Bioinspired Membrane Interfaces: Controlling Actomyosin Architecture and Contractility.

The creation of biologically inspired artificial lipid bilayers on planar supports provides a unique platform to study membrane-confined processes in a well-controlled setting. At the plasma membrane of mammalian cells, the linkage of the filamentous (F)-actin network is of pivotal importance leading to cell-specific and dynamic F-actin architectures, which are essential for the cell's shape, mechanical resilience, and biological function. These networks are established through the coordinated action of diverse actin-binding proteins and the presence of the plasma membrane. Here, we established phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2)-doped supported planar lipid bilayers to which contractile actomyosin networks were bound via the membrane-actin linker ezrin. This membrane system, amenable to high-resolution fluorescence microscopy, enabled us to analyze the connectivity and contractility of the actomyosin network. We found that the network architecture and dynamics are not only a function of the PtdIns[4,5]P2 concentration but also depend on the presence of negatively charged phosphatidylserine (PS). PS drives the attached network into a regime, where low but physiologically relevant connectivity to the membrane results in strong contractility of the actomyosin network, emphasizing the importance of the lipid composition of the membrane interface.

Membrane fusion studied by colloidal probes.

Membrane-coated colloidal probes combine the benefits of solid-supported membranes with a more complex three-dimensional geometry. This combination makes them a powerful model system that enables the visualization of dynamic biological processes with high throughput and minimal reliance on fluorescent labels. Here, we want to review recent applications of colloidal probes for the study of membrane fusion. After discussing the advantages and disadvantages of some classical vesicle-based fusion assays, we introduce an assay using optical detection of fusion between membrane-coated glass microspheres in a quasi two-dimensional assembly. Then, we discuss free energy considerations of membrane fusion between supported bilayers, and show how colloidal probes can be combined with atomic force microscopy or optical tweezers to access the fusion process with even greater detail.

Rheology of Membrane-Attached Minimal Actin Cortices.

The actin cortex is a thin cross-linked network attached to the plasma membrane, which is responsible for the cell's shape during migration, division, and growth. In a reductionist approach, we created a minimal actin cortex (MAC) attached to a lipid membrane to correlate the filamentous actin architecture with its viscoelastic properties. The system is composed of a supported 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine bilayer doped with the receptor lipid phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P2) to which a constitutively active mutant of ezrin, which is a direct membrane-cytoskeleton linker, is bound. The formation of the MAC on the supported lipid bilayer is analyzed as a function of increasing PtdIns(4,5)P2/ezrin pinning points, revealing an increase in the intersections between actin filaments, that is, the node density of the MAC. Bead tracking microrheology on the membrane-attached actin network provides information about its viscoelastic properties. The results show that ezrin serves as a dynamic cross-linker for the actin cortex attached to the lipid bilayer and that the stiffness of the network is influenced by the pinning point density, relating the plateau storage modulus G0 to the node density of the MAC.

Size and mobility of lipid domains tuned by geometrical constraints.

In the plasma membrane of eukaryotic cells, proteins and lipids are organized in clusters, the latter ones often called lipid domains or "lipid rafts." Recent findings highlight the dynamic nature of such domains and the key role of membrane geometry and spatial boundaries. In this study, we used porous substrates with different pore radii to address precisely the extent of the geometric constraint, permitting us to modulate and investigate the size and mobility of lipid domains in phase-separated continuous pore-spanning membranes (PSMs). Fluorescence video microscopy revealed two types of liquid-ordered (lo) domains in the freestanding parts of the PSMs: (i) immobile domains that were attached to the pore rims and (ii) mobile, round-shaped lo domains within the center of the PSMs. Analysis of the diffusion of the mobile lo domains by video microscopy and particle tracking showed that the domains' mobility is slowed down by orders of magnitude compared with the unrestricted case. We attribute the reduced mobility to the geometric confinement of the PSM, because the drag force is increased substantially due to hydrodynamic effects generated by the presence of these boundaries. Our system can serve as an experimental test bed for diffusion of 2D objects in confined geometry. The impact of hydrodynamics on the mobility of enclosed lipid domains can have great implications for the formation and lateral transport of signaling platforms.

Geometry of the Contact Zone between Fused Membrane-Coated Beads Mimicking Cell-Cell Fusion.

The fusion of lipid membranes is a key process in biology. It enables cells and organelles to exchange molecules with their surroundings, which otherwise could not cross the membrane barrier. To study such complex processes we use simplified artificial model systems, i.e., an optical fusion assay based on membrane-coated glass spheres. We present a technique to analyze membrane-membrane interactions in a large ensemble of particles. Detailed information on the geometry of the fusion stalk of fully fused membranes is obtained by studying the diffusional lipid dynamics with fluorescence recovery after photobleaching experiments. A small contact zone is a strong obstruction for the particle exchange across the fusion spot. With the aid of computer simulations, fluorescence-recovery-after-photobleaching recovery times of both fused and single-membrane-coated beads allow us to estimate the size of the contact zones between two membrane-coated beads. Minimizing delamination and bending energy leads to minimal angles close to those geometrically allowed.

Size, Kinetics, and Free Energy of Clusters Formed by Ultraweak Carbohydrate-Carbohydrate Bonds.

Weak noncovalent intermolecular interactions play a pivotal role in many biological processes such as cell adhesion or immunology, where the overall binding strength is controlled through bond association and dissociation dynamics as well as the cooperative action of many parallel bonds. Among the various molecules participating in weak bonds, carbohydrate-carbohydrate interactions are probably the most ancient ones allowing individual cells to reversibly enter the multicellular state and to tell apart self and nonself cells. Here, we scrutinized the kinetics and thermodynamics of small homomeric Lewis X-Lewis X ensembles formed in the contact zone of a membrane-coated colloidal probe and a solid supported membrane ensuring minimal nonspecific background interactions. We used an atomic force microscope to measure force distance curves at Piconewton resolution, which allowed us to measure the force due to unbinding of the colloidal probe and the planar membrane as a function of contact time. Applying a contact model, we could estimate the free binding energy of the formed adhesion cluster as a function of dwell time and thereby determine the precise size of the contact zone, the number of participating bonds, and the intrinsic rates of association and dissociation in the presence of calcium ions. The unbinding energy per bond was found to be on the order of 1 kBT. Approximately 30 bonds were opened simultaneously at an off-rate of koff = 7 ± 0.2 s(-1).

Resolving single membrane fusion events on planar pore-spanning membranes.

Even though a number of different in vitro fusion assays have been developed to analyze protein mediated fusion, they still only partially capture the essential features of the in vivo situation. Here we established an in vitro fusion assay that mimics the fluidity and planar geometry of the cellular plasma membrane to be able to monitor fusion of single protein-containing vesicles. As a proof of concept, planar pore-spanning membranes harboring SNARE-proteins were generated on highly ordered functionalized 1.2 µm-sized pore arrays in Si3N4. Full mobility of the membrane components was demonstrated by fluorescence correlation spectroscopy. Fusion was analyzed by two color confocal laser scanning fluorescence microscopy in a time resolved manner allowing to readily distinguish between vesicle docking, intermediate states such as hemifusion and full fusion. The importance of the membrane geometry on the fusion process was highlighted by comparing SNARE-mediated fusion with that of a minimal SNARE fusion mimetic.

Optical fusion assay based on membrane-coated spheres in a 2D assembly.

A major goal in neurophysiology and research on enveloped viruses is to understand and control the biology and physics of membrane fusion and its inhibition as a function of lipid and protein composition. This poses an experimental challenge in the realization of fast and reliable assays that allow us, with a minimal use of fluorescent or radioactive labels, to identify the different stages of membrane-membrane interaction ranging from docking to complete membrane merging. Here, an optical two-dimensional fusion assay based on monodisperse membrane-coated microspheres is introduced, allowing unequivocal assignment of docking and membrane fusion. The hard-sphere fluid captures and quantifies relevant stages of membrane fusion and its inhibition without interference from aggregation, liposome rupture, extensive fluorescence labeling, and light scattering. The feasibility of the approach is demonstrated by using an established model system based on coiled-coil heterodimers formed between two opposing membrane-coated microspheres.

Ultra-slow dynamics in low density amorphous ice revealed by deuteron NMR: indication of a glass transition.

The postulated glass-liquid transition of low density amorphous ice (LDA) is investigated with deuteron NMR stimulated echo experiments. Such experiments give access to ultra-slow reorientations of water molecules on time scales expected for structural relaxation of glass formers close to the glass-liquid transition temperature. An involved data analysis is necessary to account for signal contributions originating from a gradual crystallization to cubic ice. Even if some ambiguities remain, our findings support the view that pressure amorphized LDA ices are of glassy nature and undergo a glass-liquid transition before crystallization.

Collective behavior of Dictyostelium discoideum monitored by impedance analysis.

Dictyostelium discoideum cells respond to periodic signals of extracellular cAMP by collective changes of cell-cell and cell-substrate contacts. This was confirmed by dielectric analysis employing electric cell-substrate impedance sensing (ECIS) and impedance measurements involving cell-filled micro channels in conjunction with optical microscopy providing a comprehensive picture of chemotaxis under conditions of starvation.

Chemotaxis of Dictyostelium discoideum: collective oscillation of cellular contacts.

Chemotactic responses of Dictyostelium discoideum cells to periodic self-generated signals of extracellular cAMP comprise a large number of intricate morphological changes on different length scales. Here, we scrutinized chemotaxis of single Dictyostelium discoideum cells under conditions of starvation using a variety of optical, electrical and acoustic methods. Amebas were seeded on gold electrodes displaying impedance oscillations that were simultaneously analyzed by optical video microscopy to relate synchronous changes in cell density, morphology, and distance from the surface to the transient impedance signal. We found that starved amebas periodically reduce their overall distance from the surface producing a larger impedance and higher total fluorescence intensity in total internal reflection fluorescence microscopy. Therefore, we propose that the dominant sources of the observed impedance oscillations observed on electric cell-substrate impedance sensing electrodes are periodic changes of the overall cell-substrate distance of a cell. These synchronous changes of the cell-electrode distance were also observed in the oscillating signal of acoustic resonators covered with amebas. We also found that periodic cell-cell aggregation into transient clusters correlates with changes in the cell-substrate distance and might also contribute to the impedance signal. It turned out that cell-cell contacts as well as cell-substrate contacts form synchronously during chemotaxis of Dictyostelium discoideum cells.

Limits of metastability in amorphous ices: 2H-NMR relaxation.

The high-frequency reorientation dynamics of O-(2)H bonds is investigated in various amorphous ices including eHDA (expanded high density amorphous ice), LDA-II (low density amorphous ice II) and HGW (hyperquenched glassy water) using (2)H-NMR spin-lattice relaxation as a local probe. Both low density forms, HGW and LDA-II, show similar spin-lattice relaxation but differ in the thermal stability with respect to the transition into crystalline cubic ice I(c). HGW already transforms slightly above 135 K whereas LDA-II crystallizes at 150 K. eHDA is distinguishable from other high density amorphous ices in its thermal stability and spin-lattice relaxation. Its relaxation times are much larger compared to those of VHDA (very high density amorphous ice) and uHDA (unrelaxed high density amorphous ice). eHDA does not show annealing effects, transforms sharply into LDA-II above 123 K and provides higher thermal stability as compared to other high density forms.

Energetics of adhesion cluster formation in the context of biological membranes.

The orchestration of cellular adhesion is an intricate process that involves a multitude of specialized proteins but at the same time follows a simple physical plan. It is still not totally known why cells create clusters of bonds to adhere on a substrate. Besides experimental methods to study cellular adhesion there exist several different theoretical models to describe the stability of focal adhesion clusters. The model proposed in this work makes it possible to understand the formation of adhesion bond clusters. It explains the emergence of a long-range bond-bond attraction that originates from the finite membrane elasticity as the driving force of cluster growth. In combination with a thermally driven bond disintegration such a model will eventually be able to explain the kinetic stability of finite size bond clusters. The model extends conventional two-state models where bonds are either open or closed by introducing a rich scenario of metastable states associated with hysteretic behavior in the bond closure and rupture dynamics.

Limits of metastability in amorphous ices: the neutron scattering Debye-Waller factor.

Recently, it became clear that relaxation effects in amorphous ices play a very important role that has previously been overlooked. The thermodynamic history of amorphous samples strongly affects their transition behavior. In particular, well-relaxed samples show higher thermal stability, thereby providing a larger window to investigate their glass transitions. We here present neutron scattering experiments using fixed elastic window scans on relaxed forms of amorphous ice, namely expanded high density amorphous ice (eHDA), a variant of low density amorphous ice (LDA-II) and hyperquenched glassy water (HGW). These amorphous ices are expected to be true glassy counterparts of deeply supercooled liquid water, therefore fast precursor dynamics of structural relaxation are expected to appear below the calorimetric glass transition temperature. The Debye-Waller factor shows a very weak sub-T(g) anomaly in some of the samples, which might be the signature of such fast precursor dynamics. However, we cannot find this behavior consistently in all samples at all reciprocal length scales of momentum transfer.

Colloidal probe microscopy of membrane-membrane interactions: from ligand-receptor recognition to fusion events.

A versatile model system to study membrane-membrane interactions in great detail is introduced. Based on colloidal probe microscopy with membrane covered spherical probes attached to tip-less cantilevers the interaction forces and adhesion energies are quantified down to single molecule resolution. Two opposing membranes equipped with ligands on one side and receptors on the other side were brought in contact at a defined load and pulled apart at constant velocity. Ni-NTA functionalized lipids served as receptors on the probe, while lipopeptides displaying short His-tags (CGGH(6) or CGWH(6)) were incorporated in the planar supporting membrane on a silicon substrate. The rather intricate force distance curves were scrutinized in terms of breakthrough events upon contact of the probe with the surface, the overall work of adhesion, maximum adhesion force, as well as formation frequency, lifetime, and force of membrane tethers suggesting that hemifusion of the two opposing bilayers takes place.

Statistics of reversible bond dynamics observed in force-clamp spectroscopy.

We present a detailed analysis of two-state trajectories obtained from force-clamp spectroscopy (FCS) of reversibly bonded systems. FCS offers the unique possibility to vary the equilibrium constant in two-state kinetics, for instance, the unfolding and refolding of biomolecules, over many orders of magnitude due to the force dependence of the respective rates. We discuss two different kinds of counting statistics, the event counting usually employed in the statistical analysis of two-state kinetics and additionally the so-called cycle counting. While in the former case all transitions are counted, cycle counting means that we focus on one type of transitions. This might be advantageous in particular if the equilibrium constant is much larger or much smaller than unity because in these situations the temporal resolution of the experimental setup might not allow to capture all transitions of an event-counting analysis. We discuss how an analysis of FCS data for complex systems exhibiting dynamic disorder might be performed yielding information about the detailed force dependence of the transition rates and about the time scale of the dynamic disorder. In addition, the question as to which extent the kinetic scheme can be viewed as a Markovian two-state model is discussed.

Mistimed stimulated echoes and distorted spin-alignment spectra of powdered solids.

It is commonplace that NMR echo maxima appear at times for which the dephasing and the rephasing periods of a pulse sequence are equally long. However, for stimulated echoes a significant time shift from this naively expected echo position can be observed if the dephasing times are smaller than the inverse line width of the NMR spectrum. This effect, which will be observable for any line shape, is evaluated quantitatively for Gaussian and for Pake-like patterns. Comparison of the calculations is made with experimental results from (6)Li- and from (2)H-NMR and excellent agreement is found. In the simultaneous presence of broad and narrow lines, the apparent time shift can give rise to characteristic distortions in spin-alignment spectra. This explains some features previously observed using (7)Li-NMR.

Stimulated echoes and two-dimensional nuclear magnetic resonance spectra for solids with simple line shapes.

Nuclear magnetic resonance (NMR) experiments on ion conductors often yield rather unstructured spectra, which are hard to interpret if the relation between the actual translational motion of the mobile species and the changes of the NMR frequencies is not known. In order to facilitate a general analysis of experiments on solids with such spectra, different models for the stochastic evolution of the NMR frequencies are considered. The treated models involve random frequency jumps, diffusive evolutions, or approximately fixed frequency jumps. Two-dimensional nuclear magnetic resonance spectra as well as stimulated-echo functions for the study of slow and ultraslow translational dynamics are calculated for Gaussian equilibrium line shapes. The results are compared with corresponding ones from rotational models and with experimental data.

On the problem of field-gradient NMR measurements of intracrystalline diffusion in small crystallites--water in NaA zeolites as an example.

Necessary conditions for measuring intracrystalline diffusion in small crystal size systems via field-gradient NMR are discussed. As an illustrative case self-diffusion coefficients of water adsorbed in NaA zeolites (average crystal diameter about 1 microm) have been measured by 1H-NMR stimulated echoes in static magnetic field gradients of up to 180 T/m in the temperature range of 254-344 K. Obtaining intracrystalline diffusion coefficients necessitates a sufficiently high spatial resolution only provided by such large field gradients.