn’t obvious during my graduate and postdoctoral studies, as my mentors were always there for me and proofread and edited my writing. Now I have to stand up for myself. I spend most of my time writing; I’ve improved my writing skills but it’s still an ongoing process.Reconstruction of the nerve system of C. elegans by confocal microscopy. Green corresponds to YFP-labeled neuronal specific marker Q40, and red labels germline specific mitochondrial outer membrane protein TOMM-20::mkate2. Image courtesy of Ye Tian’s lab.What has been the biggest accomplishment in your career so far?My very first PhD student, Qian Zhang, graduated with two first-author papers and decided to pursue a research career in academia. Being responsible for someone else’s career is challenging but also rewarding.What has been the biggest challenge in your career so far?I use the model organism C. elegans for my research in aging, so from time to time, peers criticize the relevance of my work to human health. I’m used to justifying my scientific approach to funding agencies and peers in other fields, but sometimes it’s exhausting or not pleasant.Who were your key influences early in your career?My PhD mentor, Hong Zhang. He is very passionate about the science he does, and he is courageous to shift his research directions to answer new biological questions.What is the best advice you have been given?I think the best advice I’ve gotten is that “tomorrow is another day.” It reminds me to keep going and be optimistic.What hobbies do you have?I love art and music. When I was in San Diego, I used to play in the Chinese Music Band; I miss my musician friends over there. In my teens, I used to hike mountainside trails along the river with my parents. Now, running has become my new favorite hobby. I enjoy the tranquility and peace of mind while running; it’s soothing.     

Chemical biology approaches in plant stress research

In addition to their importance as essential agrochemicals and life-saving drugs, small molecules serve as powerful research tools to address questions at all levels of biological complexity from protein function to plant biotic interactions. In certain contexts, chemical tools are complementary or even preferred to genetic analysis, since not all experimental systems are amenable for genetic dissection. For example, mutants impaired in oxygen sensing cannot easily be recovered. Pharmacological and chemical genetics approaches have come to the rescue of biologists in unraveling such genetically intractable systems. In this review, I have discussed my own efforts to analyze oxygen deprivation signaling in plants to illustrate the validity of small molecular approaches in elucidating an essential pathway such as oxygen sensing. Chemical biology is also a potent approach to tease out genetically redundant biological processes. The recent breakthrough in identifying the elusive abscisic acid receptors has clearly demonstrated the power of chemical tools in dissecting redundant pathways and led to the blossoming of this area as a distinct discipline of plant biology research. I present a summary of this work and conclude the review with potential challenges in using chemical tools.     

A career pathway in protein folding: from model peptides to postreductionist protein science

This review discusses the inherent challenge of linking "reductionist" approaches to decipher the information encoded in protein sequences with burgeoning efforts to explore protein folding in native environments-"postreductionist" approaches. Because the invitation to write this article came as a result of my selection to receive the 2010 Dorothy Hodgkin Award of the Protein Society, I use examples from my own work to illustrate the evolution from the reductionist to the postreductionist perspective. I am incredibly honored to receive the Hodgkin Award, but I want to emphasize that it is the combined effort, creativity, and talent of many students, postdoctoral fellows, and collaborators over several years that has led to any accomplishments on which this selection is based. Moreover, I do not claim to have unique insight into the topics discussed here; but this writing opportunity allows me to illustrate some threads in the evolution of protein folding research with my own experiences and to point out to those embarking on careers how the twists and turns in anyone's scientific path are influenced and enriched by the scientific context of our research. The path my own career has taken thus far has been shaped by the timing of discoveries in the field of protein science; together with our contemporaries, we become part of a knowledge evolution. In my own case, this has been an epoch of great discovery in protein folding and I feel very fortunate to have participated in it.     

Hongyuan Yang: Tracking lipids,one droplet at a time

Hongyuan Yang investigates lipid trafficking and lipid droplet biogenesis.

Hongyuan Yang grew up in a small city east of Beijing, China. From his childhood, Hongyuan recalls that “food was not abundant, so I was hungry at times, but education was free and good.” Driven by his curiosity for science, after completing his undergraduate studies at Peking University Health Science Center, China, he enrolled at Columbia University, NY, for his doctoral training. Under the guidance of his advisor, Dr. Stephen Sturley, Hongyuan studied lipids in budding yeast. The laboratory’s research department fostered a strong interest in lipids and atherosclerosis, and after earning his PhD, Hongyuan obtained a faculty position at the National University of Singapore (NUS) in 1999. In 2007, he moved to the University of New South Wales (UNSW) in Sydney, Australia, to continue his scientific journey exploring lipids. We contacted Hongyuan to learn more about his career and interests.Hongyuan Robert Yang. Photo courtesy of UNSW.What interested you about lipids?My five-year doctoral study focused entirely on the enzymes Sterol O-Acyltransferases (SOAT, also known as ACAT, Acyl-CoA Cholesterol Acyltransferases), which catalyze the formation of sterol esters from sterols/cholesterol and fatty acyl CoAs (1). SOATs, integral membrane proteins of the ER, are potential therapeutic targets for heart disease and Alzheimer’s disease. Since then, I have been fascinated by two things related to SOAT: first, what happens upstream of SOAT, i.e., how exogenous cholesterol reaches SOAT/ER; and second, what happens downstream of SOAT, i.e., how its product—cholesterol esters—is stored in cells in the form of lipid droplets (LDs).These are fundamental questions in cell biology. While reading on how cholesterol arrives at the ER for esterification by SOAT/ACAT in the late 1990s, I realized that the trafficking of most lipids was poorly characterized with little molecular insight. Significant progress has been made in the last 20 years, but the lack of tools that track the movement of lipids has hampered our understanding of the selectivity, efficacy, and kinetics of lipid trafficking. Few cell biologists cared about LDs ∼20 years ago, even though LDs are prominent cellular structures in many disease conditions. Each LD comprises a hydrophobic core of storage lipids (triglycerides and sterol esters) wrapped by a monolayer of phospholipids. Largely considered inert lipid granules, LDs originate from the ER and are relatively simple cellular structures as compared with other organelles (see image). Now, we know that LDs are not that simple: their biogenesis is tightly regulated, they actively interact with other organelles, and they regulate many aspects of cellular function as well as disease progression. Astonishingly, we still have little understanding of how LDs originate from the ER. I am very much intrigued by the complexity of these two seemingly simple cellular processes, i.e., lipid trafficking and LD biogenesis.What are some of the scientific questions currently of interest in your laboratory?We are currently focusing on how LDs originate from the ER. The first significant paper from my own laboratory was the discovery of seipin as a key regulator of LD formation (2). Results from many groups have demonstrated that seipin can organize the formation of LDs; however, the exact molecular function of seipin remains mysterious. Our data suggest that seipin may directly impact the level and/or distribution of lipids such as phosphatidic acid near sites of LD biogenesis, and the effect of seipin deficiency on LD formation is secondary to changes in local lipids. We are now working hard to test this hypothesis. Moreover, data from my laboratory and others indicated that nonbilayer lipids may have a greater impact on the biogenesis of LDs than that of other ER-derived structures, such as COPII vesicles. This may result from the monolayer nature of the LD surface. We hope to dissect the dynamic changes of lipids at ER domains where LDs are born. More broadly, the ER is a fascinating organelle to me. The simple division of ER into sheets and tubules does not reflect the dynamic nature of this organelle. Dissecting the composition and organization of lipids and proteins of the ER would help answer key questions relating to LD biogenesis, and it is therefore one of our future directions.Another major focus is to understand how cholesterol and phosphatidylserine are moved between organelles. We have been working on how low-density lipoprotein (LDL)–derived cholesterol (LDL-C) reaches the ER for two decades. The release of LDL-C from lysosomes requires the Niemann Pick C1&2 proteins, whose malfunction causes lysosomal cholesterol accumulation and a lethal genetic disorder affecting young children. The Ara Parseghian Medical Research Foundation has led the way in supporting research into cholesterol trafficking, and I take this opportunity to thank their generous support. Once released from lysosomes, LDL-C is believed to reach the plasma membrane first and then the ER. We identified ORP2 as a possible carrier of LDL-C to the plasma membrane using a PI(4,5)P₂ gradient (3). There must be other carriers and/or pathways because ORP2 deficiency only causes a minor accumulation of cholesterol in lysosomes. Another interesting question is what prevents LDL-C from reaching the ER directly from lysosomes, given the close contact between lysosomes and the ER. We reported that ORP5 may bring LDL-C directly to the ER (4). However, it was later found that ORP5 binds and transfers phosphatidylserine, not cholesterol. Thus, our observed link between ORP5 and cholesterol is through some indirect yet unknown mechanism. We have been perplexed by these observations for many years, but a recent study demonstrated that phosphatidylserine is required for the trafficking of LDL-C, establishing a close link between cholesterol and phosphatidylserine (5). We are now trying to understand how the trafficking and distribution of cholesterol, phosphatidylserine, and PI(4,5)P₂ are interconnected. For a long time, I felt that it was impossible to figure out the molecular details governing the cellular trafficking of lipids due to redundant pathways and a lack of tools to track lipids. Recent progress in this field has given me hope.Lipid droplets in a HeLa cell are shown in red (BODIPY), with their surface in green. DAPI (blue) labels DNA. Image courtesy of Hongyuan Yang.What kind of approach do you bring to your work?Besides honesty and open-mindedness, we emphasize rigor and comprehensiveness. We often make our initial discoveries in cell-based screens. This approach has many advantages, but it also gives rise to artifacts and cell-line specific observations. We aim to complement our initial findings with biochemical and structural analyses in vitro as well as animal studies in vivo. To further establish the reproducibility of our data, I often ask my close friends and collaborators to independently repeat the key findings of a study before submission. It generally takes a long time for us to complete a study, but I believe the effort will pay off in the long run.What did you learn during your training that helped prepare you for being a group leader? What were you unprepared for?During my PhD at Columbia, I was most impressed with the general attitude of my mentors toward research. No matter how much they have achieved, they take every new experiment and every poster presentation seriously.As I did not have postdoctoral training, I was somewhat unprepared at the beginning of my independent career. One difficult challenge was knowing when to finish a paper and project. We often kept working and working. I have now gotten a lot better.You’ve done research on three continents throughout your career. Can you tell us about some of these transitions?During the last year of my doctoral studies at Columbia, I was offered a lecturer position by the Department of Biochemistry at NUS. It was a very hard decision to leave the United States, but I was excited by the prospect of starting my own laboratory at a top institution. Life at NUS was very good overall, despite some struggles. I had to make ∼700 slides for teaching during the first year and my start-up fund was 10,000 Singapore dollars (~6,000 USD). But the graduate students were fully supported by the university, and most of them are hard working and talented. The crucial screen that led to the discovery of seipin as a key regulator of LD formation was performed at NUS (2). I enjoyed my time at NUS, where I was promoted and tenured. However, my family and I could not get used to the heat and humidity. We looked for a place with better climate, and it happened that my current employer, UNSW, had an opening in 2006. Moving continents with two kids was very disruptive, and I had zero publications in 2007. Our work on seipin was delayed and almost got scooped. I was also very worried about funding in Australia since I hardly knew anyone and the funding system. It turned out that the Australian community was very supportive of our research from day one. I have also been very fortunate to receive generous support from the Ara Parseghian Medical Research Foundation, based in the United States, after my move to Sydney.Hongyuan’s “metabolism team” after a basketball game. Photo courtesy of Hongyuan Yang.What has been the biggest accomplishment in your career so far?While I am mostly recognized for discovering seipin’s role in lipid droplet formation, I am prouder of the work we have done on lipid trafficking and the oxysterol binding proteins. We struggled mightily for the first 15 years. At one point in 2015, I seriously considered abandoning this line of research. But we persisted and discovered their roles in regulating plasma membrane PI(4,5)P₂ and cholesterol, as well as in lipid droplet formation (3, 6).What has been the biggest challenge in your career so far?The biggest challenge has to do with the subject of my research topic: the fundamental cell biology of lipids. The sorting, distribution, and storage of cellular lipids are clearly very important topics in biology, but they are sometimes too fundamental to explain to funding agencies and new students. These days, lipid research is not as “sexy” as other topics. But there are so many unanswered questions in lipidology. I strongly believe that lipid research is going to be the next “big thing” as new techniques such as cryoEM now allow us to appreciate lipids and membrane proteins with unprecedented clarity.Who were your key influences early in your career?Besides mentors and teachers at Columbia, I really enjoyed reading and studying the works by Drs. Mike Brown and Joseph Goldstein, Ta-Yuan Chang, and Scott Emr. While they were not my teachers, their work inspired and impacted many young scientists, including me.What is the best advice you have been given?I have been given many pieces of great advice during my career. The best one in my view is “Less is more.” I was once told, “You would be better off with a lab of six than twelve.” Initially, I did not get it because I thought that a bigger group would allow me to explore more directions and be more productive. The reality is that, as a little-known junior researcher, few experienced people would join my laboratory. Funding is also a major limiting factor. Supervising a large number of students is fulfilling, but it also takes away some of my own time to think critically about the projects. I have largely kept my group under six, and this allows me to better supervise and guide the trainees. People say, “Once your team has more than 15 members, you become a manager instead of a scientist.” My own experience corroborates that statement because I struggled quite a bit when my group reached 12 at one point.What hobbies do you have?I am heavily into sports, especially basketball and tennis. I follow the NBA closely, and Jeremy Lin is my hero. I still play basketball at least twice a week. I am the captain of a basketball team comprised of scientists working on metabolism (see image). We play real, refereed basketball games against local teams during conferences. As I am getting older, I have also picked up tennis. I watch coaching videos on YouTube but still need a lot of work on my forehand. Through sports, I learned teamwork and the spirit of fighting to the last second. If I were not a scientist, I would probably run a sports-related business.What has been your biggest accomplishment outside of the laboratory?I got married and had children relatively early. Both of my kids are now in college and they appear to be decent human beings. I have been extremely lucky because my wife did most of the heavy lifting in looking after the kids. It was still a struggle for me to balance work and parental duties during the early days of my independent career. I am very proud and happy with where we are as a family right now.Any tips for a successful research career?Everyone is unique. Knowing your strengths and especially your weaknesses can be crucial to your success. My undergraduate training was in medicine and health management, and my PhD work focused on genetics and cell biology, so my understanding of physical chemistry is rather inadequate. I am also very bad at developing new methods. To alleviate these deficiencies, I constantly monitor new methods in my field and I purposefully look for collaborators with strong chemistry backgrounds. I have benefited immensely from such efforts.