Heavy water induces bundling in entangled actin networks.

Heavy water is known to affect many different biological systems, with the most striking effects observed at the cellular level. Many dynamic processes, such as migration or invasion, but also central processes of cell proliferation are measurably inhibited by the presence of deuterium oxide (D2O). Furthermore, individual cell deformabilities are significantly decreased upon D2O treatment. In order to understand the origin of these effects, we studied entangled filamentous actin networks, a commonly used model system for the cytoskeleton, which is considered a central functional element for dynamic cellular processes. Using bulk shear rheology to extract rheological signatures of reconstituted actin networks at varying concentrations of D2O, we found a non-monotonic behavior, which is explainable by a drastic change in the actin network architecture. Applying light scattering and fluorescence microscopy, we were able to demonstrate that the presence of deuterium oxide induces bundling in reconstituted entangled networks of filamentous actin. This constitutes an entirely novel and previously undescribed actin bundling mechanism.

Cells in Slow Motion: Apparent Undercooling Increases Glassy Behavior at Physiological Temperatures.

Solvent conditions are unexpectedly sufficient to drastically and reversibly slow down cells. In vitro on the molecular level, protein-solvent interactions drastically change in the presence of heavy water (D2 O) and its stronger hydrogen bonds. Adding D2 O to the cell medium of living cells increases the molecular intracellular viscosity. While cell morphology and phenotype remain unchanged, cellular dynamics transform into slow motion in a changeable manner. This is exemplified in the slowdown of cell proliferation and migration, which is caused by a reversible gelation of the cytoplasm. In analogy to the time-temperature superposition principle, where temperature is replaced by D2 O, an increase in viscosity slows down the effective time. Actin networks, crucial structures in the cytoplasm, switch from a power-law-like viscoelastic to a more rubber-like elastic behavior. The resulting intracellular resistance and dissipation impair cell movement. Since cells are highly adaptive non-equilibrium systems, they usually respond irreversibly from a thermodynamic perspective. D2 O induced changes, however, are fully reversible and their effects are independent of signaling as well as expression. The stronger hydrogen bonds lead to glass-like, drawn-out intramolecular dynamics, which may facilitate longer storage times of biological matter, for instance, during transport of organ transplants.

Heavy water reduces GFP expression in prokaryotic cell-free assays at the translation level while stimulating its transcription.

The in vitro proliferation of prokaryotic and eukaryotic cells is remarkably hampered in the presence of heavy water (D2O). Impairment of gene expression at the transcription or translation level can be the base for this effect. However, insights into the underlying mechanisms are lacking. Here, we employ a cell-free expression system for the quantitative analysis of the effect of increasing percentages of D2O on the kinetics of in-vitro GFP expression. Experiments are designed to discriminate the rates of transcription, translation, and protein folding using pDNA and mRNA vectors, respectively. We find that D2O significantly stimulates GFP expression at the transcription level but acts as a suppressor at translation and maturation (folding) in a linear dose-dependent manner. At a D2O concentration of 60%, the GFP expression rate was reduced to 40% of an undisturbed sample. We observed a similar inhibition of GFP expression by D2O in a recombinant Escherichia coli strain, although the inhibitory effect is less pronounced. These results demonstrate the suitability of cell-free systems for quantifying the impact of heavy water on gene expression and establish a platform to further assess the potential therapeutic use of heavy water as antiproliferative agent.

Differences in the modulation of collective membrane motions by ergosterol, lanosterol, and cholesterol: a dynamic light scattering study.

A dynamic light scattering setup was used to study the undulations of freely suspended planar lipid bilayers, the so-called black lipid membranes, over a previously inaccessible range of frequency and wave number. A pure synthetic lecithin bilayer, 1,2-dielaidoyl-sn-3-glycero-phoshatidylcholine (DEPC), and binary mixtures of DEPC with 40 mol % of cholesterol, ergosterol, or lanosterol were studied. By analyzing the dynamic light scattering data (oscillation and damping curves) in terms of transverse shear motion, we extracted the lateral tension and surface viscosity of the composite bilayers for each sterol. Cholesterol gave the strongest increase in lateral tension (approximately sixfold) with respect to the DEPC control, followed by lanosterol (approximately twofold), and ergosterol (1.7-fold). Most interestingly, only cholesterol simultaneously altered the surface viscosity of the bilayer by almost two orders of magnitude, whereas the other two sterols did not affect this parameter. We interpret this unique behavior of cholesterol as a result of its previously established out-of-plane motion which allows the molecule to cross the bilayer midplane, thereby effectively coupling the bilayer leaflets to form a highly flexible but more stable composite membrane.

Anisotropic motion and molecular dynamics of cholesterol, lanosterol, and ergosterol in lecithin bilayers studied by quasi-elastic neutron scattering.

Quasi-elastic neutron scattering (QENS) was employed to study the molecular dynamics of three structurally related sterols, namely, cholesterol, lanosterol, and ergosterol. Oriented bilayers of dipalmitoylphosphatidylcholine (DPPC) were investigated at 40 mol % sterol content and at three temperatures (20, 36, and 50 degrees C) for two energy resolutions. Data analysis was concentrated on a direct comparison of the out-of-plane and the in-plane high-frequency motions of the three sterols in terms of their rates and amplitudes. The (spatially restricted) diffusive motion of the three sterols in the two directions was characterized by diffusion constants in the range of (5-30) x 10(-12) x m(2) x s(-1), with a significantly faster rate of diffusion along the membrane normal, resulting in a diffusional anisotropy, D(a). At low temperature (20 degrees C), cholesterol showed the highest value (D(a) = 4.5), while lanosterol gave the lowest one (D(a) = 2.0). At high temperature (50 degrees C), ergosterol diffusion had the highest diffusion anisotropy (D(a) = 2.0) compared to lanosterol (D(a) = 1.8) and cholesterol (D(a) = 1.6). Most interestingly, cholesterol showed at all three temperatures an amplitude of its out-of-plane-motion of 1.0-1.1 nm, more than a factor of 3 higher than measured for the other two sterols. This finding suggests that the short alkyl chain of the cholesterol molecule may cross at high frequency the bilayer midplane, while the other two sterols remain confined within the geometrical limits of each monolayer leaflet. The results provide an example of how slight structural alterations of sterols can affect their molecular dynamics in bilayers, which in turn may be relevant to the membrane micromechanical properties.

Nanospheres from Polymerized Lipofullerenes.

Remarkably mechanically stable spherical nanostructures are formed by UV-initiated polymerization from polymerizable lipofullerene 1 in multilamellar lipid vesicles. The polymer beads can be filled or hollow. R=COO(CH2)9C≡C-C≡C(CH2)4CH3.