Biology subjects

Keller, S. L.

Publications and source records attributed to Keller, S. L..

8 recordsLinked to original sources

bioRxiv2026

Ups and downs of liquid-liquid transitions in GUV membranes from osmolarity and aspiration tensions

Lipid membranes undergo liquid-liquid phase separation to form micron-scale domains. Several physical parameters affect this phase transition. For example, a decrease in temperature causes membranes to demix, as does an increase in hydrostatic pressure. However, measurements to determine how tension affects membrane phase separation have yielded conflicting results. Experiments that have applied osmotic pressure differences to a population of vesicles have reported an increase in the membrane's miscibility transition temperature. Conversely, experiments that have applied micropipette aspiration or substrate stretching to single membranes have reported a decrease. Here, we find that both osmotic pressure and micropipette aspiration can increase transition temperatures. We discuss how membrane pores and hidden areas in membranes present challenges to researchers seeking to quantitatively convert experimentally measured osmolarity differences into membrane tensions. We show that challenges of comparing data from different osmotic pressure experiments can be mitigated by renormalizing osmolarity differences, specifically by dividing by the exterior osmolarity. We discuss our results in the context of existing theoretical predictions and in light of four known effects of increasing tension on vesicle membranes: 1) a reduction in hidden area of tubes and aggregates, 2) a reduction in out-of-plane thermal fluctuations, 3) an increase in the area per lipid, and 4) the formation of pores. First, hidden area can explain why vesicles can sustain high osmolarity differences. Next, suppression of thermal fluctuations and increases in the area per lipid may account for shifts in miscibility transition temperatures. Finally, pores can explain time dependences. Overall, our results highlight the need for new theory and simulation that unify predictions of how transition temperatures vary over all four regimes of membrane tension.

biophysics↗

bioRxiv2025

Micron-scale, liquid-liquid phase separation in ternary lipid membranes containing DPPE

Micron-scale, liquid-liquid phase separation occurs in membranes of living cells, with physiological consequences. To discover which lipids might support phase separation in cell membranes and how lipids might partition between phases, miscibility phase diagrams have been mapped for model membranes. Typically, model membranes are composed of ternary mixtures of a lipid with a high melting temperature, a lipid with a low melting temperature, and cholesterol. Phospholipids in ternary mixtures are chosen primarily to favor stable membranes (phosphatidylcholines and sphingomyelins) or add charge (phosphatidylglycerols and phosphatidylserines). A major class of phospholipids missing from experimental ternary diagrams has been the phosphatidylethanolamines (PEs). PE-lipids constitute up to 20 mol% of common biological membranes, where they influence protein function and facilitate membrane fusion. These biological effects are often attributed to PEs smaller headgroup, which leads to higher monolayer spontaneous curvatures and higher melting temperatures. Taken alone, the higher melting points of saturated PE-lipids imply that liquid-liquid phase separation should persist to higher temperatures in membranes containing PE-lipids. Here, we tested that hypothesis by substituting a saturated PE-lipid (DPPE) for its corresponding PC-lipid (DPPC) in two well-studied ternary membranes (DOPC/DPPC/cholesterol and DiphyPC/DPPC/cholesterol). We used fluorescence microscopy to map full ternary phase diagrams for giant vesicles over a range of temperatures. Surprisingly, we found no micron-scale, liquid-liquid phase separation in vesicles of the first mixture (DOPC/DPPE/cholesterol), and only a small region of liquid-liquid phase separation in the second mixture (DiphyPC/DPPE/cholesterol). Instead, coexisting solid and liquid phases were widespread, with the solid phase enriched in DPPE. An unusual feature of these ternary membranes is that solid and liquid-ordered phases can be distinguished by fluorescence microscopy, so tie-line directions can be estimated throughout the phase diagram, and transition temperatures to the 3-phase region (containing a liquid-disordered phase, a liquid-ordered phase, and a solid phase) can be accurately measured. SIGNIFICANCE STATEMENTUnder physiological conditions, yeast vacuole membranes phase separate into liquid phases. The resulting domains are microns in size, and they are important for the cells function. Yeast membranes contain a significant fraction of lipids with phosphatidylethanolamine headgroups. It was unknown whether these lipids enhanced liquid-liquid phase separation or hindered it. Here, we produced model membranes containing a saturated phosphatidylethanolamine lipid, we mapped miscibility phase diagrams over broad temperature ranges, and we compared our results to existing diagrams for membranes of other lipid types. We were surprised to find that the new lipid suppressed liquid-liquid phase separation compared to a lipid with a larger, phosphatidylcholine headgroup. Previous simulations incorporated phosphatidylethanolamine lipids, and our results provide an experimental basis for future simulations.

biophysics↗

bioRxiv2024

Common methods of making giant vesicles (except emulsion techniques) capture intended lipid ratios

Researchers choose different methods of making giant unilamellar vesicles in order to satisfy different constraints of their experimental designs. A challenge of using a variety of methods is that each may produce vesicles of different lipid compositions, even if all vesicles are made from a common stock mixture. Here, we use mass spectrometry to investigate ratios of lipids in vesicles made by five common methods: electroformation on indium tin oxide slides, electroformation on platinum wires, gentle hydration, emulsion transfer, and extrusion. We made vesicles from either 5-component or binary mixtures of lipids chosen to span a wide range of physical properties: di(18:1)PC, di(16:0)PC, di(18:1)PG, di(12:0)PE, and cholesterol. For a mixture of all five of these lipids, ITO electroformation, Pt electroformation, gentle hydration, and extrusion methods result in only minor shifts ([≤] 5 mol%) in lipid ratios of vesicles relative to a common stock solution. In contrast, emulsion transfer results in [~]80% less cholesterol than expected from the stock solution, which is counterbalanced by a surprising overabundance of saturated PC-lipid relative to all other phospholipids. Experiments using binary mixtures of some of the lipids largely support results from the 5-component mixture. Exact values of lipid ratios variations likely depend on the details of each method, so a broader conclusion is that experiments that increment lipid ratios in small steps will be highly sensitive to the method of lipid formation and to sample-to-sample variations, which are low (roughly {+/-}2 mol% in the 5-component mixture and either scale proportionally with increasing mole fraction or remain low). Experiments that increment lipid ratios in larger steps or that seek to explain general trends or new phenomena will be less sensitive to the method used. SIGNIFICANCE STATEMENTSmall changes to the amounts and types of lipids in membranes can drastically affect the membranes behavior. Unfortunately, it is unknown whether (or to what extent) different methods of making vesicles alter the ratios of lipids in membranes, even when identical stock solutions are used. This presents challenges for researchers when comparing data with colleagues who use different methods. Here, we measure ratios of lipid types in vesicle membranes produced by five methods. We assess each methods reproducibility and compare resulting vesicle compositions across methods. In doing so, we provide a quantitative basis that the scientific community can use to estimate whether differences between their results can be simply attributed to differences between methods or to sample-to-sample variations.

biophysics↗

bioRxiv2024

Sterol-lipids enable large-scale, liquid-liquid phase separation in membranes of only 2 components

Despite longstanding excitement and progress toward understanding liquid-liquid phase separation in natural and artificial membranes, fundamental questions have persisted about which molecules are required for this phenomenon. Except in extraordinary circumstances, the smallest number of components that has produced large-scale, liquid-liquid phase separation in bilayers has stubbornly remained at three: a sterol, a phospholipid with ordered chains, and a phospholipid with disordered chains. This requirement of three components is puzzling because only two components are required for liquid-liquid phase separation in lipid monolayers, which resemble half of a bilayer. Inspired by reports that sterols interact closely with lipids with ordered chains, we tested whether phase separation would occur in bilayers in which a sterol and lipid were replaced by a single, joined sterol-lipid. By evaluating a panel of sterol-lipids, some of which are found in bacteria, we discovered a minimal bilayer of only two components (PChemsPC and diPhyPC) that robustly demixes into micron-scale, liquid phases. It suggests a new role for sterol-lipids in nature, and it reveals a membrane in which tie-lines (and, therefore, the lipid composition of each phase) are straightforward to determine and will be consistent across multiple laboratories. Significance StatementA wide diversity of bilayer membranes, from those with hundreds of lipids (e.g., vacuoles of living yeast cells) to those with very few (e.g., artificial vesicles) phase separate into micron-scale liquid domains. The number of components required for liquid-liquid phase separation has been perplexing: only two should be necessary, but more are required except in extraordinary circumstances. What minimal set of molecular characteristics leads to liquid-liquid phase separation in bilayer membranes? This question inspired us to search for single, joined "sterol-lipid" molecules to replace both a sterol and a phospholipid in membranes undergoing liquid-liquid phase separation. By producing phase-separating membranes with only two components, we mitigate experimental challenges in determining tie-lines and in maintaining constant chemical potentials of lipids.

biophysics↗

bioRxiv2022

Remodeling of yeast vacuole membrane lipidomes from the log (1-phase) to stationary stage (2-phases)

Upon nutrient limitation, budding yeast of Saccharomyces cerevisiae shift from fast growth (the log stage) to quiescence (the stationary stage). This shift is accompanied by liquid-liquid phase separation in the membrane of the vacuole, an endosomal organelle. Recent work indicates that the resulting micron-scale domains in vacuole membranes enable yeast to survive periods of stress. An outstanding question is which molecular changes might cause this membrane phase separation. Here, we conduct lipidomics of vacuole membranes in both the log and stationary stages. Isolation of pure vacuole membranes is challenging in the stationary stage, when lipid droplets are in close contact with vacuoles. Immuno-isolation has previously been shown to successfully purify log-stage vacuole membranes with high organelle specificity, but it was not previously possible to immuno-isolate stationary stage vacuole membranes. Here, we develop Mam3 as a bait protein for vacuole immuno-isolation, and demonstrate low contamination by non-vacuolar membranes. We find that stationary stage vacuole membranes contain surprisingly high fractions of phosphatidylcholine lipids ([~]50%), roughly twice as much as log-stage membranes. Moreover, in the stationary stage these lipids have higher melting temperatures, due to longer and more saturated acyl chains. Another surprise is that no significant change in sterol content is observed. These results fit within the predominant view that phase separation in membranes requires at least three types of molecules to be present: lipids with high melting temperatures, lipids with low melting temperatures, and sterols. SIGNIFICANCE STATEMENTWhen budding yeast shift from growth to quiescence, the membrane of one of their organelles (the vacuole) undergoes liquid-liquid phase separation. What changes in the membranes lipids cause this phase transition? Here, we conduct lipidomics of immuno-isolated vacuole membranes. We analyze our data in the context of lipid melting temperatures, inspired by observations that liquid-liquid phase separation in model membranes requires a mixture of lipids with high melting temperatures, lipids with low melting temperatures, and sterols. We find that phase-separated vacuole membranes have higher concentrations of PC lipids, and that those lipids have higher melting temperatures. To conduct our experiments, we developed a tagged version of a protein (Mam3) for immuno-isolation of vacuole membranes.

biophysics↗

bioRxiv2021

Yeast cells actively tune their membranes to phase separate at temperatures that scale with growth temperatures

Membranes of vacuoles, the lysosomal organelles in yeast, undergo extraordinary changes during the cells normal growth cycle. The cycle begins with a stage of rapid cell growth. Then, as glucose becomes scarce, growth slows, and the vacuole membranes phase-separate into micron-scale liquid domains. Recent studies suggest that these domains are important for yeast survival by laterally organizing membrane proteins that play a key role in a central signaling pathway conserved among eukaryotes (TORC1). An outstanding question in the field has been whether yeast stringently regulate the phase transition and how they respond to new physical conditions. Here, we measure transition temperatures - an increase of roughly 15{degrees}C returns vacuole membranes to a state that appears uniform across a range of growth temperatures. We find that broad populations of yeast grown at a single temperature regulate the transition to occur over a surprisingly narrow temperature range. Moreover, the transition temperature scales linearly with the growth temperature, demonstrating that the cells physiologically adapt to maintain proximity to the transition. Next, we ask how yeast adjust their membranes to achieve phase separation. Specifically, we test how levels of ergosterol, the main sterol in yeast, induce or eliminate membrane domains. We isolate vacuoles from yeast during their rapid stage of growth, when their membranes do not natively exhibit domains. We find that membrane domains materialize when ergosterol is depleted, contradicting the assumption that increases in ergosterol cause membrane phase separation in vivo, and in agreement with prior studies that use artificial and cell-derived membranes. SIGNIFICANCE STATEMENTPhase separation in membranes creates domains enriched in specific components. To date, the best example of micron-scale phase separation in the membrane of an unperturbed, living cell occurs in a yeast organelle called the vacuole. Recent studies indicate that the phases are functionally important, enabling yeast survival during periods of cellular stress. We have discovered that yeast regulate this phase transition; the temperature at which membrane components mix into a single phase is ~15{degrees}C above the growth temperature. To maintain this offset, yeast may tune the level of ergosterol (a molecule that is structurally similar to cholesterol) in their membranes. We find that reducing sterol levels in vacuole membranes causes them to phase separate, in contrast to previous assumptions.

biophysics↗

bioRxiv2020

Ripples at edges of blooming lilies and torn plastic sheets

Ripples arise at edges of petals of blooming Lilium casablanca flowers and at edges of torn plastic sheets. In both systems, ripples are a consequence of excess length along the edge of a sheet. Through the use of both time-lapse videos of blooming lilies and still images of torn plastic sheets, we find that ripples in both systems are well-described by the scaling relationship [Formula], where a is amplitude, w is wavelength, and L is arc length. By approximating that the arc length is proportional to the wavelength, we recover a phenomenological relationship previously reported for self-similar ripple patterns, namely [<]a[>] {propto} [<]w[>]. Our observations imply that a broad class of systems in which morphological changes are driven by excess length along an edge will produce ripples described by [Formula]. Significance StatementEarly in the blooming process of Lilium casablanca flowers, large ripples appear in the edges of their petals. As blooming progresses, smaller ripples arise on top of the original ones. All ripples are characterized by three variables, an amplitude, a wavelength, and an arc length. Here, we derive an equation that relates these variables. To test the equation, we collect movies of blooming lilies. We find that the equation quantitatively describes single ripples in the petals. The equation is general, so it applies to all systems in which ripples arise by the same mechanism. To illustrate this point, we show that the equation holds for single ripples that appear at the edges of torn plastic sheets.

biophysics↗

bioRxiv2020

Direct Imaging of Liquid Domains in Membranes by Cryo Electron Tomography

Images of micron-scale domains in lipid bilayers have provided the gold standard of model-free evidence to understand the domains shapes, sizes, and distributions. Corresponding techniques to directly and quantitatively assess smaller (nanoscale and submicron) liquid domains have been lacking, leading to an inability to answer key questions. For example, researchers commonly seek to correlate activities of membrane proteins with attributes of the domains in which they reside; doing so hinges on identification and characterization of membrane domains. Although some features of membrane domains can be probed by indirect methods, these methods are often constrained by the limitation that data must be analyzed in the context of models that require multiple assumptions or parameters. Here, we address this challenge by developing and testing two new methods of identifying submicron domains in biomimetic membranes. Both methods leverage cryo-electron tomograms of ternary membranes under native solution conditions. The first method is optimized for probe-free applications: domains are directly distinguished from the surrounding membrane by their thickness. This technique measures area fractions of domains with quantitative accuracy, in excellent agreement with known phase diagrams. The second method is optimized for applications in which a single label is deployed for imaging membranes by both high-resolution cryo-electron tomography and diffraction-limited optical microscopy. For this method, we test a panel of probes, find that a trimeric mCherry label performs best, and specify criteria for developing future high-performance, dual-use probes. These developments have led to the first direct and quantitative imaging of submicron membrane domains under native conditions. SIGNIFICANCE STATEMENTFluorescence micrographs that capture the sizes, shapes, and distributions of liquid domains in model membranes have provided high standards of evidence to prove (and disprove) theories of how micron-scale domains form and grow. Corresponding theories about smaller domains have remained untested, partly because experimental methods of identifying submicron domains in vesicles under native solvent conditions have not been available. Here we introduce two such methods. Both leverage cryo-electron tomography to observe membrane features far smaller than the diffraction limit of light. The first method is probe-free and identifies differences in thicknesses between liquid domains and their surrounding membranes. The second method identifies membrane regions labeled by an electron-dense, fluorescent protein, which enables direct comparison of fluorescence micrographs with cryo-electron tomograms.

biophysics↗