Automatic cryo-EM particle selection for membrane proteins in spherical liposomes

Random spherically constrained (RSC) single particle reconstruction is a method to obtain structures of membrane proteins embedded in lipid vesicles (liposomes). As in all single-particle cryo-EM methods, structure determination is greatly aided by reliable detection of protein “particles” in micrographs. After fitting and subtraction of the membrane density from a micrograph, normalized cross-correlation (NCC) and estimates of the particle signal amplitude are used to detect particles, using as references the projections of a 3D model. At each pixel position, the NCC is computed with only those references that are allowed by the geometric constraint of the particle’s embedding in the spherical vesicle membrane. We describe an efficient algorithm for computing this position-dependent correlation, and demonstrate its application to selection of membrane-protein particles, GluA2 glutamate receptors, which present very different views from different projection directions.     

The buckling of plant roots

The mechanical stresses required to buckle root tips were measured directly for seven plant species. For two of these, both seminal and primary lateral roots were measured. For four of the plant species investigated, the easier and more rapid method used to measure the buckling stresses of excised root tips gave results which agree closely with those obtained for the growing roots of intact plants. Values of buckling stress were also calculated from previously determined elastic properties of roots. The calculated and measured values of buckling stress are of the same magnitude only, and comparison for ten root types fails to show any consistent relationship between these two methods. From the results from three plant species it has been possible to define empirical functions to account for the observed changes in root buckling stress with air gap length and water stress. Buckling stresses are not significantly affected by the concentration of nitrate in the growth medium.     

Carnivorous Utricularia: The buckling scenario

We review recent results about the functioning of aquatic carnivorous traps from the genus Utricularia. The use of high speed cameras has helped to elucidate the mechanism at the origin of the ultra fast capture process of Utricularia, at a millisecond time scale. As water is pumped out of the trap, pressure decreases inside the trap and elastic energy is stored due to the change of shape of the trap body. This energy is suddenly released when the trap is fired: the trap door undergoes an elastical instability: buckling, which allows its fast and passive opening and closure. This mechanism is used by Utricularia both to catch preys touching its trigger hairs and to fire spontaneously at regular time intervals. The results leading to this interpretation are reviewed and discussed and suggestions for further work are briefly presented.     

The transfer of antioxidants between liposomes

Abstract

A quantitative study is reported on a comparison of antioxidant action of the a-tocopherol model 2,2,5,6,7-pentamethyl-6-hydroxychroman (PMHC), 2,6-di-tert-butyl-4-methoxvphenol (DBHA) and a-tocopherol when these antioxidants are delivered to peroxidizing dilinoleoylphosphatidylcholine (DLPC) bilayers by (a) intermembrane transfer between donor dimyristoylphosphatidylcholine (DMPC) liposomes and acceptor DLPC liposomes, versus (b) the conventional coevaporation methods. The profiles of inhibited oxidation using method (a) were comparable with those of method (b) during inhibited peroxidation of DLPC liposomes, initiated by lipid-soluble azo-bis-dimethylvaleronitrile (ADVN), and quantitative determinations of the rate of chain initiation, R_i, were the same for the two methods. Differences observed, between antioxidant action by methods (a) and (b), when using the water-soluble initiator azo-bis-amidinopropane hydrochloride (ABAP), are attributed to slow diffusion of ABAP through the multilamellar DLPC system and a resultant non-uniform initiation. PMHC underwent facile intermembrane transfer through a barrier of dialysis tubing, from donor DMPC liposomes to acceptor DLPC or DMPC liposomes, based on analytical and quantitative inhibition studies. α-Tocopherol was comparatively slow to undergo intermembrane transfer by direct contact between liposomes and transferred only slightly through a barrier. The slight but measurable solubility of PMHC and DBHA in the aqueous phase supports a pathway of intermembrane transfer involving a water-soluble intermediate.     

The permeability of liposomes to nonelectrolytes

The Journal of Membrane Biology - The effect of temperature on the permeability of nonelectrolytes across liposomal membranes above and below their transition temperature has been studied by using...     

The spherical deformation model

Miller et al. (1994) describe a model for representing spatial objects with no obvious landmarks. Each object is represented by a global translation and a normal deformation of a sphere. The normal deformation is defined via the orthonormal spherical-harmonic basis. In this paper we analyse the spherical deformation model in detail and describe how it may be used to summarize the shape of star-shaped three-dimensional objects with few parameters. It is of interest to make statistical inference about the three-dimensional shape parameters from continuous observations of the surface and from a single central section of the object. We use maximum-likelihood-based inference for this purpose and demonstrate the suggested methods on real data.     

Asymmetrical stress generated by the erythrocyte lipid flippase triggers multiple bud formation on the surface of spherical giant liposomes

Proteo-giant liposomes were electroformed from a mixture of lecithin vesicles and inside-out vesicles from erythrocytes. After addition of Mg-ATP in the vicinity of the proteo-giant liposomes, small buds appeared on the liposome surfaces, which—via an increase in lipids in the outer monolayer—demonstrated the active transport of lipids from the inner to the outer monolayer, indicating flippase activity.     

The influence ofPseudomonas aeruginosa on liposomes

The interaction of liposomes loaded with Ponceau red (used as a marker) withPseudomonas aeruginosa cells was observed and it resulted in marker leakage. The marker leakage from liposomes was low in physiological fluids. The interaction was independent of secreted phospholipase C level and the serotype of the tested strain. Six of 37 examined isolates did not cause any release of the marker from the liposomes. Marker release of over 50% of total encapsulated material was observed only for ten of the strains tested.     

The size of spherical virus capsomeres

The maximum size of the capsomeres of a spherical virus is discussed in the light of an analogy between this virus problem and the mathematical problem of placing equal nonoverlapping circles on a sphere.     

The characterisation of liposomes with covalently attached proteins

The problem of characterising liposomes with covalently attached proteins has been analysed theoretically in terms of a normal weight distribution of liposome diameters. The polydispersity of protein conjugation is considered in terms of the width (standard deviation) of the liposome size distribution. It is shown that the weight-average number of proteins per liposome is a convenient parameter to use to define the protein content of proteoliposomes. Two types of proteoliposome have been prepared (small unilamellar vesicles and reverse phase evaporation vesicles) in which wheat germ agglutinin is covalently coupled to the liposomal surface. The liposomes cover a range of weight average diameter from 65 to 240 nm and of polydispersity (weight to number average diameter (dw/dn) from 2.6 to 11.4. The liposomes have been characterised by chemical analysis and photon correlation spectroscopy and the results are discussed in terms of the theoretical consequences of an equivalent normal weight distribution of diameters.     

The current-voltage relationships of liposomes and mitochondria.

Current-voltage relationships were determined for various membrane systems. We show that phospholipid and mitochondrial membranes exhibit linear relations between H+ flux and pH gradients. These membranes, however, exhibited non-linear relationships when the applied voltage was a membrane potential. The current-voltage relationship approximated to an exponential function. This relationship was found to be linearized when the membranes were treated with an electrogenic proton ionophore. The incorporation of cytochrome c oxidase (EC 1.9.3.1) was found to have no effect on the current-voltage characteristics of the phospholipid membranes. When a membrane potential of more than 140 mV was imposed across vesicular and mitochondrial membranes, they exhibited reversible di-electric breakdown. This phenomenon was correlated with the requirement of a permeant ion for the experimental demonstration of proton translocation by so-called 'proton pumps'.     

The Bax pore in liposomes, Biophysics

The protein BAX of the Bcl-2-family is felt to be one of the two Bcl-2-family proteins that directly participate in the mitochondrial cytochrome c-translocating pore. We have studied the kinetics, stoichiometry and size of the pore formed by BAX in planar lipid bilayers and synthetic liposomes. Our data indicate that a cytochrome c-competent pore can be formed by in-membrane association of BAX monomers.     

The effect of surface charges of liposomes in immunopotentiation

The purpose of this study was to establish the effect of surface charges of liposomes on its adjuvant activity to an entrapped protein antigen. The immune responses of rabbits immunized subcutaneously with lysozyme entrapped in neutral negatively and positively charged liposomes and compared with complete Freund's adjuvant (CFA), showed positively charged liposomes to be a better adjuvant than neutral, negatively charged liposomes and even CFA. This was true for solid liposomes also. Interestingly, injection of positively charged liposomes led to the formation of granulomas at the sites of immunization, which was not observed with neutral and negatively charged liposomes.     

Spray-freezing of liposomes

In freeze-etch studies it was found that liposomes of some lecithins exhibited wrinkled structures on fracture faces, when quenched from above the transition temperature. The formation of this artifact can be prevented by spray-freezing the liposome suspension.