Vesicle Tumbling Research Articles

Vesicle tumbling inhibited by inertia

Vesicles under flow constitute a model system for the study of red blood cells (RBCs) dynamics and blood rheology. In the blood circulatory system the Reynolds number (at the scale of the RBC) is not always small enough for the Stokes limit to be valid. We develop a numerical method in two dimensions based on the level set approach and solve the fluid/membrane coupling by using an adaptive finite element technique. We find that a Reynolds number of order one can destroy completely the vesicle tumbling motion obtained in the Stokes regime. We analyze in details this phenomenon and discuss some of the far reaching consequences. We suggest experimental tests on vesicles.

Phosphatidylcholine “Wobble” in Vesicles Assessed by High-Resolution 13C Field Cycling NMR Spectroscopy

High resolution (13)C NMR field cycling (covering 11.7 down to 0.002 T) relaxation studies of the sn-2 carbonyl of phosphatidylcholines in vesicles provide a detailed look at the dynamics of this position of the phospholipid in vesicles. The spin-lattice relaxation rate, R(1), observed down to 0.05 T is the result of dipolar and CSA relaxation components characterized by a single correlation time tau(c), with a small contribution from a faster motion contributing to CSA relaxation. At lower fields, R(1) increases further with a correlation time consistent with vesicle tumbling. The tau(c) is particularly interesting since it is 2-3 times slower than what is observed for (31)P of the same phospholipid. However, cholesterol increases the tau(c) for both (31)P and (13)C sites to the same value, approximately 25 ns. These observations suggest faster local motion dominates the dipolar relaxation of the (31)P, while a slower rotation or wobble dominates the relaxation of the carbonyl carbon by the alpha-CH(2) group. The faster motion must be damped with the sterol present. As a general methodology, high resolution (13)C field cycling may be useful for quantifying dynamics in other complex systems as long as a (13)C label (without attached protons) can be introduced.

Vacillating Breathing and Tumbling of Vesicles under Shear Flow

The dynamics of vesicles under a shear flow are analyzed analytically in the small deformation regime. We derive two coupled nonlinear equations which describe the vesicle orientation in the flow and its shape evolution. A new type of motion is found, namely, a "vacillating-breathing" mode: the vesicle orientation undergoes an oscillation around the flow direction, while the shape executes breathing dynamics. This solution coexists with tumbling. Moreover, we provide an explicit expression for the tumbling threshold. A rheological law for a dilute vesicle suspension is outlined.

Tumbling of vesicles under shear flow within an advected-field approach.

We study dynamics of vesicles with a viscosity contrast between the interior and exterior, and subjected to a linear shear flow. It is shown that the vesicle, which assumes a steady orientation with respect to the shear flow for r identical with eta(in)/eta(out)<r(c) (where eta(in) and eta(out) are the internal and external viscosities, and r(c) is a critical value which depends upon the swelling factor), undergoes a tumbling bifurcation for r>or=r(c). Tumbling occurs as a saddle-node bifurcation. We present analytical and numerical results. We develop a powerful method, the advected-field approach. This method allows one to treat several different phenomena with great flexibility.

Structural Analysis of the Protein/Lipid Complexes Associated with Pore Formation by the Bacterial Toxin Pneumolysin

Pneumolysin, a major virulence factor of the human pathogen Streptococcus pneumoniae, is a soluble protein that disrupts cholesterol-containing membranes of cells by forming ring-shaped oligomers. Magic angle spinning and wideline static (31)P NMR have been used in combination with freeze-fracture electron microscopy to investigate the effect of pneumolysin on fully hydrated model membranes containing cholesterol and phosphatidylcholine and dicetyl phosphate (10:10:1 molar ratio). NMR spectra show that the interaction of pneumolysin with cholesterol-containing liposomes results in the formation of a nonbilayer phospholipid phase and vesicle aggregation. The amount of the nonbilayer phase increases with increasing protein concentration. Freeze-fracture electron microscopy indicates the coexistence of aggregated vesicles and free ring-shaped structures in the presence of pneumolysin. On the basis of their size and analysis of the NMR spectra it is concluded that the rings are pneumolysin oligomers (containing 30-50 monomers) complexed with lipid (each with 840-1400 lipids). The lifetime of the phospholipid in either bilayer-associated complexes or free pneumolysin-lipid complexes is > 15 ms. It is further concluded that the effect of pneumolysin on lipid membranes is a complex combination of pore formation within the bilayer, extraction of lipid into free oligomeric complexes, aggregation and fusion of liposomes, and the destabilization of membranes leading to formation of small vesicles.

Lipid bilayer dynamics from simultaneous analysis of orientation and frequency dependence of deuterium spin-lattice and quadrupolar order relaxation

Simultaneous analysis of the deuterium ${(}^{2}\mathrm{H})$ NMR spin relaxation rates of lipid bilayers as a function of both frequency and sample orientation may be decisive in evaluating different models for the dynamics of membranes. Angular dependent ${}^{2}\mathrm{H}$ spin-lattice ${(R}_{1Z})$ and quadrupolar order ${(R}_{1Q})$ ${}^{2}\mathrm{H}$ relaxation rates have been measured at 46.1 and 76.8 MHz for macroscopically oriented bilayers of 1,2-diperdeuteriomyristoyl-$\mathrm{sn}$-glycero-3-phosphocholine (DMPC-${d}_{54}),$ with perdeuterated acyl chains, in the liquid-crystalline ${(L}_{\ensuremath{\alpha}})$ state. The data have been simultaneously fitted to various dynamical models, together with frequency dependent ${}^{2}\mathrm{H}$ ${R}_{1Z}$ data for vesicles of specifically ${}^{2}\mathrm{H}$-labeled DMPC. The same mechanism for the nuclear spin relaxation in lipids has been assumed for both oriented bilayers and vesicles, except for the presence of orientational averaging and a possible contribution from vesicle tumbling in the latter case. A noncollective model describing individual molecular reorientations in the presence of a potential of mean torque is able to adequately account for the orientation dependence; however, the quality of the fits to the frequency dispersion is less satisfactory. By contrast, a three-dimensional director fluctuation model accounts for the frequency dispersion for DMPC vesicles, but does not fit the orientation dependence of the ${R}_{1Z}$ and ${R}_{1Q}$ relaxation data. Higher-order director fluctuations have also been included, which do not significantly improve the quality of the fits to the collective model. Therefore, a composite membrane model is proposed including both noncollective molecular motions and director fluctuations. The model adequately describes both the frequency and orientation dependent data along the entire acyl chain simultaneously, which suggests that both dynamical processes can be detected by analyzing the ${}^{2}\mathrm{H}$ NMR relaxation rates in the MHz range. Quantitative information about the bilayer dynamics including lipid reorientation rates, degree of molecular ordering, relative contributions from collective and noncollective motions, and director-frame spectral densities of motion has been obtained. The results suggest the bilayer dynamics in the MHz regime reflect molecular reorientations that are superimposed onto nematiclike deformations of the membrane interior in the liquid crystalline state.

Rotational dynamics of the calcium ATPase in sarcoplasmic reticulum studied by time-resolved phosphorescence anisotropy

We have investigated the microsecond rotational motions of the Ca-ATPase in rabbit skeletal sarcoplasmic reticulum (SR), by measuring the time-resolved phosphorescence anisotropy of erythrosin 5-isothiocyanate (ERITC) covalently and specifically attached to the enzyme. Over a wide range of solvent conditions and temperatures, the phosphorescence anisotropy decay was best fit by a sum of three exponentials plus a constant term. At 4 degrees C, the rotational correlation times were phi 1 = 13 +/- 3 microseconds, phi 2 = 77 +/- 11 microseconds, and phi 3 = 314 +/- 23 microseconds. Increasing the solution viscosity with glycerol caused very little effect on the correlation times, while decreasing the lipid viscosity with diethyl ether decreased the correlation times substantially, indicating that the decay corresponds to rotation of the protein within the membrane, not to vesicle tumbling. The normalized residual anisotropy (A infinity) is insensitive to viscosity and temperature changes, supporting the model of uniaxial rotation of the protein about the membrane normal. The value of A infinity (0.20 +/- .02) indicates that each of the three decay components can be analyzed as a separate rotational species, with the preexponential factor Ai equal to 1.25X the mole fraction. An empirically accurate measurement of the membrane lipid viscosity was obtained, permitting a theoretical analysis of the correlation times in terms of the sizes of the rotating species. At 4 degrees C, the dominant correlation time (phi 3) is too large for a Ca-ATPase monomer, strongly suggesting that the enzyme is primarily aggregated (oligomeric).(ABSTRACT TRUNCATED AT 250 WORDS)

High-field, high-resolution proton "magic-angle" sample-spinning nuclear magnetic resonance spectroscopic studies of gel and liquid crystalline lipid bilayers and the effects of cholesterol

We have obtained proton ('H) magic-angle sample-spinning (MASS) nuclear magnetic resonance (NMR) spectra of a variety of smectic liquid crystalline phases, including sodium decanoate (30.1 wt %)-decanol(38.9 wt %)-water, potassium oleate (72 wt %)-water, and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (lecithin)(50 wt %)-water, in addition to investigating the effects of temperature and cholesterol (CHOL) addition on the lecithin spectrum. Our results indicate that even relatively slow (-3 kHz) MASS causes averaging of the dipolar interactions causing line broadening in the static NMR spectra, at least for the non-CHOL species. All of the major proton-containing groups are well resolved, the resolution being at least as good as obtained in previous studies of oriented samples or sonicated systems. The methylene chain protons in each liquid crystalline lipid bilayer system give rise to intense, sharp, spinning sidebands (SSBs) due to the special form of the dipolar Hamiltonian. The methyl groups of the lipids, and the trimethylammonium group in lecithin, do not yield intense SSB patterns. Addition of CHOL causes attenuation of the center-band methylene peak of the lecithin, and a corresponding increase in SSB intensity. All or nearly all of the non-CHOL protons present in the samples appear to contribute to the high-resolution spectra, within our experimental error of - 10-20%. Use of a chain-deuterated lecithin allows peaks arising from the side chain of CHOL to be observed. In the gel phase of lecithin, only the trimethylammonium peak is apparent. The high-resolution afforded by MASS of the liquid crystalline phases permits rapid determination of the spin-lattice relaxation times (TI) of all resolved resonances. In addition, the observation of numerous chemically shifted peaks permits the use of two-dimensional (2-D) NMR techniques, which can give information on the spatial proximity of the various groups in the bilayer. Taken together, our results indicate a very promising future for high-field 'H MASS NMR studies of other lipid and membrane systems because of the extremely high sensitivity of the IH nucleus and the unique ability to obtain chemical shift, TI, and 2-D information from a single sample, without recourse to isotopic labeling, macroscopic sample orientation, or ultrasonic irradiation. Nuclear magnetic resonance (NMR) spectroscopy has been used to investigate the structure of model and biological mem- branes for over 20 years, and studies of molecular motion in simple lipids and hydrocarbons can be traced back even further.'S2 The earliest studies concentrated on the 'H nucleus, which because of its high sensitivity and abundance was expected to be a useful probe of membrane structure. Early studies by Cerb~jn~,~ identified mobile lipid components in Nocardia asteroides; then Chapman et al. began an extensive series of studies of model- and biological membrane systems.+l2 The early 'H NMR experiments utilized wide-line methods, and assumed that 'H-IH dipolar interactions dominated the observed line widths, since very broad lines were, in general, obtained. These line widths could be reduced by sonicating the (liquid crystalline) lipid bilayers, or membranes, reducing their particle size and permitting faster vesicle tumbling. Shortly after the first papers by Chapman et al., numerous other groups published similar studies on related sy~tems.l~-*~ Three main questions arose from the early studies. These centered around the nature of the line-broadening mechanisms in the liquid crystalline phases, the effects of sonication (does it cause line narrowing due to a change in lipid bilayer structure, or because of increased rates of particle rotation?), and whether spin diffusion occurs in the liquid crystalline phases. These questions have (at least in part) been answered over the past 15 years. Thus, Chan et a1.,26 Tidd~,~' and Oldfield et aLZ8 showed that line broadening in various liquid crystalline phases (at least up to -90 MHz) was purely dipolar in origin, since the line widths (or effective T2 values, T2*) were field independent. This view was supported by the results of 'H 'magic-angle sample-spinning (MASS) NMR,29 by multiple-pulse line narrowing,30 and by magic-angle alignment of oriented sample^.^'-^^ The effects of sonication have been widely studied, and the early view, that sonication causes line narrowing only due to increased particle tumbling, has received considerable s~pport,~~-~~ although it is

Incorporation of the cytochrome P-450 monooxygenase system into large unilamellar liposomes using octylglucoside, especially for measurements of protein diffusion in membranes

Cytochrome P-450 and NADPH cytochrome P-450 reductase were incorporated into large unilamellar lipid vesicles (200–300 nm in diameter) removing octylglucoside from mixed micelles by dialysis. The large size of the protein-containing liposomes guarantees a negligibly small vesicle tumbling. Such large vesicles are better suited for studies of protein rotation in reconstituted membranes than vesicles prepared by use of bile salts. At present the octylglucoside reconstituted monooxygenase system seems to be the most appropriate model for studying protein-protein and protein-lipid interactions in liver microsomes due to the similarity with respect to the main structural and functional properties, including size.

Cytochrome oxidase rotates in the inner membrane of intact mitochondria and submitochondrial particles.

A transient dichroism is detected after photolysis by a linearly polarized laser flash of the cytochrome oxidaseCO complex in bovine heart mitochondria, rat heart mitochondria, and bovine heart submitochondrial particles. A decay in the absorption anisotropy is characterized by a time constant of about 300 to 400 mus in both mitochondria and submitochondrial particles. Since vesicle tumbling in the time range less than 5 ms can be excluded in these experiments, we conclude that cytochrome oxidase rotates in the mitochondrial membrane with a relaxation time of several hundred microseconds. However, it is likely that only about one-half of cytochrome oxidase contributes to the observed decay, the remainder being relatively immobile.