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).

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.

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.

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.