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.

Macrophages are known to engulf small membrane fragments, or trogocytose, target cells and pathogens, rather than fully phagocytose them. However, little is known about what causes macrophages to choose trogocytosis versus phagocytosis. Here, we report that cortical tension of target cells is a key regulator of macrophage trogocytosis. At low tension, macrophages will preferentially trogocytose antibody-opsonized cells, while at high tension they tend towards phagocytosis. Using model vesicles, we demonstrate that macrophages will rapidly switch from trogocytosis to phagocytosis when membrane tension is increased. Stiffening the cortex of target cells also biases macrophages to phagocytose them, a trend that can be countered by increasing antibody surface density and is captured in a mechanical model of trogocytosis. This work suggests that the target cell, rather than the macrophage, determines phagocytosis versus trogocytosis, and that macrophages do not require a distinct molecular pathway for trogocytosis.

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 PE's 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 phosphatidylcholine 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 three-phase region (containing a liquid-disordered phase, a liquid-ordered phase, and a solid phase) can be accurately measured.

Coexisting liquid-ordered (Lo) and liquid-disordered (Ld) phases are frequently observed in the membranes of giant unilamellar vesicles composed of two phospholipid types and cholesterol. The separation of coexisting phases in a membrane varies with both temperature and composition. To date, demixing of membranes into Lo and Ld phases has been investigated in ternary systems that have primarily included cholesterol and lipids like phosphatidylcholines (PC), sphingomyelins (SM), phosphatidylserines (PS), and phosphatidylglycerols (PG). Although phosphatidylethanolamine (PE) lipids comprise about 20% of the cytoplasmic leaflet of red blood cells, little is known about the demixing of membranes containing PE lipids. In part, this is because PE lipids are cone-shaped, which drives transitions from lamellae to inverted hexagonal phases, making measurements challenging. To better understand how PE lipids affect membrane phase behavior, we study several ternary systems that contain PE lipids. Here, we map the phase diagram for a ternary membrane in which one of the lipid types is a PE lipid. We compare our data to analogous membranes containing cholesterol and PC lipids.

Hertwig's rule states that cells divide along their longest axis, usually driven by forces acting on the mitotic spindle. Here, we show that in contrast to this rule, microtubule-based pulling forces in early Caenorhabditis elegans embryos align the spindle with the short axis of the cell. We combine theory with experiments to reveal that in order to correct this misalignment, inward forces generated by the constricting cytokinetic ring rotate the entire cell until the spindle is aligned with the cell's long axis. Experiments with slightly compressed mouse zygotes indicate that this cytokinetic ring-driven mechanism of ensuring Hertwig's rule is general for cells capable of rotating inside a confining shell, a scenario that applies to early cell divisions of many systems.

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 micrometer-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 (∼40%), 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 lipidomic changes, which are largely reflected on the whole-cell level, 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.

Liquid-liquid phase separation in living biological membranes is usually described as occurring on sub-micron length scales. A stunning counterexample occurs in S. cerevisiae. When the yeast shift from the log stage of growth to the stationary stage, huge, micron-scale liquid domains appear in the membranes of the vacuole, an endosomal organelle. These phases are functionally important, enabling yeast survival during periods of stress. This talk will review recent results showing: (1) This miscibility transition is reversible as would be expected from equilibrium thermodynamics, even though it occurs in a living system. (2) Yeast actively regulate this phase transition to hold the membrane transition ∼15°C above the yeast growth temperature. (3) Yeast significantly remodel their vacuole lipidomes in the shift from the log stage to the stationary stage.

Hybrid vesicles consisting of phospholipids and block-copolymers are increasingly finding applications in science and technology. Herein, small angle X-ray scattering (SAXS) and cryo-electron tomography (cryo-ET) were used to obtain detailed structural information about hybrid vesicles with different ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and poly(1,2-butadiene-block-ethylene oxide) (PBd22-PEO14, Ms = 1800 gmol-1) . Using single particle analysis (SPA) we were able to further interpret the information gained from SAXS and cryo-ET experiments, showing that increasing PBd22-PEO14 mole fraction increases the membrane thickness from 52 Å for a pure lipid system to 97 Å for pure PBd22-PEO14 vesicles. We find two vesicle populations with different membrane thicknesses in hybrid vesicle samples. As these lipids and polymers are known to homogeneously mix, bistability is inferred between weak and strong interdigitation regimes of PBd22-PEO14 within the hybrid membranes. It is hypothesized that membranes of intermediate structure are not energetically favourable. Therefore, upon formation, each vesicle selects one of these two membrane structures, which are assumed to have comparable free energies. We conclude that, by combining biophysical methods, an accurate determination of the influence of composition on the structural properties of hybrid membranes is achieved, revealing that two distinct membranes structures can coexist in homogeneously mixed lipid-polymer hybrid vesicles.

ABSTRACT Membranes of vacuoles, the lysosomal organelles in yeast, undergo extraordinary changes during the cell’s 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°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 STATEMENT Phase 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°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.

The first cell membranes were likely composed of single-chain amphiphiles such as fatty acids. An open question is whether fatty acid membranes could have functioned within evaporative lakes on the early Earth, which have been hypothesized to concentrate prebiotic reactants. Evaporation also concentrates monovalent salts, which in turn cause fatty acid membrane vesicles to flocculate; significant loss of encapsulated contents during flocculation would have impeded early cell evolution. Here, we tested whether fatty acid vesicles retain encapsulated contents after flocculation and after drying. We found that vesicles composed of 2:1 decanoic acid:decanol encapsulate calcein dye throughout a process of flocculation in saturated salt solution and subsequent disaggregation of vesicles by dilution of the salt. However, 30 minutes of complete dehydration disrupted encapsulation by fatty acid vesicles. In contrast, phospholipid vesicles maintained encapsulation. Our results reveal a selective pressure for protocells to incorporate phospholipids: while fatty acid membranes can retain encapsulated contents during periods of dilute and saturating salt, phospholipids are necessary for encapsulation during dry periods. Our results are consistent with the hypothesis that evaporative lakes were productive sites for prebiotic chemistry and the origin of cells.

Phagocytosis, the process by which a cell engulfs a target, is a key effector function of the mammalian immune system. Macrophages are phagocytes responsible for detecting and eliminating immunological threats, such as bacterial and parasitic pathogens. In addition, macrophages must phagocytose targets along a spectrum of human physiology and disease, varying from atherosclerotic cholesterol crystals to cancerous and apoptotic cells. To initiate phagocytosis, macrophages must integrate specific biochemical and physical properties of these diverse targets.

The first cellular compartments were likely composed of fatty acids. Saturated fatty acids are widely considered to have been available on the early Earth due to their presence on carbonaceous meteorites and potential abiotic synthesis via Fischer-Tropsch reactions, and they spontaneously self-assemble into membranes across a range of pH and temperature conditions. However, high concentrations of monovalent salts cause fatty acid membranes to flocculate. If flocculation causes the loss of membrane contents, it would have impeded early cell evolution. This problem is potentially serious because evaporative lake environments (where dissolved salts reach saturating concentrations due to evaporation) are otherwise attractive sites for prebiotic chemistry. We employed size-exclusion chromatography and fluorescence microscopy to determine whether flocculated vesicles retain their contents. We show that membranes composed of decanoic acid maintain encapsulation of calcein during salt-induced flocculation and subsequent regeneration of unaggregated vesicles by dilution. The ability to retain encapsulated contents during periods of salt-induced flocculation would have been critical to the development of the earliest cells in evaporative lake environments, and strengthens the case for evaporative environments as productive hotspots for prebiotic chemistry.

Membrane invagination and vesicle formation are key steps in endocytosis and cellular trafficking. Here, we show that endocytic coat proteins with prion-like domains (PLDs) form hemispherical puncta in the budding yeast, Saccharomyces cerevisiae These puncta have the hallmarks of biomolecular condensates and organize proteins at the membrane for actin-dependent endocytosis. They also enable membrane remodeling to drive actin-independent endocytosis. The puncta, which we refer to as endocytic condensates, form and dissolve reversibly in response to changes in temperature and solution conditions. We find that endocytic condensates are organized around dynamic protein-protein interaction networks, which involve interactions among PLDs with high glutamine contents. The endocytic coat protein Sla1 is at the hub of the protein-protein interaction network. Using active rheology, we inferred the material properties of endocytic condensates. These experiments show that endocytic condensates are akin to viscoelastic materials. We use these characterizations to estimate the interfacial tension between endocytic condensates and their surroundings. We then adapt the physics of contact mechanics, specifically modifications of Hertz theory, to develop a quantitative framework for describing how interfacial tensions among condensates, the membrane, and the cytosol can deform the plasma membrane to enable actin-independent endocytosis.

Images of micrometer-scale domains in lipid bilayers have pro-vided the gold standard of model-free evidence to understand the domains' shapes, sizes, and distributions. Corresponding tech-niques to directly and quantitatively assess smaller (nanoscale and submicron) liquid domains have been limited. Researchers com-monly seek to correlate activities of membrane proteins with at-tributes of the domains in which they reside; doing so hinges on identification and characterization of membrane domains. Al-though some features of membrane domains can be probed by indirect methods, these methods are often constrained by the lim-itation 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 methods of identifying submicron domains in biomimetic membranes. Both methods le-verage cryo-electron tomograms of ternary membranes under vit-rified, hydrated conditions. The first method is optimized for probe-free applications: Domains are directly distinguished from the surrounding membrane by their thickness. This technique quantitatively and accurately measures area fractions of domains, 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 direct and quantitative imaging of submicron mem-brane domains in vitrified, hydrated vesicles.

Direct imaging of membrane domains has provided evidence necessary to answer a wide range of scientific questions; direct imaging has been used to prove that vacuole membranes phase separate in living yeast cells and to show that current theories of modulated phases are incomplete. Many deep questions about membrane domains remain extremely challenging to answer because our current arsenal of direct imaging techniques is incomplete. For example, the existence of sub-micron domains in model membranes (and in some cases, the cell plasma membrane) has been inferred by indirect methods such as FRET, neutron scattering, and NMR. In cases where indirect methods require assumption-dependent models, it is important to verify the results directly with microscopy. To overcome current limitations, new methods of imaging sub-micron membrane domains are needed. Here, we present two novel cryo-EM methods of imaging sub-micron lipid domains in ternary model membranes. The first method employs a monovalent probe that is both fluorescent and electron-dense, enabling direct, and model-free comparisons for a single vesicle analyzed by both fluorescence microscopy and electron microscopy. The second method is entirely label-free; it identifies domains that are thicker than the surrounding membrane. To validate this method, we leverage existing phase diagrams (obtained through fluorescence microscopy) and AFM data to identify compositions with a membrane thickness difference between the Lo phase and Ld phase of ≥ 1 nm. We benchmark our techniques against known phase diagrams and demonstrate that our new methods accurately quantify the area fractions of each phase.

A major challenge in understanding how biological cells arose on the early Earth is explaining how RNA and membranes originally colocalized. We propose that the building blocks of RNA (nucleobases and ribose) bound to self-assembled prebiotic membranes. We have previously demonstrated that the bases bind to membranes composed of a prebiotic fatty acid, but evidence for the binding of sugars has remained a technical challenge. Here, we used pulsed-field gradient NMR spectroscopy to demonstrate that ribose and other sugars bind to membranes of decanoic acid. Moreover, the binding of some bases is strongly enhanced when they are linked to ribose to form a nucleoside or - with the addition of phosphate - a nucleotide. This enhanced binding could have played a role in the molecular evolution leading to the production of RNA.

Membranes of vacuoles, the lysosomal organelle in yeast, phase separate into two coexisting liquid phases. This phenomenon provides a physical mechanism for lateral organization of biological membranes. Recent studies show that these domains play a key role in a central eukaryotic signaling pathway. Howthe cell adapts and tunes its vacuole membrane to invoke phase separation is still largely unknown. Here we examine how membrane composition changes due to two different environmental stimuli: growth temperature and nutrient depletion. If it is important for cells to regulate phase separation of their membranes, then we expect yeast cells to acclimate to external conditions. We test this hypothesis by growing yeast at 25°C and 30°C and find that the yeast adapts to this change by dramatically shifting the demixing temperature of vacuole membranes (to 29.7°C and 42.7°C respectively). This stark change in vacuole miscibility temperatures scales with growth temperature both in live cells and in isolated vacuoles, showing that yeast alter their membrane compositions to maintain phase separation. We also directly investigate the role of ergosterol, the main sterol in yeast, in domain formation in vacuoles. The yeast growth cycle starts with a logarithmic phase of rapid growth followed by a stationary phase in which glucose becomes scarce and vacuole membranes exhibit coexisting liquid domains. We isolate vacuoles from yeast in the logarithmic phase of growth, in which the membranes do not natively exhibit domains. Using fluorescence microscopy, we observe changes in vacuole phase separation as a function of ergosterol content. We find the surprising result that domains appear in vacuole membranes with reduced ergosterol.