On the Physical Basis of Biological Signaling by Interface Pulses.

Currently, biological signaling is envisaged as a combination of activation and movement, triggered by local molecular interactions and molecular diffusion, respectively. However, here, we suggest that other fundamental physical mechanisms might play an at least equally important role. We have recently shown that lipid interfaces permit the excitation and propagation of sound pulses. Here, we demonstrate that these reversible perturbations can control the activity of membrane-embedded enzymes without a requirement for molecular transport. They can thus facilitate rapid communication between distant biological entities at the speed of sound, which is here on the order of 1 m/s within the membrane. The mechanism described provides a new physical framework for biological signaling that is fundamentally different from the molecular approach that currently dominates the textbooks.

Chemical and mechanical impact of silica nanoparticles on the phase transition behavior of phospholipid membranes in theory and experiment.

The interaction of nanoparticles (NPs) with lipid membranes is an integral step in the interaction of NPs and living cells. During particle uptake, the membrane has to bend. Due to the nature of their phase diagram, the modulus of compression of these membranes can vary by more than one order of magnitude, and thus both the thermodynamic and mechanical aspects of the membrane have to be considered simultaneously. We demonstrate that silica NPs have at least two independent effects on the phase transition of phospholipid membranes: 1), a chemical effect resulting from the finite instability of the NPs in water; and 2), a mechanical effect that originates from a bending of the lipid membrane around the NPs. Here, we report on recent experiments that allowed us to clearly distinguish both effects, and present a thermodynamic model that includes the elastic energy of the membranes and correctly predicts our findings both quantitatively and qualitatively.

Propagation of 2D pressure pulses in lipid monolayers and its possible implications for biology.

The existence and propagation of acoustic pressure pulses on lipid monolayers at the air-water interface are directly observed by simple mechanical detection. The pulses are excited by small amounts of solvents added to the monolayer. Controlling the state of the lipid interface, we show that the pulses propagate at velocities c following the lateral compressibility κ. This is manifested by a pronounced minimum in c (∼0.3 m/s) within the transition regime. The role of interface density pulses in biology is discussed, in particular, in the context of communicating localized alterations in protein function (signaling) and nerve pulse propagation.

Transport, separation, and accumulation of proteins on supported lipid bilayers.

Transport, separation, and accumulation of proteins in their natural environment are central goals in protein biotechnology. Miniaturized assays of supported lipid bilayers (SLBs) have been proposed as promising candidates to realize such technology on a chip, but a modular system for the controlled transport of membrane proteins does not exist. In this letter, we demonstrate that standing surface acoustic waves drive the in-plane redistribution of proteins on planar SLBs over macroscopic distances (3.5 mm). Accumulation of proteins in periodic patterns of about 10-fold protein concentration difference is accomplished and shown to relax into the homogeneous state by diffusion. Different proteins separate in individual fractions from a homogeneous distribution and are transported and accumulated into clusters using beats. The modular planar setup has the potential of integrating other lab-on-a-chip tools, for monitoring the membrane-protein integrity or adding microfluidic features for blood screening or DNA analysis.

Wave propagation in lipid monolayers.

Sound waves are excited on lipid monolayers using a set of planar electrodes aligned in parallel with the excitable medium. By measuring the frequency-dependent change in the lateral pressure, we are able to extract the sound velocity for the entire monolayer phase diagram. We demonstrate that this velocity can also be directly derived from the lipid monolayer compressibility, and consequently displays a minimum in the phase transition regime. This minimum decreases from v(0) = 170 m/s for one-component lipid monolayers down to v(m) = 50 m/s for lipid mixtures. No significant attenuation can be detected confirming an adiabatic phenomenon. Finally, our data propose a relative lateral density oscillation of Deltarho/rho approximately 2%, implying a change in all area-dependent physical properties. Order-of-magnitude estimates from static couplings therefore predict propagating changes in surface potential of 1-50 mV, 1 unit in pH (electrochemical potential), and 0.01 K in temperature, and fall within the same order of magnitude as physical changes measured during nerve pulse propagation. These results therefore strongly support the idea of propagating adiabatic sound waves along nerves as first thoroughly described by Kaufmann in 1989 and recently by Heimburg and Jackson, but already claimed by Wilke in 1912.

Phase-state dependent current fluctuations in pure lipid membranes.

Current fluctuations in pure lipid membranes have been shown to occur under the influence of transmembrane electric fields (electroporation) as well as a result from structural rearrangements of the lipid bilayer during phase transition (soft perforation). We demonstrate that the ion permeability during lipid phase transition exhibits the same qualitative temperature dependence as the macroscopic heat capacity of a D15PC/DOPC vesicle suspension. Microscopic current fluctuations show distinct characteristics for each individual phase state. Although current fluctuations in the fluid phase show spikelike behavior of short timescales (approximately 2 ms) with a narrow amplitude distribution, the current fluctuations during lipid phase transition appear in distinct steps with timescales of approximately 20 ms. We propose a theoretical explanation for the origin of timescales and permeability based on a linear relationship between lipid membrane susceptibilities and relaxation times near the phase transition.

Thermodynamic relaxation drives expulsion in giant unilamellar vesicles.

We investigated the thermodynamic relaxation of giant unilamellar vesicles (GUVs) which contained small vesicles within their interior. Quenching these vesicles from their fluid phase (T > T(m)) through the phase transition in the gel state (T < T(m)) drives the inner vesicles to be expelled from the larger mother vesicle via the accompanying decrease in the vesicle area by approximately 25% which forces a pore to open in the mother vesicle. We demonstrate that the proceeding time evolution of the resulting efflux follows the relaxation of the membrane area and describes the entire relaxation process using an Onsager-like non-equilibrium thermodynamics ansatz. As a consequence of the volume efflux, internal vesicles are expelled from the mother vesicle. Although complete sealing of the pore may occur during the expulsion, the global relaxation dynamics is conserved. Finally, comparison of these results to morphological relaxation phenomena found in earlier studies reveals a universal relaxation behaviour in GUVs. When quenched from the fluid to gel phase the typical time scale of relaxation shows little variation and ranges between 4 and 5 s.

Activity-dependent nuclear translocation and intranuclear distribution of NFATc in adult skeletal muscle fibers.

TTranscription factor nuclear factor of activated T cells NFATc (NFATc1, NFAT2) may contribute to slow-twitch skeletal muscle fiber type-specific gene expression. Green fluorescence protein (GFP) or FLAG fusion proteins of either wild-type or constitutively active mutant NFATc [NFATc(S-->A)] were expressed in cultured adult mouse skeletal muscle fibers from flexor digitorum brevis (predominantly fast-twitch). Unstimulated fibers expressing NFATc(S-->A) exhibited a distinct intranuclear pattern of NFATc foci. In unstimulated fibers expressing NFATc-GFP, fluorescence was localized at the sarcomeric z-lines and absent from nuclei. Electrical stimulation using activity patterns typical of slow-twitch muscle, either continuously at 10 Hz or in 5-s trains at 10 Hz every 50 s, caused cyclosporin A-sensitive appearance of fluorescent foci of NFATc-GFP in all nuclei. Fluorescence of nuclear foci increased during the first hour of stimulation and then remained constant during a second hour of stimulation. Kinase inhibitors and ionomycin caused appearance of nuclear foci of NFATc-GFP without electrical stimulation. Nuclear translocation of NFATc-GFP did not occur with either continuous 1 Hz stimulation or with the fast-twitch fiber activity pattern of 0.1-s trains at 50 Hz every 50 s. The stimulation pattern-dependent nuclear translocation of NFATc demonstrated here could thus contribute to fast-twitch to slow-twitch fiber type transformation.

IP3 receptor: localization to plasma membrane of T cells and cocapping with the T cell receptor.

Immune responses in lymphocytes require cellular accumulation of large amounts of calcium (Ca2+) from extracellular sources. In the T cell tumor line Jurkat, receptors for the Ca(2+)-releasing messenger inositol 1,4,5-trisphosphate (IP3) were localized to the plasma membrane (PM). Capping of the T cell receptor-CD3 complex, which is associated with signal transduction, was accompanied by capping of IP3 receptors. The IP3 receptor on T cells appears to be responsible for the entry of Ca2+ that initiates proliferative responses.

Release-activated Ca2+ transport in neurons of frog sympathetic ganglia.

Frog sympathetic ganglion neurons exhibit a novel Ca2+ uptake mechanism, release-activated calcium transport or RACT, which is manifest in both cytosolic and store [Ca2+] signals as greatly accelerated Ca2+ uptake after Ca2+ release from internal stores. RACT is activated by Ca2+ release but not by Ca2+ entry and serves to selectively refill Ca2+ stores after release. RACT lowers cytosolic [Ca2+] with a rate constant about 1.6 times that of the SERCA pump with empty ER. RACT is thapsigargin-insensitive, was eliminated by ryanodine, but was not affected by blocking mitochondrial or plasma membrane Ca2+ transport. A Ca2+ flux model with RACT in the ER membrane reproduced the cytosolic and store [Ca2+] responses to all stimuli.

Relaxation of ultralarge VWF bundles in a microfluidic-AFM hybrid reactor.

The crucial role of the biopolymer "Von Willebrand factor" (VWF) in blood platelet binding is tightly regulated by the shear forces to which the protein is exposed in the blood flow. Under high-shear conditions, VWFs ability to immobilize blood platelets is strongly increased due to a change in conformation which at sufficient concentration is accompanied by the formation of ultra large VWF bundles (ULVWF). However, little is known about the dynamic and mechanical properties of such bundles. Combining a surface acoustic wave (SAW) based microfluidic reactor with an atomic force microscope (AFM) we were able to study the relaxation of stretched VWF bundles formed by hydrodynamic stress. We found that the dynamical response of the network is well characterized by stretched exponentials, indicating that the relaxation process proceeds through hopping events between a multitude of minima. This finding is in accordance with current ideas of VWF self-association. The longest relaxation time does not show a clear dependence on the length of the bundle, and is dominated by the internal conformations and effective friction within the bundle.

Origin sites of calcium release and calcium oscillations in frog sympathetic neurons.

In many neurons, Ca(2+) signaling depends on efflux of Ca(2+) from intracellular stores into the cytoplasm via caffeine-sensitive ryanodine receptors (RyRs) of the endoplasmic reticulum. We have used high-speed confocal microscopy to image depolarization- and caffeine-evoked increases in cytoplasmic Ca(2+) levels in individual cultured frog sympathetic neurons. Although caffeine-evoked Ca(2+) wave fronts propagated throughout the cell, in most cells the initial Ca(2+) release was from one or more discrete sites that were several micrometers wide and located at the cell edge, even in Ca(2+)-free external solution. During cell-wide cytoplasmic [Ca(2+)] oscillations triggered by continual caffeine application, the initial Ca(2+) release that began each Ca(2+) peak was from the same subcellular site or sites. The Ca(2+) wave fronts propagated with constant amplitude; the spread was mostly via calcium-induced calcium release. Propagation was faster around the cell periphery than radially inward. Local Ca(2+) levels within the cell body could increase or decrease independently of neighboring regions, suggesting independent action of spatially separate Ca(2+) stores. Confocal imaging of fluorescent analogs of ryanodine and thapsigargin, and of MitoTracker, showed potential structural correlates to the patterns of Ca(2+) release and propagation. High densities of RyRs were found in a ring around the cell periphery, mitochondria in a broader ring just inside the RyRs, and sarco-endoplasmic reticulum Ca(2+) ATPase pumps in hot spots at the cell edge. Discrete sites at the cell edge primed to release Ca(2+) from intracellular stores might preferentially convert Ca(2+) influx through a local area of plasma membrane into a cell-wide Ca(2+) increase.

Linear electrical properties of the transverse tubules and surface membrane of skeletal muscle fibers.

With the use of two intracellular microelectrodes and a circuit designed to compensate for the effects of stray capacitances around the electrodes, transfer impedance measurements were made at frequencies from 0.5 to 1000 c/s on frog sartorius muscle fibers bathed in 7.5 mM K Ringer solution. Complete AC cable analyses performed at 46, 100, 215, 464, and 1000 c/s showed that the fibers behaved as ideal one-dimensional cables having purely resistive internal impedances (R(i) = 102 +/- 11 Omega cm). Two circuits were considered for fiber inside-outside impedance, a four lumped parameter circuit and a parallel resistance and capacitance shunted by the input impedance of a lattice model for the T-system. Least squares fits to fiber input impedance phase angles were better with the latter circuit than with the former. With the use of the lattice model the specific capacitance of both the surface and transverse tubule membranes was found to be 1 microF/cm(2) and the internal resistivity of the tubules to be about 300 Omega cm.

Kinetics of intramembrane charge movement and conductance activation of batrachotoxin-modified sodium channels in frog node of Ranvier.

Sodium current and intramembrane gating charge movement (Q) were monitored in voltage-clamped frog node of Ranvier after modification of all sodium channels by batrachotoxin (BTX). Sodium current activation followed a single-exponential time course, provided a delay was interposed between the onset of the step ON depolarization and that of the current change. The delay decreased with increased ON depolarization and, for a constant ON depolarization, increased with prehyperpolarization. ON charge movement followed a single-exponential time course with time constants tau Q,ON slightly larger than tau Na, ON. For pulses between -70 and -50 mV, tau Q,ON/tau Na,ON = 1.14 +/- 0.08. The OFF charge movement and OFF sodium current tails after a depolarizing pulse followed single-exponential time courses, with tau Q, OFF larger than tau Na, OFF. tau Q,OFF/tau Na,OFF increased with OFF voltage from 1 near -100 mV to 2 near -160 mV. At a set OFF potential (-120 mV), both tau Q,OFF and tau Na,OFF increased with ON pulse duration. The delay in INa activation and the effect of ON pulse duration on tau Q,OFF and tau Na,OFF are inconsistent with a simple two-state, single-transition model for the gating of batrachotoxin-modified sodium channels.

Effects of low myoplasmic Mg2+ on calcium binding by parvalbumin and calcium uptake by the sarcoplasmic reticulum in frog skeletal muscle.

The effects of low intracellular free Mg2+ on the myoplasmic calcium removal properties of skeletal muscle were studied in voltage-clamped frog skeletal muscle fibers by analyzing the changes in intracellular calcium and magnesium due to membrane depolarization under various conditions of internal free [Mg2+]. Batches of fibers were internally equilibrated with cut end solutions containing two calcium indicators, antipyrylazo III (AP III) and fura-2, and different concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor [Ca2+] and [Mg2+] transients, whereas fura-2 fluorescence was mostly used to monitor resting [Ca2+]. Shortly after applying an internal solution containing less than 60 microM free Mg2+ to the cut ends of depolarized fibers most of the fibers exhibited spontaneous repetitive movements, suggesting that free internal Mg2+ might affect the activity of the sarcoplasmic reticulum (SR) calcium channels at rest. The spontaneous contractions generally subsided. In polarized fibers the maximal amplitude of the calcium transient elicited by a depolarizing pulse was about the same whatever the internal [Mg2+], but its decay after the end of the pulse slower in low [Mg2+]. In low [Mg2+] (less than 0.14 mM), the mean rate constant of decay obtained from fitting a single exponential plus a constant to the decay of the calcium transients was approximately 30% of its value in the control fibers (1 mM internal [Mg2+]). A model characterizing the main calcium removal properties of a frog skeletal muscle fiber, including the SR pump and the Ca-Mg sites on parvalbumin, was fitted to the decay of the calcium transients. Results of the fits show that in low internal [Mg2+] the slowing of the decay of the calcium transient can be well predicted by both a decreased rate of SR calcium uptake and an expected decreased resting magnesium occupancy of parvalbumin leading to a reduced contribution of parvalbumin to the overall rate of calcium removal. These results are thus consistent with the known properties of parvalbumin as a Ca-Mg buffer and furthermore suggest that in an intact portion of a muscle fiber, the activity of the SR calcium pump can be affected by the level of free Mg2+.

Low myoplasmic Mg2+ potentiates calcium release during depolarization of frog skeletal muscle fibers.

The role of intracellular free magnesium concentration ([Mg2+]) in modulating calcium release from the sarcoplasmic reticulum (SR) was studied in voltage-clamped frog cut skeletal muscle fibers equilibrated with cut end solutions containing two calcium indicators, fura-2 and antipyrylazo III (AP III), and various concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor calcium transients, whereas fura-2 fluorescence was used to monitor resting calcium. The rate of release (Rrel) of calcium from the SR was calculated from the calcium transient and found to be increased in low internal [Mg2+]. After correcting for effects of calcium depletion from the SR and normalization to SR content, the mean values of the inactivatable and noninactivatable components of Rrel were increased by 163 and 46%, respectively, in low Mg2+. Independent of normalization to SR content, the ratio of inactivatable to noninactivatable components of Rrel was increased in low internal [Mg2+]. Both observations suggest that internal [Mg2+] preferentially modulates the inactivatable component of Rrel, which is thought to be due to calcium-induced calcium release from the SR. This could also explain the observation that, in low internal [Mg2+], the time to the peak of the calcium transient for a 5-ms depolarizing pulse was not very different from the time to the peak of the delta [Ca2+] for a 10-ms pulse of the same amplitude. Finally, in low internal [Mg2+], the calcium transient elicited by a short depolarizing pulse was in some cases clearly followed by a very slow rise of calcium after the end of the pulse. The observed effects of reduced [Mg2+] on calcium release are consistent with a removal of the inhibition that the normal 1 mM myoplasmic [Mg2+] exerts on calcium release in skeletal muscle fibers.

Kinetics of intramembrane charge movement and sodium current in frog node of Ranvier.

Intramembrane charge movement (Q) and sodium current (INa) were monitored in isolated voltage-clamped frog nodes of Ranvier, ON charge movements (QON) for pulses from the holding potential (-100 mV) to potentials V less than or equal to 0 mV followed single exponential time courses, whereas two exponentials were found for pulses to V greater than or equal to 20 mV. The voltage dependence of both QON and its time constant tauON indicated that the two ON components resolved at V greater than or equal to 20 mV were also present, though not resolvable, for pulses to V less than or equal to 0 mV. OFF charge movements (QOFF) monitored at various potentials were well described by single exponentials. When QOFF was monitored at -30 or -40 mV after a 200-microsecond pulse to +20 mV and QON was monitored at the same potential using pulses directly from -100 mV, tauON/tauOFF = 2.5 +/- 0.3. At a set OFF potential (-90 to -70 mV), tauOFF first increased with increasing duration tON of the preceding pulse to a given potential (0 to +30 mV) and then decreased with further increases in tON. The declining phase of tauOFF followed a time course similar to that of the decline in QOFF with tON. For the same pulse protocol, the OFF time constant tauNa for INA also first increased with tON but then remained constant over the tON interval during which tauOFF and QOFF were declining. After 200- or 300-microsecond pulses to +20, +20, or +50 mV, tauOFF/tauNa at -70 to -90 mV was 1.2 +/- 0.1. Similar tauOFF/tauNa ratios were predicted by channel models having three identical charged gating particles that can rapidly and reversibly form an immobile dimer or trimer after independently crossing the membrane from their OFF to their ON locations.

Effects of membrane potential on the capacitance of skeletal muscle fibers.

A method for measuring muscle fiber capacitance using small test pulses applied with the three-microelectrode voltage clamp is presented. Using this method, three membrane potential-dependent changes in capacitance were observed: (a) Capacitance of polarized fibers increased by 5--15% with depolarization from V less then -100 mV to voltages slightly below the contraction threshold. (b) Capacitance of fibers depolarized to -30 mV by 100 mM Rb solution decreased by roughly 8% with further depolarization to about +50 mV and increased with repolarization, exhibiting a maximum increase of about 10% at -80 to -90 mV. (c) Capacitance of fibers depolarized to -15 mV by 100 mM K solution increased by about 19% with further depolarization to +43 mV and decreased by about 23% with repolarization to -62 mV. Effects a and b are attributed to changes in specific membrane capacitance due to voltage-dependent redistribution of mobile charged groups within surface of T-tubule membranes. Effect c is caused by changes in the T-system space constant lambdaT due to the voltage dependence of K conductance (inward rectification). Analysis of c showed that in 100 mM K solution lambdaT congruent to 30 mum when inward rectification was fully activated by hyperpolarization and that the density of inward rectifier channels is about the same in surface and tubular membranes. Fiber internal resistance was found to be independent of voltage, a necessary condition for the interpretation of the capacitance measurements.

Time-course of potential spread along a skeletal muscle fiber under voltage clamp.

The equations describing the time-course of potential spread into a terminated segment of muscle fiber are given for the condition that a step of voltage is applied at x - 2l. Measurements of V(2l) - V(l) were made at 16.7-19.5 degrees C, using a three-microelectrode voltage clamp, to compare with the theory. Best least squares fits of calculated curves to data obtained in Ringer's solution (5 mM K) gave GL = 10 mumho/cm and Cm' = 1.6 muF/cm2. Similar measurements in 100 mM K solution, with the inward rectifier shut off by a positive prepulse, gave GL = 20 mumho/cm and Cm' = 2.0 muF/cm2. The time-course of V(2l) - V(l), measured when the inward rectifier was fully activated by a negative prepulse, was in good agreement with the curve calculated assuming no change in GL and Cm' and that the only effect of the negative prepulse was to increase the conductance of surface and tubular membranes.

Decreased K+ conductance produced by Ba++ in frog sartorius fibers.

The action of Ba(++) on membrane potential (E(m)) and resistance (R(m)) of frog (R. pipiens) sartorius fibers was studied. In normal Cl(-) Ringer's, Ba(++) (<9 mM) did not depolarize or induce contractions, but increased R(m) slightly above the control value of 3.8 +/- 0.6 K ohm-cm(2). In Cl(-)-free Ringer's (methane sulfonate) R(m) was 28.8 +/- 2.8 K ohm-cm(2), and low concentrations of Ba(++) (0.05-5.0 mM) depolarized and induced spontaneous contractions (fibrillation), even in tetrodotoxin. To stop disturbance of the microelectrodes, contractions were prevented by using two Cl(-)-free solutions: (a) twice hypertonic with sucrose (230 mM), or (b) high K(+) (83 mM) partially replacing Na(+). In the hypertonic solution, the fiber diameters decreased, E(m) increased slightly, and R(m) decreased to 9.0 +/- 0.6 K ohm-cm(2) (perhaps due to swelling of sarcotubules). Ba(++) (0.5 mM) rapidly increased R(m) to 31.3 +/- 3.8, decreased E(m) (e.g., to -30 mv), and induced spontaneous "action potentials;" Sr(++) had no effect. In the high K(+) solution, the fibers were nearly completely depolarized, and R(m) was decreased markedly to 1.5 +/- 0.2 K ohm-cm(2); Ba(++) increased R(m) to 6.7 +/- 0.5 K ohm-cm(2). The Ba(++) actions usually began within 0.5 min and reached a maximum within 5 min. Addition of SO(4) (=), to precipitate the Ba(++), rapidly reversed the increase in R(m). Ba(++) must act by decreasing K(+) conductance (g(K)). In Cl(-) Ringer's, the high g(Cl)/g(K) ratio masked the effect of Ba(++) on g(K). Thus, small concentrations of Ba(++) specifically and rapidly decrease g(K).