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.

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.

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.

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 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.

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.

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.

Surface deformation during an action potential in pearled cells.

Electric pulses in biological cells (action potentials) have been reported to be accompanied by a propagating cell-surface deformation with a nanoscale amplitude. Typically, this cell surface is covered by external layers of polymer material (extracellular matrix, cell wall material, etc.). It was recently demonstrated in excitable plant cells (Chara braunii) that the rigid external layer (cell wall) hinders the underlying deformation. When the cell membrane was separated from the cell wall by osmosis, a mechanical deformation, in the micrometer range, was observed upon excitation of the cell. The underlying mechanism of this mechanical pulse has, to date, remained elusive. Herein we report that Chara cells can undergo a pearling instability, and when the pearled fragments were excited even larger and more regular cell shape changes were observed (∼10-100µm in amplitude). These transient cellular deformations were captured by a curvature model that is based on three parameters: surface tension, bending rigidity, and pressure difference across the surface. In this paper these parameters are extracted by curve-fitting to the experimental cellular shapes at rest and during excitation. This is a necessary step to identify the mechanical parameters that change during an action potential.

On the excitation of action potentials by protons and its potential implications for cholinergic transmission.

One of the most conserved mechanisms for transmission of a nerve pulse across a synapse relies on acetylcholine (ACh). Ever since the Nobel Prize-winning works of Dale and Loewi, it has been assumed that ACh-subsequent to its action on a postsynaptic cell-is split into inactive by-products by acetylcholinesterase (AChE). Herein, the widespread assumption of inactivity of ACh's hydrolysis products is falsified. Excitable cells (Chara braunii internodes), which had previously been unresponsive to ACh, became ACh-sensitive in the presence of AChE. The latter was evidenced by a striking difference in cell membrane depolarization upon exposure to 10 mM intact ACh (∆V = -2 ± 5 mV) and its hydrolysate (∆V = 81 ± 19 mV), respectively, for 60 s. This pronounced depolarization, which also triggered action potentials, was clearly attributed to one of the hydrolysis products: acetic acid (∆V = 87 ± 9 mV at pH 4.0; choline ineffective in the range 1-10 mM). In agreement with our findings, numerous studies in the literature have reported that acids excite gels, lipid membranes, plant cells, erythrocytes, as well as neurons. Whether excitation of the postsynaptic cell in a cholinergic synapse is due to protons or due to intact ACh is a most fundamental question that has not been addressed so far.

On the Temperature Behavior of Pulse Propagation and Relaxation in Worms, Nerves and Gels.

The effect of temperature on pulse propagation in biological systems has been an important field of research. Environmental temperature not only affects a host of physiological processes e.g. in poikilotherms but also provides an experimental means to investigate the thermodynamic phenomenology of nerves and muscle. In the present work, the temperature dependence of blood vessel pulsation velocity and frequency was studied in the annelid Lumbriculus variegatus. The pulse velocity was found to vary linearily between 0°C and 30°C. In contrast, the pulse frequency increased non-linearly in the same temperature range. A heat block ultimately resulted in complete cessation of vessel pulsations at 37.2±2.7°C (lowest: 33°C, highest: 43°C). However, quick cooling of the animal led to restoration of regularly propagating pulses. This experimentally observed phenomenology of pulse propagation and frequency is interpreted without any assumptions about molecules in the excitable membrane (e.g. ion channels) or their temperature-dependent behaviour. By following Einstein's approach to thermodynamics and diffusion, a relation between relaxation time τ and compressibility κ of the excitable medium is derived that can be tested experimentally (for κT ∼ κS). Without fitting parameters this theory predicts the temperature dependence of the limiting (i.e. highest) pulse frequency in good agreement with experimental data. The thermodynamic approach presented herein is neither limited to temperature nor to worms nor to living systems. It describes the coupling between pulse propagation and relaxation equally well in nerves and gels. The inherent consistency and universality of the concept underline its potential to explain the dependence of pulse propagation and relaxation on any thermodynamic observable.

Temperature and excitable cells: Testable predictions from a thermodynamic perspective.

Temperature affects a host of biological processes, one of which is the conduction velocity of action potentials (AP). The velocity-temperature profile of APs has remained remarkably conserved across excitable animal and plant cells. Herein, we will not analyze this behavior in terms of temperature sensitivities of single molecules (e.g., ion channels), but rather we present a phenomenological thermodynamic interpretation. By assuming that APs are acoustic phenomena, one arrives at testable predictions about the temperature-dependence of the macroscopic material properties of the excitable cell membrane. These material properties set constraints on the excitability of a cell membrane and allow us to hypothesize about its typical relaxation timescales. The presented approach-by virtue of its thermodynamic nature-is by no means limited to temperature. It applies equally well to all thermodynamic variables (e.g., mechanical stretch, pH, ion concentrations, etc.) and to underline this argument we discuss some implications and predictions for sensory physiology.

A multichannel acoustically driven microfluidic chip to study particle-cell interactions.

Microfluidic devices have emerged as important tools for experimental physiology. They allow to study the effects of hydrodynamic flow on physiological and pathophysiological processes, e.g., in the circulatory system of the body. Such dynamic in vitro test systems are essential in order to address fundamental problems in drug delivery and targeted imaging, such as the binding of particles to cells under flow. In the present work an acoustically driven microfluidic platform is presented in which four miniature flow channels can be operated in parallel at distinct flow velocities with only slight inter-experimental variations. The device can accommodate various channel architectures and is fully compatible with cell culture as well as microscopy. Moreover, the flow channels can be readily separated from the surface acoustic wave pumps and subsequently channel-associated luminescence, absorbance, and/or fluorescence can be determined with a standard microplate reader. In order to create artificial blood vessels, different coatings were evaluated for the cultivation of endothelial cells in the microchannels. It was found that 0.01% fibronectin is the most suitable coating for growth of endothelial monolayers. Finally, the microfluidic system was used to study the binding of 1 µm polystyrene microspheres to three different types of endothelial cell monolayers (HUVEC, HUVECtert, HMEC-1) at different average shear rates. It demonstrated that average shear rates between 0.5 s(-1) and 2.25 s(-1) exert no significant effect on cytoadhesion of particles to all three types of endothelial monolayers. In conclusion, the multichannel microfluidic platform is a promising device to study the impact of hydrodynamic forces on cell physiology and binding of drug carriers to endothelium.

Improving oral delivery.

It is estimated that 90% of all medicines are oral formulations and their market share is still increasing, due to sound advantages for the patient, the pharmaceutical industry and healthcare systems. Considering biopharmaceutical issues such as physicochemical requirements of the drug and physiological conditions, however, oral delivery is one of the most challenging routes. Recognising solubility, permeability and residence time in the gastrointestinal milieu as key parameters, different characteristics of drugs and their delivery systems such as size, pH, density, diffusion, swelling, adhesion, degradation and permeability can be adjusted to improve oral delivery. Future developments will focus on further improvement in patient compliance as well as the feasibility of administering biotech drugs via the oral route.

An acoustically-driven biochip - impact of flow on the cell-association of targeted drug carriers.

The interaction of targeted drug carriers with epithelial and endothelial barriers in vivo is largely determined by the dynamics of the body fluids. To simulate these conditions in binding assays, a fully biocompatible in vitro model was developed which can accurately mimic a wide range of physiological flow conditions on a thumbnail-format cell-chip. This acoustically-driven microfluidic system was used to study the interaction characteristics of protein-coated particles with cells. Poly(D,L-lactide-co-glycolide) (PLGA) microparticles (2.9 +/- 1 microm) were conjugated with wheat germ agglutinin (WGA-MP, cytoadhesive protein) or bovine serum albumin (BSA-MP, non-specific protein) and their binding to epithelial cell monolayers was investigated under stationary and flow conditions. While mean numbers of 1500 +/- 307 mm(-2) WGA-MP and 94 +/- 64 mm(-2) BSA-MP respectively were detected to be cell-bound in the stationary setup, incubation at increasing flow velocities increasingly antagonized the attachment of both types of surface-modified particles. However, while binding of BSA-MP was totally inhibited by flow, grafting with WGA resulted in a pronounced anchoring effect. This was indicated by a mean number of 747 +/- 241 mm(-2) and 104 +/- 44 mm(-2) attached particles at shear rates of 0.2 s(-1) and 1 s(-1) respectively. Due to the compactness of the fluidic chip which favours parallelization, this setup represents a highly promising approach towards a screening platform for the performance of drug delivery vehicles under physiological flow conditions. In this regard, the flow-chip is expected to provide substantial information for the successful design and development of targeted micro- and nanoparticulate drug carrier systems.

Bionanoprobes to study particle-cell interactions.

A variety of research reports have provided evidence that the interplay between nanoparticles and biological systems is strongly dependent on the composition as well as on the size of the particles. However, irrespective of that a fine tuning of the interaction characteristics can be attained by functionalisation of the particle surface. In order to be able to monitor such interactions, an analytically accessible model system for potentially target-specific therapeutically relevant nanoparticles (NP) was generated by encapsulation of the highly hydrophobic fluorophore BODIPY 493/503 into poly(D,L-lactide-co-glycolide) (PLGA) nanodroplets. Analyses of the mean particle size and zeta potential revealed that plain and human serum albumin (HSA) conjugated NP can be stored without agglomeration for at least 28 days at 4 degrees C as well as -80 degrees C. Although transfer of the particles into commonly used buffers and cell culture medium did not affect the system's stability, protein-containing dispersants should not be used for experiments demanding high sensitivity as distinct quenching effects were observed. Concerning liberation of the fluorescent marker, no release occurred when incubating the suspension for 7 days at 4 degrees C, while an onset of release was expectedly detected after 3 h at 37 degrees C. These experimental limitations were taken into account for the particle-cell interaction studies which, as a consequence of the more hydrophilic particle surface, showed an enhanced adhesion of HSA-conjugated colloids to Caco-2 cells by means of flow cytometry and fluorescence microscopy.

Fluorescent bionanoprobes to characterize cytoadhesion and cytoinvasion.

To overcome current limitations in diagnostic imaging and targeted drug delivery, a highly versatile tool is presented that can be used to representatively investigate the effects of submicroparticles intended for the use in biological systems. An effective approach to render colloids trackable is developed by stable attachment of the fluorescent probe BODIPY 493/503 (BOD: 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene) to a biodegradable and biocompatible particle core matrix. BOD submicroparticles are shown to be stable, can be surface modified, and exhibit high fluorescence emission. Upon conjugation with human serum albumin (nonspecific) and wheat germ agglutinin (biorecognitive) as model ligands explicit differences are found in the cytoadhesive and cytoinvasive characteristics of the submicroparticles using Caco-2 cells. These results demonstrate the potency of BOD-labeled colloids as a versatile analytical platform for a multifaceted investigation of cell-particle interactions in biological systems.

Stabilizer-induced viscosity alteration biases nanoparticle sizing via dynamic light scattering.

The size dependent features of colloids at the nanometer scale have been issues of increasingly intensive research. In order to be able to correctly relate characteristics to certain size-populations, accurate and reliable particle sizing by dynamic light scattering (DLS) is a main prerequisite. So far, the complexity of the systems due to the presence of surfactants, proteins, and so forth in the nanoparticle suspensions has not been accounted for. In this work, practically relevant quantities of the frequently used PEO-PPO triblock copolymer surfactant Pluronic F-68 were studied for their effect on the size determination of nanoparticles by DLS. Induced changes in the tenside-content of the nanosphere suspension were monitored using a photometric assay and were correlated to the respective variances in mean particle size. These measurements showed that alterations in the range from 0.005 to 2% of Pluronic content are associated with shifts in diameter of 200 nm-particles by as much as 65 nm. The considerable changes that were found have been attributed to the surfactant-concentration-dependent fluctuations in the viscosity of the nanoparticle suspension, which affect the dimensions of colloids calculated according to the Stokes-Einstein relation.