Microfluidic measurement of the size and shape of lipid-anchored proteins.

The surface of a cell is crowded with membrane proteins. The size, shape, density, and mobility of extracellular surface proteins mediate cell surface accessibility to external molecules, viral particles, and other cells. However, predicting these qualities is not always straightforward, even when protein structures are known. We previously developed an experimental method for measuring flow-driven lateral transport of neutravidin bound to biotinylated lipids in supported lipid bilayers. Here, we use this method to detect hydrodynamic force applied to a series of lipid-anchored proteins with increasing size. We find that the measured force reflects both protein size and shape, making it possible to distinguish these features of intact, folded proteins in their undisturbed orientation and proximity to the lipid membrane. In addition, our results demonstrate that individual proteins are transported large distances by flow forces on the order of femtoNewtons, similar in magnitude to the shear forces resulting from blood circulation or from the swimming motion of microorganisms. Similar protein transport across living cells by hydrodynamic force may contribute to biological flow sensing.

Passive and reversible area regulation of supported lipid bilayers in response to fluid flow.

Biological and model membranes are frequently subjected to fluid shear stress. However, membrane mechanical responses to flow remain incompletely described. This is particularly true of membranes supported on a solid substrate, and the influences of membrane composition and substrate roughness on membrane flow responses remain poorly understood. Here, we combine microfluidics, fluorescence microscopy, and neutron reflectivity to explore how supported lipid bilayer patches respond to controlled shear stress. We demonstrate that lipid membranes undergo a significant, passive, and partially reversible increase in membrane area due to flow. We show that these fluctuations in membrane area can be constrained, but not prevented, by increasing substrate roughness. Similar flow-induced changes to membrane structure may contribute to the ability of living cells to sense and respond to flow.

Measuring flow-mediated protein drift across stationary supported lipid bilayers.

Fluid flow near biological membranes influences cell functions such as development, motility, and environmental sensing. Flow can laterally transport extracellular membrane proteins located at the cell-fluid interface. To determine whether this transport contributes to flow signaling in cells, quantitative knowledge of the forces acting on membrane proteins is required. Here, we demonstrate a method for measuring flow-mediated lateral transport of lipid-anchored proteins. We rupture giant unilamellar vesicles to form discrete patches of supported membrane inside rectangular microchannels and then allow proteins to bind to the upper surface of the membrane. While applying flow, we observe the formation of protein concentration gradients that span the membrane patch. By observing how these gradients dynamically respond to changes in applied shear stress, we determine the flow mobility of the lipid-anchored protein. We use simplified model membranes and proteins to demonstrate our method's sensitivity and reproducibility. Our intention was to design a quantitative, reliable method and analysis for protein mobility that we will use to compare flow transport for a variety of proteins, lipid anchors, and membranes in model systems and on living cells.

Systematic measurements of interleaflet friction in supported bilayers.

When lipid membranes curve or are subjected to strong shear forces, the two apposed leaflets of the bilayer slide past each other. The drag that one leaflet creates on the other is quantified by the coefficient of interleaflet friction, b. Existing measurements of this coefficient range over several orders of magnitude, so we used a recently developed microfluidic technique to measure it systematically in supported lipid membranes. Fluid shear stress was used to force the top leaflet of a supported membrane to slide over the stationary lower leaflet. Here, we show that this technique yields a reproducible measurement of the friction coefficient and is sensitive enough to detect differences in friction between membranes made from saturated and unsaturated lipids. Adding cholesterol to saturated and unsaturated membranes increased interleaflet friction significantly. We also discovered that fluid shear stress can reversibly induce gel phase in supported lipid bilayers that are close to the gel-transition temperature.

Structure, Dynamics, and Interactions of GPI-Anchored Human Glypican-1 with Heparan Sulfates in a Membrane.

Glypican-1 and its heparan sulfate (HS) chains play important roles in modulating many biological processes including growth factor signaling. Glypican-1 is bound to a membrane surface via a glycosylphosphatidylinositol (GPI)-anchor. In this study, we used all-atom molecular modeling and simulation to explore the structure, dynamics, and interactions of GPI-anchored glypican-1, three HS chains, membranes, and ions. The folded glypican-1 core structure is stable, but has substantial degrees of freedom in terms of movement and orientation with respect to the membrane due to the long unstructured C-terminal region linking the core to the GPI-anchor. With unique structural features depending on the extent of sulfation, high flexibility of HS chains can promote multi-site interactions with surrounding molecules near and above the membrane. This study is a first step toward all-atom molecular modeling and simulation of the glycocalyx, as well as its modulation of interactions between growth factors and their receptors.

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.

Expanded phenotypes and outcomes among 256 LGI1/CASPR2-IgG-positive patients.

OBJECTIVE: To describe an expanded phenotypic spectrum and longitudinal outcome in 256 LGI1-IgG-seropositive and/or CASPR2-IgG-seropositive patients. METHODS: Patients were identified through service neural autoantibody evaluation. Ninety-five had longitudinal follow-up (7-456 months; median = 35). RESULTS: Among 3,910 patients tested, 196 were LGI1-IgG positive, 51 were CASPR2-IgG positive, and 9 were dual positive. Cerebrospinal fluid testing was less sensitive than serum testing, detecting only 24 of 38 (63%) LGI1-IgG-positive and 5 of 6 (83%) CASPR2-IgG-positive patients. LGI1-IgG-positive specimens had higher voltage-gated potassium channel-IgG immunoprecipitation values (0.33nmol/l, range = 0.02-5.14) than CASPR2-IgG-positive specimens (0.10nmol/l, range = 0.00-0.45, p < 0.001). Of patients presenting with pain or peripheral nervous system (PNS) manifestations, 39% were LGI1-IgG seropositive (7% had solely neuropathy or pain). Multivariate analysis identified age as the only significant predictor of central nervous system (CNS) versus PNS involvement (>50 years; odds ratio = 15, p < 0.001). Paroxysmal dizziness spells (PDS), a unique LGI1-IgG accompaniment (14% of patients), frequently delayed the diagnosis. T2-mesiotemporal hyperintensity was more common in LGI1-IgG-positive (41%) than in CASPR2-IgG-positive patients (p = 0.033). T1-bright basal ganglia were confined to LGI1-IgG-positive patients with faciobrachial-dystonic seizures (9 of 39, 31%). Cancer was found in 44% of LGI1-IgG/CASPR2-IgG dual seropositive patients (one-third thymoma). Response to initial immunotherapy was favorable in 97%; mean modified Rankin score was 3 (range = 1-5) at onset and 1.74 (range = 0-6) at last follow-up, with 9% having severe refractory disability, 20% being asymptomatic, 28% receiving immunotherapy, and 58% receiving antiepileptic medication. INTERPRETATION: Older age is a strong predictor of CNS involvement in patients seropositive for CASPR2-IgG or LGI1-IgG. Pain, peripheral manifestations, and stereotypic paroxysmal dizziness spells are common with LGI1-IgG. Response to initial immunotherapy is often favorable, but some patients remain severely disabled, requiring long-term immunotherapy and/or antiepileptic medications. Ann Neurol 2017;82:79-92.

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.

Dynamics of a Volvox embryo turning itself inside out.

Deformations of cell sheets are ubiquitous in early animal development, often arising from a complex and poorly understood interplay of cell shape changes, division, and migration. Here, we explore perhaps the simplest example of cell sheet folding: the "inversion" process of the algal genus Volvox, during which spherical embryos turn themselves inside out through a process hypothesized to arise from cell shape changes alone. We use light sheet microscopy to obtain the first three-dimensional visualizations of inversion in vivo, and develop the first theory of this process, in which cell shape changes appear as local variations of intrinsic curvature, contraction and stretching of an elastic shell. Our results support a scenario in which these active processes function in a defined spatiotemporal manner to enable inversion.

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.

Imaging Giant Vesicle Membrane Domains with a Luminescent Europium Tetracycline Complex.

Microdomains in lipid bilayer membranes are routinely imaged using organic fluorophores that preferentially partition into one of the lipid phases, resulting in fluorescence contrast. Here, we show that membrane microdomains in giant unilamellar vesicles (GUVs) can be visualized with europium luminescence using a complex of europium III (Eu3+) and tetracycline (EuTc). EuTc is unlike typical organic lipid probes in that it is a coordination complex with a unique excitation/emission wavelength combination (396/617 nm), a very large Stokes shift (221 nm), and a very narrow emission bandwidth (8 nm). The probe preferentially interacts with liquid disordered domains in GUVs, which results in intensity contrast across the surface of phase-separated GUVs. Interestingly, EuTc also alters GM1 ganglioside partitioning. GM1 typically partitions into liquid ordered domains, but after labeling phase-separated GUVs with EuTc, cholera toxin B-subunit (CTxB), which binds GM1, labels liquid disordered domains. We also demonstrate that EuTc, but not free Eu3+ or Tc, significantly reduces lipid diffusion coefficients. Finally, we show that EuTc can be used to label cellular membranes similar to a traditional membrane probe. EuTc may find utility as a membrane imaging probe where its large Stokes shift and sharp emission band would enable multicolor imaging.

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.

Cellular organization in lab-evolved and extant multicellular species obeys a maximum entropy law.

The prevalence of multicellular organisms is due in part to their ability to form complex structures. How cells pack in these structures is a fundamental biophysical issue, underlying their functional properties. However, much remains unknown about how cell packing geometries arise, and how they are affected by random noise during growth - especially absent developmental programs. Here, we quantify the statistics of cellular neighborhoods of two different multicellular eukaryotes: lab-evolved 'snowflake' yeast and the green alga Volvox carteri. We find that despite large differences in cellular organization, the free space associated with individual cells in both organisms closely fits a modified gamma distribution, consistent with maximum entropy predictions originally developed for granular materials. This 'entropic' cellular packing ensures a degree of predictability despite noise, facilitating parent-offspring fidelity even in the absence of developmental regulation. Together with simulations of diverse growth morphologies, these results suggest that gamma-distributed cell neighborhood sizes are a general feature of multicellularity, arising from conserved statistics of cellular packing.

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 membrane's 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.