Photonic emitter manipulation to sample nanoscale topography.

We demonstrate that photonic emitter manipulation can be used to image the nanoscale topography of a fluorescently labeled layer in confocal imaging. We exploit the fact that a metallic probe manipulates a fluorophore's photonic environment, and thereby its fluorescent lifetime, in a strongly distance-dependent manner. To image surface topography, a metallic probe that is not in contact with the surface is rasterscanned over a fluorescently labeled sample. The axial position of the probe is kept constant. At each lateral probe position, the fluorescence decay is recorded and analyzed to obtain probe - sample distances and hence, the topography of the sample. We present images resolving a microfabricated step of 14 nm in topography, with the probe positioned at different axial positions.

Exogenous α-synuclein hinders synaptic communication in cultured cortical primary rat neurons.

Amyloid aggregates of the protein α-synuclein (αS) called Lewy Bodies (LB) and Lewy Neurites (LN) are the pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. We have previously shown that high extracellular αS concentrations can be toxic to cells and that neurons take up αS. Here we aimed to get more insight into the toxicity mechanism associated with high extracellular αS concentrations (50-100 µM). High extracellular αS concentrations resulted in a reduction of the firing rate of the neuronal network by disrupting synaptic transmission, while the neuronal ability to fire action potentials was still intact. Furthermore, many cells developed αS deposits larger than 500 nm within five days, but otherwise appeared healthy. Synaptic dysfunction clearly occurred before the establishment of large intracellular deposits and neuronal death, suggesting that an excessive extracellular αS concentration caused synaptic failure and which later possibly contributed to neuronal death.

Tryptophan fluorescence reveals structural features of alpha-synuclein oligomers.

Oligomeric alpha-synuclein (alphaS) is considered to be the potential toxic species responsible for the onset and progression of Parkinson's disease, possibly through the disruption of lipid membranes. Although there is evidence that oligomers contain considerable amounts of secondary structure, more detailed data on the structural characteristics and how these mediate oligomer-lipid binding are critically lacking. This report is, to our knowledge, the first study that aimed to address the structure of oligomeric alphaS on a more detailed level. We have used tryptophan (Trp) fluorescence spectroscopy to gain insight into the structural features of oligomeric alphaS and the structural basis for oligomer-lipid interactions. Several single Trp mutants of alphaS were used to gain site-specific information about the microenvironments of monomeric alphaS, oligomeric alphaS and lipid-bound oligomeric alphaS. Acrylamide quenching and spectral analyses indicate that the Trp residues are considerably more solvent protected in the oligomeric form compared with the monomeric protein. In the oligomers, the negatively charged C-terminus was the most solvent exposed part of the protein. Upon lipid binding, a blue shift in fluorescence was observed for alphaS mutants where the Trp is located within the N-terminal region. These results suggest that, as in the case of monomeric alphaS, the N-terminus is critical in determining oligomer-lipid binding.

Cytoskeletal polymer networks: viscoelastic properties are determined by the microscopic interaction potential of cross-links.

Although the structure of cross-linking molecules mainly determines the structural organization of actin filaments and with that the static elastic properties of the cytoskeleton, it is largely unknown how the biochemical characteristics of transiently cross-linking proteins (actin-binding proteins (ABPs)) affect the viscoelasticity of actin networks. In this study, we show that the macroscopic network response of reconstituted actin networks can be traced back to the microscopic interaction potential of an individual actin/ABP bond. The viscoelastic response of cross-linked actin networks is set by the cross-linker off-rate, the binding energy, and the characteristic bond length of individual actin/ABP interactions.

Transient binding and dissipation in cross-linked actin networks.

In contrast with entangled actin solutions, transiently cross-linked actin networks can provide highly elastic properties while still allowing for local rearrangements in the microstructure-on biological relevant time scales. Here, we show that thermal unbinding of transient cross-links entails local stress relaxation and energy dissipation in an intermediate elasticity dominated frequency regime. We quantify the viscoelastic response of an isotropically cross-linked actin network by experimentally tuning the off rate of the transiently cross-linking molecules, their density, and the solvent viscosity. We reproduce the measured frequency response by a semiphenomenological model that is predicated on microscopic unbinding events.

Helical twist controls the thickness of F-actin bundles.

In the presence of condensing agents such as nonadsorbing polymer, multivalent counter ions, and specific bundling proteins, chiral biopolymers typically form bundles with a finite thickness, rather than phase-separating into a polymer-rich phase. Although short-range repulsive interactions or geometrical frustrations are thought to force the equilibrium bundle size to be limited, the precise mechanism is yet to be resolved. The importance of the tight control of biopolymer bundle size is illustrated by the ubiquitous cytoskeletal actin filament bundles that are crucial for the proper functioning of cells. Using an in vitro model system, we show that size control relies on a mismatch between the helical structure of individual actin filaments and the geometric packing constraints within bundles. Small rigid actin-binding proteins change the twist of filamentous actin (F-actin) in a concentration-dependent manner, resulting in small, well defined bundle thickness up to approximately 20 filaments, comparable to those found in filopodia. Other F-actin cross-linking proteins can subsequently link these small, well organized bundles into larger structures of several hundred filaments, comparable to those found in, for example, Drosophila bristles. The energetic tradeoff between filament twisting and cross-linker binding within a bundle is suggested as a fundamental mechanism by which cells can precisely adjust bundle size and strength.

Osmotic shrinkage and reswelling of giant vesicles composed of dioleoylphosphatidylglycerol and cholesterol.

The osmotic shrinkage of giant unilamellar dioleoylphosphatidylglycerol (DOPG) vesicles in a hypertonic osmotic solution is investigated. The volume reduction for given membrane area leads to a vesiculation of the bilayer into the interior of the giant. The size of the daughter vesicles that appear inside the giant is uniform and an increasing function of the cholesterol content, but independent of the osmotic gradient applied. The radius of the daughter vesicles increases from 0.2 microm to 3.0 microm when the cholesterol content is changed from 0 to 40%. It is argued that the size of the daughter vesicles is regulated by the membrane persistence length, which is an exponential function of the mean bending modulus. From the kinetics of shrinkage it follows that approximately 14% of the daughter vesicles remain attached to the mother giant. This is in reasonable agreement with osmotic swelling experiments which show that approximately 11% of the daughter vesicles is available for area expansion.

Mechanics of bundled semiflexible polymer networks.

While actin bundles are used by living cells for structural fortification, the microscopic origin of the elasticity of bundled networks is not understood. Here, we show that above a critical concentration of the actin binding protein fascin, a solution of actin filaments organizes into a pure network of bundles. While the elasticity of weakly cross-linked networks is dominated by the affine deformation of tubes, the network of bundles can be fully understood in terms of nonaffine bending undulations.

Bending rigidity of mixed phospholipid bilayers and the equilibrium radius of corresponding vesicles.

In spite of the large mean bending moduli observed for phospholipid bilayers, stable vesicle phases were recently observed for dilute solutions of charged phospholipids. A correspondingly large negative Gaussian bending modulus associated with charged membranes results in an overall curvature energy that is so low that entropic stabilization is possible. The mean bending modulus determines the membrane persistence length and therefore it is reasonable that there is a correlation between the membrane rigidity and the size of the lipid vesicles. Here we show that in mixtures of the anionic phospholipid dioleoylphosphatidylglycerol and the zwitterionic phospholipid dioleoylphosphatidylcholine the radius of vesicles produced by repetitive freeze-thaw cycles is considerably smaller than expected from the rigidities of the corresponding pure lipid bilayers. Self-consistent field calculations indicate that the changes in the equilibrium radius of mixed bilayers can be attributed to the dependences of the mean bending modulus k(c) on lipid mixing and the average surface charge density.

Opposing effects of cation binding and hydration on the bending rigidity of anionic lipid bilayers.

We correlate the molecularly realistic self-consistent field predictions for the mean bending modulus kc of charged lipid vesicles with experimental observations of the size R of corresponding vesicles that are produced by the freeze-thaw method. We elaborate on the Ansatz that the bending modulus is related to the membrane persistence length and that this length scale sets the radius of the vesicles. Alkali cations have a remarkable effect on the mean bending modulus and thus on the equilibrium radius of negatively charged entropically stabilized dioleoylphosphatidylglycerol (DOPG) vesicles. Where cation hydration typically results in thicker and thus stiffer membranes, specific adsorption to the bilayer surface results in a decrease of the surface charge density and the thickness of the membrane-associated electric double layer. As a result of these opposing effects on kc and R, the largest DOPG vesicles are found in the presence of K+, which combines an intermediate hydration enthalpy and PG-binding affinity.

Entropic stabilization and equilibrium size of lipid vesicles.

We have studied the phase behavior of zwitterionic phospholipid dioleoylphosphatidylcholine (DOPC) vesicles (membranes) and interpreted our results using scaling arguments in combination with molecular realistic self-consistent field (SCF) calculations. DOPC membranes acquire a partial negative charge per lipid molecule at intermediate NaBr concentrations. As a result of this, dilute DOPC solutions form stable unilamellar vesicles. Both at low and high salt concentrations phase separation into a lamellar and a vesicular phase is observed. The vesicle radius decreases as a power law with decreasing lipid concentration. This power-law concentration dependence indicates that the vesicle phase is entropically stabilized; the size of the DOPC vesicles result from a competition between the bending energy and translation and undulation entropy. This scaling behavior breaks down for very small vesicles. This appears to be consistent with SCF predictions that point to the fact that in this regime the mean bending modulus kc increases with curvature. The SCF theory predicts that, at low ionic strength, the membrane stability improves when there is more charge on the lipids. Upon a decrease of the ionic strength, lipids with a full negative charge form vesicles that grow exponentially in size because the mean bending modulus increases with decreasing ionic strength. At the same time the Gaussian bending modulus becomes increasingly negative such that the overall bending energy tends to zero. This indicates that small micelles become the dominant species. The SCF theory thus predicts a catastrophic break down of giant vesicles in favor of small micelles at sufficiently low ionic strength and high charge density on the lipids.

Viscoelasticity of isotropically cross-linked actin networks.

Despite their importance for the proper function of living cells, the physical properties of cross-linked actin networks remain poorly understood as the occurrence of heterogeneities hamper a quantitative physical description. The isotropic homogeneously cross-linked actin network presented here enables us to quantitatively relate the network response to a single filament model by determining the dominating length scale. The frequency dependence of the linear response and nonuniversal form of the nonlinear response reveal the importance of cross-linker unbinding events.

Micro- and macrorheological properties of actin networks effectively cross-linked by depletion forces.

The structure and rheology of cytoskeletal networks are regulated by actin binding proteins. Aside from these specific interactions, depletion forces can also alter the properties of cytoskeletal networks. Here we demonstrate that the addition of poly(ethylene glycol) (PEG) as a depletion agent results not only in severe structural changes, but also in alterations in mechanical properties of actin solutions. In the plateau of the elastic modulus two regimes can be distinguished by micro and macrorheological methods. In the first, the elastic modulus increases only slightly with increasing depletion agent, whereas above a critical concentration c*, a strong increase of cPEG6k3.5 is observed in a distinct second regime. Microrheological data and electron microscopy images show a homogenous network of actin filaments in the first regime, whereas at higher PEG concentrations a network of actin bundles is observed. The concentration dependence of the plateau modulus G0, the shift in entanglement time taue, and the nonlinear response indicate that below c* the network becomes effectively cross-linked, whereas above c* G0(cPEG6k) is primarily determined by the network of bundles that exhibits a linearly increasing bundle thickness.

Charged lipid vesicles: effects of salts on bending rigidity, stability, and size.

The swelling behavior of charged phospholipids in pure water is completely different from that of neutral or isoelectric phospholipids. It was therefore suggested in the past that, instead of multilamellar phases, vesicles represent the stable structures of charged lipids in excess water. In this article, we show that this might indeed be the case for dioleoylphosphatidylglycerol and even for dioleoylphosphatidylcholine in certain salts. The size of the vesicles formed by these lipids depends on the phospholipid concentration in a way that has been predicted in the literature for vesicles of which the curvature energy is compensated for by translational entropy and a renormalization of the bending moduli (entropic stabilization). Self-consistent field calculations on charged bilayers show that the mean bending modulus kc and the Gaussian bending modulus k have opposite sign and /k/>kc, especially at low ionic strength. This has the implication that the energy needed to curve the bilayer into a closed vesicle Eves=4pi(2kc+k) is much less than one would expect based on the value of kc alone. As a result, Eves can relatively easily be entropically compensated. The radii of vesicles that are stabilized by entropy are expected to depend on the membrane persistence length and thus on kc. Experiments in which the vesicle size is studied as a function of the salt and the salt concentration correlate well with self-consistent field predictions of kc as a function of ionic strength.

Quantitative NMR microscopy of osmotic stress responses in maize and pearl millet.

The effect of osmotic stress (-0.35 MPa) on the cell water balance and apical growth was studied non-invasively for maize (Zea mays L., cv. LG 11) and pearl millet (Pennisetum americanum L., cv. MH 179) by (1)H NMR microscopy in combination with water uptake measurements. Single parameter images of the water content and the transverse relaxation time (T(2)) were used to discriminate between the different tissues and to follow the water status of the apical region during osmotic stress. The T(2) values of non-stressed stem tissue turned out to be correlated to the cell dimensions as determined by optical microscopy. Growth was found to be strongly inhibited by mild stress in both species, whereas the water uptake was far less affected. During the experiment hardly any changes in water content or T(2) in the stem region of maize were observed. In contrast, the apical tissue of pearl millet showed a decrease in T(2) within 48 h of stress. This decrease in T(2) is interpreted as an increase in the membrane permeability for water.

Supernormal left ventricular diastolic function in triathletes.

We studied the structural and functional heart adaptations of 52 male triathletes compared with those of 22 active, nonathletic men, by 2-dimensional Doppler echocardiography. Left ventricular diastolic function was evaluated by recording transmitral flow velocities. To exclude the influences of preload, left atrial pressure, and aortic pressure, left ventricular diastolic function was also evaluated by pulsed Doppler tissue imaging. Significant differences in cardiac structure and function were observed between the 2 groups. In the triathletes, the left ventricular diastolic function was completely normal, despite signs of mixed eccentric and concentric left ventricular hypertrophy, and this function was better than that in the control group. We measured 2 aspects of the late passive diastolic filling period in the triathletes: ASEAC value (the amplitude of excursion of the interventricular septal endocardium at the end of left ventricular diastole just after atrial contraction); and the time between onset of the P wave on the electrocardiographic tracing and onset of systolic septal movement on M-mode echocardiography. Pulsed Doppler tissue imaging confirmed these results. The E/A ratios (peak early left ventricular diastolic motion velocity divided by the peak atrial systolic motion velocity), measured by pulsed Doppler tissue imaging, yielded even more evidence for supernormal left ventricular diastolic function in the triathletes. Left ventricular relaxation and filling properties were measured along the longitudinal and transverse axes by pulsed Doppler tissue imaging, which was useful for evaluating left ventricular diastolic function. We determined that triathletes may develop supernormal left ventricular diastolic function with increased diastolic reserves.