Sharp, localized phase transitions in single neuronal cells.

The origin of nonlinear responses in cells has been suggested to be crucial for various cell functions including the propagation of the nervous impulse. In physics, nonlinear behavior often originates from phase transitions. Evidence for such transitions on the single-cell level, however, has so far not been provided, leaving the field unattended by the biological community. Here, we demonstrate that single cells of a human neuronal cell line display all optical features of a sharp, highly nonlinear phase transition within their membrane. The transition is reversible and does not originate from protein denaturation. Triggered by temperature and modified by pH here, a thermodynamic approach strongly suggests that similar nonlinear state changes can be induced by other variables such as calcium or mechanical stress. At least in lipid membranes, such state changes are accompanied by significant changes in permeability, enzyme activity, elastic, and electrical properties.

Specific Regulation of Enzymatic Activity by Interface Pulses.

The thermodynamic state of the interface in which an enzyme is embedded can regulate the enzymatic activity. Indeed, it has been demonstrated by others and us that close to the maximum in compressibility, the activity of the enzyme is at a maximum as well. Pulses propagating along the interface can modulate the interface state and were demonstrated to be able to modulate the activity of interface-associated acetylcholinesterase (AChE). Here, we demonstrate that enzyme activity modulation by interface pulses depends specifically on the pulse type. Using membrane-embedded enzyme phospholipase A2 (PLA2), enzyme activity can be monitored by detecting the lateral pressure without an additional assay required. We show that pulses that shift the state toward higher pressure and higher lateral density increase the enzymatic activity, while pulses that reduce the pressure induce the opposite effect. These results further support a physical mechanism for enzyme-enzyme communication where compressibility, lateral density, and pressure (thermodynamic state) and not specific molecular modifications regulate enzymatic activity.

Unitary Response of Solvatochromic Dye to Pulse Excitation in Lipid and Cell Membranes.

The existence of acoustic pulse propagation in lipid monolayers at the air-water interface is well known. These pulses are controlled by the thermodynamic state of the lipid membrane. Nevertheless, the role of acoustic pulses for intra- and inter-cellular communication is still a matter of debate. Herein, we used the dye di-4-ANEPPDHQ, which is known to be sensitive to the physical state and transmembrane potential of membranes, in order to gain insights into compression waves in lipid-based membrane interfaces. The dye was incorporated into lipid monolayers made of phosphatidylserine or phosphatidylcholine at the air-water-interface. A significant blue shift of the emission spectrum was detected when the state of the monolayer was changed from the liquid-expanded (LE) to the liquid-condensed (LC) phase. This "transition sensitivity" of di-4-ANEPPDHQ was generalized in experiments with the bulk solvent dimethyl sulfoxide (DMSO). Upon crystallization of solvent, the emission spectrum also underwent a blue shift. During compression pulses in lipid monolayers, a significant fluorescence response was only observed when the main transition is crossed. The optical signature of these waves─in terms of sign and magnitude─was identical to the response of di-4-ANEPPDHQ during action potentials in neurons and excitable plant cells. These findings corroborated the suggestion that action potentials are nonlinear state changes that propagate in the cell membrane.

Nonlinear pulses at the interface and its relation to state and temperature.

Environmental temperature has a well-conserved effect on the pulse velocity and excitability of excitable biological systems. The consistency suggests that the cause originates from a fundamental principle. A physical (hydrodynamic) approach has proposed that the thermodynamic state of the hydrated interface (e.g., plasma membrane) determines the pulse behavior. This implies that the temperature effect happens because the environmental temperature affects the state of the interface in any given system. To test the hypothesis, we measured temperature-dependent phase diagrams of a lipid monolayer and studied the properties of nonlinear acoustic pulses excited along the membrane. We observed that the membrane in the fluid-gel transition regime exhibited lower compressibility (i.e., stiffer) overall with increasing temperature. Nonlinear pulses excited near the transition state propagated with greater velocity with increasing temperature, and these observations were consistent with the compressibility profiles. Excitability was suppressed significantly or ceased completely when the state departed too far from the transition regime either by cooling or by heating. The overall correlation between the pulses in the membrane and in living systems as a function of temperature supports the view that the thermodynamic state of the interface and phase transition are the key to understanding pulse propagation in excitable systems.

Cell Surface Deformation during an Action Potential.

The excitation of many cells and tissues is associated with cell mechanical changes. The evidence presented herein corroborates that single cells deform during an action potential. It is demonstrated that excitation of plant cells (Chara braunii internodes) is accompanied by out-of-plane displacements of the cell surface in the micrometer range (∼1-10 µm). The onset of cellular deformation coincides with the depolarization phase of the action potential. The mechanical pulse: 1) propagates with the same velocity as the electrical pulse (within experimental accuracy, ∼10 mm s-1), 2) is reversible, 3) in most cases is of biphasic nature (109 out of 152 experiments), and 4) is presumably independent of actin-myosin-motility. The existence of transient mechanical changes in the cell cortex is confirmed by micropipette aspiration experiments. A theoretical analysis demonstrates that this observation can be explained by a reversible change in the mechanical properties of the cell surface (transmembrane pressure, surface tension, and bending rigidity). Taken together, these findings contribute to the ongoing debate about the physical nature of cellular excitability.

On measuring the acoustic state changes in lipid membranes using fluorescent probes.

Ultrasound is increasingly being used to modulate the properties of biological membranes for applications in drug delivery and neuromodulation. While various studies have investigated the mechanical aspects of the interaction such as acoustic absorption and membrane deformation, it is not clear how these effects transduce into biological functions, for example, changes in the permeability or the enzymatic activity of the membrane. A critical aspect of the activity of an enzyme is the thermal fluctuations of its solvation or hydration shell. Thermal fluctuations are also known to be directly related to membrane permeability. Here solvation shell changes of lipid membranes subject to an acoustic impulse were investigated using a fluorescence probe, Laurdan. Laurdan was embedded in multi-lamellar lipid vesicles in water, which were exposed to broadband pressure impulses of the order of 1 MPa peak amplitude and 10 µs pulse duration. An instrument was developed to monitor changes in the emission spectrum of the dye at two wavelengths with sub-microsecond temporal resolution. The experiments show that changes in the emission spectrum, and hence the fluctuations of the solvation shell, are related to the changes in the thermodynamic state of the membrane and correlated with the compression and rarefaction of the incident sound wave. The results suggest that acoustic fields affect the state of a lipid membrane and therefore can potentially modulate the kinetics of channels and enzymes embedded in the membrane.

Collision of two action potentials in a single excitable cell.

BACKGROUND: It is a common incident in nature, that two waves or pulses run into each other head-on. The outcome of such an event is of special interest, because it allows conclusions about the underlying physical nature of the pulses. The present experimental study dealt with the head-on meeting of two action potentials (AP) in a single excitable plant cell (Chara braunii internode). METHODS: The membrane potential was monitored with multiple sensors along a single excitable cell. In control experiments, an AP was excited electrically at either end of the cell cylinder. Subsequently, stimuli were applied simultaneously at both ends of the cell in order to generate two APs that met each other head-on. RESULTS: When two action potentials propagated into each other, the pulses did not penetrate but annihilated (N=26 experiments in n=10 cells). CONCLUSIONS: APs in excitable plant cells did not penetrate upon meeting head-on. In the classical electrical model, this behavior is specifically attributed to relaxation of ion channel proteins. From an acoustic point of view, annihilation can be viewed as a result of nonlinear material properties (e.g. a phase change). GENERAL SIGNIFICANCE: The present results suggest that APs in excitable animal and plant cells belong to a similar class of nonlinear phenomena. Intriguingly, other excitation waves in biology (intracellular waves, cortical spreading depression, etc.) also annihilate upon collision and are thus expected to follow the same underlying principles as the observed action potentials.

Controlled surface-induced flows from the motion of self-assembled colloidal walkers.

Biological flows at the microscopic scale are important for the transport of nutrients, locomotion, and differentiation. Here, we present a unique approach for creating controlled, surface-induced flows inspired by a ubiquitous biological system, cilia. Our design is based on a collection of self-assembled colloidal rotors that "walk" along surfaces in the presence of a rotating magnetic field. These rotors are held together solely by magnetic forces that allow for reversible assembly and disassembly of the chains. Furthermore, rotation of the magnetic field allows for straightforward manipulation of the shape and motion of these chains. This system offers a simple and versatile approach for designing microfluidic devices as well as for studying fundamental questions in cooperative-driven motion and transport at the microscopic level.

Evidence for a transition in the cortical membranes of Paramecium.

Ever since the pioneering studies in the 1960s and 70s, the importance of order transitions for cell membrane functions has remained a matter of debate. Recently, it has been proposed that the nonlinear stimulus-response curve of excitable cells, which manifests in all-or-none pulses (action potentials (AP)), is due to a transition in the cell membrane. Indeed, evidence for transitions has accumulated in plant cells and neurons, but studies with other excitable cells are expedient in order to show if this finding is of a general nature. Herein, we investigated intact, motile specimens of the "swimming neuron" Paramecium. The cellular membranes were labelled with the solvatochromic fluorophores LAURDAN or Di-4-ANEPPDHQ. Subsequently, a cell was trapped in a microfluidic channel and investigated by fluorescence spectroscopy. The generalized polarization (GP) of the fluorescence emission from cell cortical membranes (probably plasma and alveolar membranes) was extracted by an edge-finding algorithm. The thermo-optical state diagram, i.e. the dependence of GP on temperature, exhibited clear indications for a reversible transition. This transition had a width of ~10-15 °C and a midpoint that was located ~4 °C below the growth temperature. The state diagrams with LAURDAN and Di-4-ANEPPDHQ had widely identical characteristics. These results suggested that the cortical membranes of Paramecium reside in an order transition regime under physiological growth conditions. Based on these findings, membrane potential fluctuations, spontaneous depolarizing spikes, and thermal excitation of Paramecium was interpreted.

Reversible single cell trapping of Paramecium caudatum to correlate swimming behavior and membrane state.

Single cell measurements with living specimen like, for example, the ciliated protozoan Paramecium caudatum can be a challenging task. We present here a microfluidic trapping mechanism for measurements with these micro-organisms that can be used, e.g., for optical measurements to correlate cellular functions with the phase state of the lipid membrane. Here, we reversibly trap single cells in small compartments. Furthermore, we track and analyze the swimming behavior of single cells over several minutes. Before and after reversible trapping the swimming speed is comparable, suggesting that trapping does not have a large effect on cell behavior. Last, we demonstrate the feasibility of membrane order measurements on living cells using the fluorescent dye 6-lauryl-2-dimethylaminonaphthalene (Laurdan).

DNA concentration modulation on supported lipid bilayers switched by surface acoustic waves.

Spatially addressable arrays of molecules embedded in or anchored to supported lipid bilayers are important for on-chip screening and binding assays; however, methods to sort or accumulate components in a fluid membrane on demand are still limited. Here we apply in-plane surface acoustic shear waves (SAWs) to laterally accumulate double-stranded DNA segments electrostatically bound to a cationic supported lipid bilayer. The fluorescently labeled DNA segments are found to segregate into stripe patterns with a spatial frequency corresponding to the periodicity of the standing SAW wave (~10 µm). The DNA molecules are accumulated 10-fold in the regions of SAW antinodes. The superposition of two orthogonal sets of SAW sources creates checkerboard like arrays of DNA demonstrating the potential to generate arrayed fields dynamically. The pattern relaxation time of 0.58 s, which is independent of the segment length, indicates a sorting and relaxation mechanism dominated by lipid diffusion rather than DNA self-diffusion.

Living systems approached from physical principles.

This article attempts to review our work in the field since 2008, attempts to put it in a coherent framework and takes a courageous look vis-à-vis the bigger picture. It summarizes our approach, successes and open questions to start from physical principles when approaching living systems. It stresses the importance of conservation laws versus material and/or structural approaches to living systems commonly taken in (molecular) biology. Indeed, we claim that the crucial system in biology isn't a molecule or a molecular class whatsoever, but the interface created by biomolecules in water. It is the physical or thermodynamic state of this 2D interface and the action of conservation laws on it, which determines biological function, an approach I refer to as the "state-to-function-approach" in stark contrast to the structure-function approach.Three key ideas, all based on physical principles, particularly the 2nd law of thermodynamics and momentum conservation, are presented and experimentally confirmed. In Idea One we bridge physical state and biological function directly, e.g. by demonstrating the control of enzymatic activity and ion conductivity via thermodynamic state. Idea Two presents the role of momentum conservation in biological communication specifically applied to the principles of nerve pulse propagation. Idea Three finally introduces a physical concept of specificity, which is free of structural requirements and includs the specific interaction between pulses and enzymes.We finally discuss the extend of applicability and the universality of the mentioned ideas by presenting some impressive similarities between fairly remotely appearing biological processes, such as cell growth and pulse propagation. We close with the question in how far a thermodynamic approach can bring insight in the concept of cell adaptation, the evolution of organs or a deeper understanding of health and disease?

Sound pulses in lipid membranes and their potential function in biology.

Experimental observations in lipid monolayers at the air-water interface have demonstrated that solitary sound pulses can be excited. These pulses propagate electrical, chemical, and thermal variations in addition to the mechanical changes in lateral pressure and lipid density, and can interact with nearby ions, polymers, and water. In addition, it was demonstrated that sound pulses that reversibly traverse the melting transition between the so-called liquid-expanded and liquid-condensed phases display unusual nonlinear properties that are strikingly similar to those of action potentials in living cells. This review describes recent experimental and theoretical investigations of sound in lipid membranes and their potential function in biology.

The living state: How cellular excitability is controlled by the thermodynamic state of the membrane.

The thermodynamic (TD) properties of biological membranes play a central role for living systems. It has been suggested, for instance, that nonlinear pulses such as action potentials (APs) can only exist if the membrane state is in vicinity of a TD transition. Herein, two membrane properties in living systems - excitability and velocity - are analyzed for a broad spectrum of conditions (temperature (T), 3D-pressure (p) and pH-dependence). Based on experimental data from Characean cells and a review of literature we predict parameter ranges in which a transition of the membrane is located (15-35°C below growth temperature; 1-3pH units below pH7; at ∼800atm) and propose the corresponding phase diagrams. The latter explain: (i) changes of AP velocity with T,p and pH.(ii) The existence and origin of two qualitatively different forms of loss of nonlinear excitability ("nerve block", anesthesia). (iii) The type and quantity of parameter changes that trigger APs. Finally, a quantitative comparison between the TD behavior of 2D-lipid model membranes with living systems is attempted. The typical shifts in transition temperature with pH and p of model membranes agree with values obtained from cell physiological measurements. Taken together, these results suggest that it is not specific molecules that control the excitability of living systems but rather the TD properties of the membrane interface. The approach as proposed herein can be extended to other quantities (membrane potential, calcium concentration, etc.) and makes falsifiable predictions, for example, that a transition exists within the specified parameter ranges in excitable cells.

Lipid Membrane State Change by Catalytic Protonation and the Implications for Synaptic Transmission.

In cholinergic synapses, the neurotransmitter acetylcholine (ACh) is rapidly hydrolyzed by esterases to choline and acetic acid (AH). It is believed that this reaction serves the purpose of deactivating ACh once it has exerted its effect on a receptor protein (AChR). The protons liberated in this reaction, however, may by themselves excite the postsynaptic membrane. Herein, we investigated the response of cell membrane models made from phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidic acid (PA) to ACh in the presence and absence of acetylcholinesterase (AChE). Without a catalyst, there were no significant effects of ACh on the membrane state (lateral pressure change ≤0.5 mN/m). In contrast, strong responses were observed in membranes made from PS and PA when ACh was applied in presence of AChE (>5 mN/m). Control experiments demonstrated that this effect was due to the protonation of lipid headgroups, which is maximal at the pK (for PS: pKCOOH≈5.0; for PA: pKHPO4-≈8.5). These findings are physiologically relevant, because both of these lipids are present in postsynaptic membranes. Furthermore, we discussed evidence which suggests that AChR assembles a lipid-protein interface that is proton-sensitive in the vicinity of pH 7.5. Such a membrane could be excited by hydrolysis of micromolar amounts of ACh. Based on these results, we proposed that cholinergic transmission is due to postsynaptic membrane protonation. Our model will be falsified if cholinergic membranes do not respond to acidification.

The activity of the intrinsically water-soluble enzyme ADAMTS13 correlates with the membrane state when bound to a phospholipid bilayer.

Membrane-associated enzymes have been found to behave differently qualitatively and quantitatively in terms of activity. These findings were highly debated in the 1970s and many general correlations and reaction specific models have been proposed, reviewed, and discarded. However, new biological applications brought up the need for clarification and elucidation. To address literature shortcomings, we chose the intrinsically water-soluble enzyme a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) and large unilamellar vesicles with a relative broad phase transition. We here present activity measurements of ADAMTS13 in the freely dissolved state and the membrane associated state for phosphocholine lipids with different acyl-chain lengths (13:0, 14:0 and 15:0) and thus main phase transition temperatures. While the freely dissolved enzyme shows a simple Arrhenius behavior, the activity of membrane associated ADAMTS13 in addition shows a peak. This peak temperature correlates with the main phase transition temperature of the used lipids. These findings support an alternative theory of catalysis. This theory predicts a correlation of the membrane associated activity and the heat capacity, as both are susceptibilities of the same surface Gibb's free energy, since the enzyme is attached to the membrane.

Optical studies of membrane state during action potential propagation.

One of the most striking phenomena in biology is the action potential (AP), a nonlinear pulse with threshold and amplitude saturation (all-or-none-behavior) that propagates along neurons and other cells. In the classical interpretation the AP is considered to be an electrical phenomenon - a regenerating current flowing in a "biological cable". In contrast, the thermodynamic interpretation has emphasized that conservation laws necessitate pulses and that pulses must manifest as transient changes of all observables of the system (electrical, mechanical, thermal, etc.). It is a key prediction of the latter approach that the cell membrane must undergo thermodynamic state changes during an AP. In order to characterize the thermodynamic state of an excitable membrane, plant cells (Chara australis) were stained with Di-4-ANEPPDHQ. The location of the dye in the cell membrane was confirmed by confocal microscopy and changes of fluorescence emission were investigated as a function of temperature and extracellular pH. In parallel, emission of the dye was studied in artificial lipid vesicles (DMPC, DMPS) in the vicinity of the main transition temperature. In all these systems, the emission spectrum shifted as a function of membrane state. This shift became nonlinear and was maximal when the membrane underwent a transition (∂λ∂T∼(6-10)nm°C-1). In the excitable cell Di-4-ANEPPDHQ exhibited a transient blueshift by ∼7 nm during an AP. A blueshift also occurred upon cooling and extracellular acidification. These results provided evidence for a sequence of state changes during an AP in which the cellular membrane condenses followed by expansion. This finding is in line with the thermodynamic interpretation of cellular excitability. Future studies should confirm/falsify these findings with other fluorescent dyes or state-sensitive techniques.

Interaction of a charged polymer with zwitterionic lipid vesicles.

The interaction between polyethylenimine (PEI) and phospholipid bilayers plays an important role in several biophysical applications such as DNA transfection of target cells. Despite considerable investigation into the nature of the interaction between PEI and phospholipid bilayers, the physical process remains poorly understood. In this paper, we study the impact of PEI on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles as a function of salt concentration using several techniques including dynamic (DLS) and static (SLS) light scattering, differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR). At low salt concentration, vesicles aggregate, leading to the formation of stable clusters whose final size depends on the PEI concentration. At high salt concentration the system does not aggregate; DSC and NMR data reveal that the PEI penetrates into the bilayer, and SLS measurements are consistent with PEI crossing the bilayer. The transfectional ability of PEI is discussed in terms of these results.

Platelet adhesion and aggregate formation controlled by immobilised and soluble VWF.

BACKGROUND: It has been demonstrated that von Willebrand factor (VWF) mediated platelet-endothelium and platelet-platelet interactions are shear dependent. The VWF's mobility under dynamic conditions (e.g. flow) is pivotal to platelet adhesion and VWF-mediated aggregate formation in the cascade of VWF-platelet interactions in haemostasis. RESULTS: Combining microfluidic tools with fluorescence and reflection interference contrast microscopy (RICM), here we show, that specific deletions in the A-domains of the biopolymer VWF affect both, adhesion and aggregation properties independently. Intuitively, the deletion of the A1-domain led to a significant decrease in both adhesion and aggregate formation of platelets. Nevertheless, the deletion of the A2-domain revealed a completely different picture, with a significant increase in formation of rolling aggregates (gain of function). We predict that the A2-domain effectively 'masks' the potential between the platelet glycoprotein (GP) Ib and the VWF A1-domain. Furthermore, the deletion of the A3-domain led to no significant variation in either of the two functional characteristics. CONCLUSIONS: These data demonstrate that the macroscopic functional properties i.e. adhesion and aggregate formation cannot simply be assigned to the properties of one particular domain, but have to be explained by cooperative phenomena. The absence or presence of molecular entities likewise affects the properties (thermodynamic phenomenology) of its neighbours, therefore altering the macromolecular function.