For Biophysics Week, members of the Publications Committee selected a few influential articles from Biophysical Journal to highlight as well as the people who wrote them. This is the first in the series.
by Andrea Gohlke, AstraZeneca, Cambridge UK
Phase separation, generally occurring in lipid membranes and cell cytoplasms, has become a major focus when studying membrane-associated processes in the cell. Part of biophysicists' excitement about phase separation arises because it has the potential to drive sorting of proteins. Within membranes, the first images of micron-scale phase separation of lipids were published nearly 20 years ago, using membranes composed of high- and low-melting-temperature lipids with cholesterol (or other sterols). An early, highly influential article in the Biophysical Journal that explored the ground rules of phase separation in these types of membranes was “Separation of Liquid Phases in Giant Vesicles of Ternary Mixtures of Phospholipids and Cholesterol,” by Sarah Veatch and Sarah Keller (Biophys J., Nov 2003, DOI: 10.1016/S0006-3495(03)74726-2).
In this article, Veatch and Keller investigated giant unilamellar vesicles of ternary mixtures (e.g., high Tm (DPPC), low Tm (DOPC) and cholesterol) over a wide range of lipid compositions and temperature by fluorescence microscopy. Their resulting phase diagram was (and continues to be) widely used in the field. In this study, Veatch and Keller also found that liquid-ordered phases are enriched in saturated lipids, whereas liquid-disordered phases are enriched in unsaturated lipids. Furthermore, they explored the necessity of ternary vs. binary mixtures for micron-scale liquid phase coexistence, concluding that binary mixtures do not result in liquid phase separation. Their findings laid a foundation for future studies of ternary lipid mixtures and the influence of lipid phase separation on proteins. Their 2003 article is one of the most cited papers in the Biophysical Journal; it has inspired membrane biophysicists as well as scientists across wider fields.
The Impact
When I asked Sarah Keller and Sarah Veatch about how this paper impacted their career, Sarah Keller commented: “I was an assistant professor and Sarah Veatch was the first graduate student in my lab when we mapped the full, ternary phase diagram of a membrane with coexisting liquid phases. Professors and students bring different skills to projects. Professors bring experience. Much more importantly, grad students bring fresh eyes! Whenever I called the Lo phase the 'cholesterol-rich' phase (based on my prior experience with monolayers in Harden McConnell's lab), Sarah Veatch insisted that bilayers were different. She asserted that the Lo and Ld phases differed most in their unsaturated and saturated lipid content, and only slightly in their cholesterol content. In this paper, we demonstrated that she was right.
In retrospect, this paper has had huge impact. … We've heard from cell biologists that it inspired them to recast temperature changes in terms of miscibility. We've heard from physicists that the paper stimulated them to write new theory. …“
Sarah Veatch notes about her experience of writing the paper: "This was my first full-length paper and I was proud of it for sure — especially by how many conditions and key caveats it included, but I had no idea at the time that it would have such an impact. We started the project thinking that only a few lipid membranes might phase separate into Lo and Ld phases. We ended with a huge list of phase-separating systems, a linear relationship between melting temperature and demixing temperature, and a rubric that membranes that exhibit Lo and Ld phases must contain three types of components: a lipid with a high melting temperature, a lipid with a low melting temperature, and a sterol. Observations first mentioned in this paper led to major projects in our lab to measure critical point exponents, domain diffusion, coarsening rates, and the effect of changing lipid types. Looking at the data in this way also made it possible to draw connections to biological membranes, which contain many different protein and lipid components. “
Sarah Keller adds to the feedback they received for this paper: “Of course, impact is always clearer in hindsight. After presenting the very first, qualitative tie-lines of this paper at a Biophysical Society Meeting, I remember being intensely frustrated that so many speakers at the following BPS meeting still discussed Lo phases as arising solely from an increase in cholesterol. Sarah and I channelled the energy of that frustration into gathering incontrovertible, quantitative tie-lines. We used the outstanding networking opportunity of BPS meetings to initiate a long and fruitful collaboration with Klaus Gawrish and his NMR lab, which pushed us in directions we would not otherwise have pursued.
Today, when I re-read our paper, and I smile at our conclusion that 'clear, quantitative comparisons between different methods are needed, particularly since so many operational definitions and length-scales for 'rafts' are currently used.' This sentiment is as relevant now as it was in 2003. If anyone else wants to read our paper, it is freely accessible from the Biophysical Journal online!”
The Authors
Sarah L. Veatch is a membrane biophysicist who obtained her BSc at MIT, where she was also played on the rugby team. She was the first graduate student in the group of Keller Group at the University of Washington. After that she did postdoctoral research, most notably as a Miller Fellow at Cornell. She is now an Associate Professor at the University of Michigan focussing on how the composition of lipid membranes influences cellular functions. Her work has been recognized through several awards, for example the 2014 Margaret Oakley Dayhoff Award "for her substantial contributions to the field of membrane physical chemistry as it translates into biological systems."
Sarah L. Keller is the Duane and Barbara LaViolette Professor of Chemistry at the University of Washington, Seattle. She earned her PhD in physics from Princeton University. Her main research interest encompasses the study of complex phase behaviors within lipid membranes. Her research was greatly acknowledged through election as a Fellow of the American Physical Society and the Washington State Academy of Science. Within the Biophysical Society, she has been awarded the 2005 Margaret Oakley Dayhoff Award, the 2014 Tom Thompson Award, and the 2017 Avanti Award.
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