Aurelia R. Honerkamp-Smith

Critical Fluctuations in Plasma Membrane Vesicles

We demonstrate critical behavior in giant plasma membrane vesicles (GPMVs) that are isolated directly from living cells. GPMVs contain two liquid phases at low temperatures and one liquid phase at high temperatures and exhibit transition temperatures in the range of 15 to 25 degrees C. In the two-phase region, line tensions linearly approach zero as temperature is increased to the transition. In the one-phase region, micrometer-scale composition fluctuations occur and become increasingly large and long-lived as temperature is decreased to the transition. These results indicate proximity to a critical point and are quantitatively consistent with established theory. Our observations of robust critical fluctuations suggest that the compositions of mammalian plasma membranes are tuned to reside near a miscibility critical point and that heterogeneity corresponding to < 50 nm-sized compositional fluctuations are present in GPMV membranes at physiological temperatures. Our results provide new insights for plasma membrane heterogeneity that may be related to functional lipid raft domains in live cells.

Line Tensions, Correlation Lengths, and Critical Exponents in Lipid Membranes Near Critical Points

Membranes containing a wide variety of ternary mixtures of high chain-melting temperature lipids, low chain-melting temperature lipids, and cholesterol undergo lateral phase separation into coexisting liquid phases at a miscibility transition. When membranes are prepared from a ternary lipid mixture at a critical composition, they pass through a miscibility critical point at the transition temperature. Since the critical temperature is typically on the order of room temperature, membranes provide an unusual opportunity in which to perform a quantitative study of biophysical systems that exhibit critical phenomena in the two-dimensional Ising universality class. As a critical point is approached from either high or low temperature, the scale of fluctuations in lipid composition, set by the correlation length, diverges. In addition, as a critical point is approached from low temperature, the line tension between coexisting phases decreases to zero. Here we quantitatively evaluate the temperature dependence of line tension between liquid domains and of fluctuation correlation lengths in lipid membranes to extract a critical exponent, nu. We obtain nu = 1.2 +/- 0.2, consistent with the Ising model prediction nu = 1. We also evaluate the probability distributions of pixel intensities in fluorescence images of membranes. From the temperature dependence of these distributions above the critical temperature, we extract an independent critical exponent of beta = 0.124 +/- 0.03, which is consistent with the Ising prediction of beta = 1/8.

An introduction to critical points for biophysicists; observations of compositional heterogeneity in lipid membranes

Scaling laws associated with critical points have the power to greatly simplify our description of complex biophysical systems. We first review basic concepts and equations associated with critical phenomena for the general reader. We then apply these concepts to the specific biophysical system of lipid membranes. We recently reported that lipid membranes can contain composition fluctuations that behave in a manner consistent with the two-dimensional Ising universality class. Near the membranes critical point, these fluctuations are micron-sized, clearly observable by fluorescence microscopy. At higher temperatures, above the critical point, we expect to find submicron fluctuations. In separate work, we have reported that plasma membranes isolated directly from cells exhibit the same Ising behavior as model membranes do. We review other models describing submicron lateral inhomogeneity in membranes, including microemulsions, nanodomains, and mean field critical fluctuations, and we describe experimental tests that may distinguish these models.

Coarsening Dynamics of Domains in Lipid Membranes

We investigate isothermal diffusion and growth of micron-scale liquid domains within membranes of free-floating giant unilamellar vesicles with diameters between 80 and 250 μm. Domains appear after a rapid temperature quench, when the membrane is cooled through a miscibility phase transition such that coexisting liquid phases form. In membranes quenched far from a miscibility critical point, circular domains nucleate and then progress within seconds to late stage coarsening in which domains grow via two mechanisms 1), collision and coalescence of liquid domains, and 2), Ostwald ripening. Both mechanisms are expected to yield the same growth exponent, α = 1/3, where domain radius grows as time(α). We measure α = 0.28 ± 0.05, in excellent agreement. In membranes close to a miscibility critical point, the two liquid phases in the membrane are bicontinuous. A quench near the critical composition results in rapid changes in morphology of elongated domains. In this case, we measure α = 0.50 ± 0.16, consistent with theory and simulation.

Membrane Viscosity Determined from Shear-Driven Flow in Giant Vesicles

The viscosity of lipid bilayer membranes plays an important role in determining the diffusion constant of embedded proteins and the dynamics of membrane deformations, yet it has historically proven very difficult to measure. Here we introduce a new method based on quantification of the large-scale circulation patterns induced inside vesicles adhered to a solid surface and subjected to simple shear flow in a microfluidic device. Particle image velocimetry based on spinning disk confocal imaging of tracer particles inside and outside of the vesicle and tracking of phase-separated membrane domains are used to reconstruct the full three-dimensional flow pattern induced by the shear. These measurements show excellent agreement with the predictions of a recent theoretical analysis, and allow direct determination of the membrane viscosity.

Experimental Observations of Dynamic Critical Phenomena in a Lipid Membrane

Near a critical point, the time scale of thermally induced fluctuations diverges in a manner determined by the dynamic universality class. Experiments have verified predicted three-dimensional dynamic critical exponents in many systems, but similar experiments in two dimensions have been lacking for the case of conserved order parameter. Here we analyze the time-dependent correlation functions of a quasi-two-dimensional lipid bilayer in water to show that its critical dynamics agree with a recently predicted universality class. In particular, the effective dynamic exponent z(eff) crosses over from ~2 to ~3 as the correlation length of fluctuations exceeds a hydrodynamic length set by the membrane and bulk viscosities.

Transbilayer Colocalization of Lipid Domains Explained via Measurement of Strong Coupling Parameters

When micron-scale compositional heterogeneity develops in membranes, the distribution of lipids on one face of the membrane strongly affects the distribution on the other. Specifically, when lipid membranes phase separate into coexisting liquid phases, domains in each monolayer leaflet of the membrane are colocalized with domains in the opposite leaflet. Colocalized domains have never been observed to spontaneously move out of registry. This result indicates that the lipid compositions in one leaflet are strongly coupled to compositions in the opposing leaflet. Predictions of the interleaflet coupling parameter, Λ, vary by a factor of 50. We measure the value of Λ by applying high shear forces to supported lipid bilayers. This causes the upper leaflet to slide over the lower leaflet, moving domains out of registry. We find that the threshold shear stress required to deregister domains in the upper and lower leaflets increases with the inverse length of domains. We derive a simple, closed-form expression relating the threshold shear to Λ, and find Λ = 0.016 ± 0.004 kBT/nm2.

Solubility limits of cholesterol, lanosterol, ergosterol, stigmasterol, and β-sitosterol in electroformed lipid vesicles

Here we use nuclear magnetic resonance to measure the solubility limit of several biologically relevant sterols in electroformed giant unilamellar vesicle membranes containing phosphatidylcholine (PC) lipids in ratios of 1:1:X DOPC:DPPC:sterol. We find solubility limits of cholesterol, lanosterol, ergosterol, stigmasterol, and β-sitosterol to be 65-70%, ~35%, 30-35%, 20-25%, and ~40%, respectively. The low solubilities of stigmasterol and β-sitosterol, which differ from cholesterol only in their alkyl tails, show that subtle differences in tail structure can strongly affect sterol solubility. Below the solubility limits, the fraction of sterol to PC-lipid in electroformed vesicles linearly reflects the fraction in the original stock solutions used in the electroformation process.

The noisy basis of morphogenesis: Mechanisms and mechanics of cell sheet folding inferred from developmental variability

Variability is emerging as an integral part of development. It is therefore imperative to ask how to access the information contained in this variability. Yet most studies of development average their observations and, discarding the variability, seek to derive models, biological or physical, that explain these average observations. Here, we analyse this variability in a study of cell sheet folding in the green alga Volvox, whose spherical embryos turn themselves inside out in a process sharing invagination, expansion, involution, and peeling of a cell sheet with animal models of morphogenesis. We generalise our earlier, qualitative model of the initial stages of inversion by combining ideas from morphoelasticity and shell theory. Together with three-dimensional visualisations of inversion using light sheet microscopy, this yields a detailed, quantitative model of the entire inversion process. With this model, we show how the variability of inversion reveals that two separate, temporally uncoupled processes drive the initial invagination and subsequent expansion of the cell sheet. This implies a prototypical transition towards higher developmental complexity in the volvocine algae and provides proof of principle of analysing morphogenesis based on its variability.

Using in vivo Optical Sectioning to Investigate Mechanical Aspects of Volvox Development

Volvox is a genus of swimming algae consisting of a spherical single sheet of cells. At the end of cell division, embryos form a sphere with their flagella pointing the wrong way (to the inside) and must complete their development by turning themselves inside out. Although this phenomenon was observed hundreds of years ago and has been the subject of extensive study, no quantification of the mechanics has been performed. The simple geometry and connectivity of the cells makes these organisms a tractable example for studying morphogenic processes, while their development still shares features with more complicated mechanisms of gastrulation in animals. Previous study of embryo shapes during inversion required chemical fixation, so that individuals could not be followed through all stages and dynamics were lost. An open-source selective plane illumination microscope (SPIM) [1], has enabled accurate recording of the shapes of embryos as they progress through their inversion process. Unprecedented views of the progress of cell division and the growth of mature spheroids are also within reach. With this dynamic, three-dimensional data, new analysis of embryo and tissue mechanics become possible.[1] Pitrone P. G., Schindelin J., Stuyvenberg L., Preibisch S., Weber M.; Eliceiri K. W., Huisken J., Tomancak P. OpenSPIM: an open access light sheet microscopy platform Nature Methods 10, 598-599 (2013).