Magainin 2 belongs to the family of peptides, which interacts with the lipid membranes. The present work deals with the effect of this peptide on the mechanical properties of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine Giant Unilamellar Vesicle, characterized by the bending stiffness modulus. The bending elastic modulus is measured by Vesicle Fluctuation Analysis at biologically relevant pH and physiological buffer conditions and shows a dramatic decrease with increasing peptide concentration. The observed bilayer softening is interpreted in terms of a continuum model describing perturbations on the membrane organization. Our analysis suggests that the adsorbed peptides give rise to considerable local curvature disruptions of the membrane.
Fluorescent probes are used in membrane biophysics studies to provide information about physical properties such as lipid packing, polarity and lipid diffusion or to visualize membrane domains. However, our understanding of the effects the dyes themselves may induce on the membrane structure and properties are sparse. As mechanical properties like bending elasticity were already shown to be highly sensitive to the addition of "impurities" into the membranes, we have investigated the impact of six different commonly used fluorescent membrane probes (LAURDAN, TR-DPPE, Rh-DPPE, DiIC18, Bodipy-PC and NBD-PC) on the bending elasticity of dye containing POPC GUVs as compared to single component POPC GUVs. Small changes in the membrane bending elasticity compared to single POPC bilayers are observed when 2 mol% of Rh-DPPE, Bodipy-PC or NBD-PC are added in POPC membranes. These binary membranes are showing non reproducible mechanical properties attributed to a photo-induced peroxidation processes that may be controlled by a reduction of the fluorescent dye concentration. For TR-DPPE, a measurable decrease of the bending elasticity is detected with reproducible bending elasticity measurements. This is a direct indication that this dye, when exposed to illumination by a microscope lamp and contrary to Rh-DPPE, does not induce chemical degradation. At last, LAURDAN and DiIC18 probes mixed with POPC do not significantly affect the bending elasticity of pure POPC bilayers, even at 2 mol%, suggesting these latter probes do not induce major perturbations on the structure of POPC bilayers.
We show how to greatly improve precision when determining bending elasticity of giant unilamellar vesicles. Taking advantage of the well-known quasi-spherical model of liposome flickering, we analyze the full probability distributions of the configurational fluctuations instead of limiting the analysis to the second moment measurements only as usually done in previously published works. This leads to objective criteria to reject vesicles that do not behave according to the model. As a result, the confidence in the bending elasticity determination of individual vesicles that fit the model is improved and, consequently, the reproducibility of this measurement for a given membrane system. This approach uncovers new possibilities for bending elasticity studies like detection of minute influences by solutes in the buffer or into the membrane. In the same way, we are now able to detect the inhomogeneous behavior of giant vesicle systems such as the hazardous production of peroxide in bilayers containing fluorescent dyes.
In biophysical and biochemical studies of lipid bilayers the influence of the used buffer is often ignored or assumed to be negligible on membrane structure, elasticity, or physical properties. However, we here present experimental evidence, through bending rigidity measurements performed on giant vesicles, of a more complex behavior, where the buffering molecules may considerably affect the bending rigidity of phosphatidylcholine bilayers. Furthermore, a synergistic effect on the bending modulus is observed in the presence of both salt and buffer molecules, which serves as a warning to experimentalists in the data interpretation of their studies, since typical lipid bilayer studies contain buffer and ion molecules.
Interaction between integral membrane proteins and the lipidbilayer component of biological membranes is expected to mutually influence the proteins and the membrane. We present quantitative evidence of a manifestation of the lipid-protein interactions in liposomal membranes, reconstituted with actively pumping Na + ,K + -ATPase, in terms of nonequilibrium shape fluctuations that contain a relaxation time, τ, which is robust and independent of the specific fluctuation modes of the membrane. In the case of pumping Na + -ions, analysis of the flicker-noise temporal correlation spectrum of the liposomes leads to τ ≃ 0.5 s, comparing favorably with an intrinsic reaction-cycle time of about 0.4 s from enzymology.sodium pump reconstituted in giant vesicles | vesicle fluctuation analysis | membrane dynamics T he interaction between integral proteins and lipids in biological membranes is a key to unraveling the molecular organization and the biological functioning of cell membranes (1, 2). Quantitative studies of lipid-protein interactions in membranes proceed most conveniently by means of well-defined biophysical model systems (3). Fundamental questions involve how lipids influence protein function, on the one hand, and how proteins and protein function impact on the properties of the lipids and membrane organization, on the other hand. Generally, biophysical model studies are performed under some kind of equilibrium conditions, and it is difficult to quantitatively read out membrane properties under the conditions of fully functional proteins. We present here a model study of a lipid-protein reconstitution in giant unilamellar vesicles (GUVs) from which it is possible not only to read out a well-defined nonequilibrium property, the dynamic flicker spectrum, of an active membrane but also to analyze this property in terms of a molecular time scale of an active protein, Na + ,K + -ATPase, while pumping sodium ions.It has been known for a long time that thermal noise establishes itself in equilibrium bending fluctuations and the characteristic flickering motion of erythrocytes (4), and it has been proposed that protein activity contributes to an enhancement of the flickering in erythrocytes (5-8) and lymphocytes (9). The physical picture is that protein activity, in particular the pumping of ions across the membrane, couples to the lipid-bilayer component of the membrane or the cytoskeleton and hence renormalizes the effective bending rigidity. Quantitative interpretation of experimental data for complex real biological membranes is, however, difficult and questionable (10). For this reason, model studies of so-called active membranes, being lipid bilayers with reconstituted active proteins, such as bacteriorhodopsin (11) and Ca 2+ -ATPase (12), have been advanced, showing, by use of micropipette aspiration techniques, that the membranes become softer when the proteins were active, an observation that was interpreted as due to enhanced bending fluctuations and flickering. We demonstrate here, in the case of active membra...
During asymmetric division of the Caenorhabditis elegans zygote, to properly distribute cell fate determinants, the mitotic spindle is asymmetrically localized by a combination of centering and cortical-pulling microtubule-mediated forces, the dynamics of the latter being regulated by mitotic progression. Here, we show a, to our knowledge, novel and additional regulation of these forces by spindle position itself. For that, we observed the onset of transverse spindle oscillations, which reflects the burst of anaphase pulling forces. After delaying anaphase onset, we found that the position at which the spindle starts to oscillate was unchanged compared to control embryos and uncorrelated to anaphase onset. In mapping the cortical microtubule dynamics, we measured a steep increase in microtubule contact density after the posterior centrosome reached the critical position of 70% of embryo length, strongly suggesting the presence of a positional switch for spindle oscillations. Expanding a previous model based on a force-generator temporal control, we implemented this positional switch and observed that the large increase in microtubule density accounted for the pulling force burst. Thus, we propose that the spindle position influences the cortical availability of microtubules on which the active force generators, controlled by cell cycle progression, can pull. Importantly, we found that this positional control relies on the polarity-dependent LET-99 cortical band, the boundary of which could be probed by microtubules. This dual positional and temporal control well accounted for our observation that the oscillation onset position resists changes in cellular geometry and moderate variations in the active force generator number. Finally, our model suggests that spindle position at mitosis end is more sensitive to the polarity factor LET-99, which restricts the region of active force generators to a posterior-most region, than to microtubule number or force generator number/activity. Overall, we show that robustness in spindle positioning originates in cell mechanics rather than biochemical networks.
Contaminants taken up by living organisms in the environment as a result of anthropogenic contamination can reduce the tolerance of natural stressors, e.g., low temperatures, but the physiological mechanisms behind these interactions of effects are poorly understood. The tolerance to low temperatures of organisms that cannot regulate their body temperature (ectotherms) depends on their ability to increase the fluidity of their cellular membranes at low temperatures. Our study shows that contaminants accumulating in lipids of organisms alter the physical state of their membranes simply by being present. Contaminants of varying chemical structures can alter the membrane fluidity in either direction and correspondingly modulate the cold tolerance of intact animals.
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