Tadeusz Gulik-Krzywicki

Inverse micellar phases of phospholipids and glycolipids. Invited Lecture

We here review the current knowledge of lyotropic n liquid-crystalline phases having structures based upon n periodic three-dimensional packings of discrete inverse n micellar aggregates. These have been shown during the last decade to n be rather common in lipid systems, particularly in lipid mixtures. n The most frequently observed such structure is a cubic phase of n crystallographic spacegroup Fd3m. We have n previously determined the structure of this inverse micellar cubic n phase, both by X-ray diffraction (V. Luzzati, R. Vargas, A. n Gulik, P. Mariani, J. M. Seddon and E. Rivas, Biochemistry, n 1992, 31, 279) and by freeze fracture electron microscopy n (FFEM) (H. Delacroix, T. Gulik-Krzywicki and J. M. Seddon, J. n Mol. Biol., 1996, 258, 88), and have n determined the effect of pressure on its stability (P. M. Duesing, J. n M. Seddon, R. H. Templer and D. A. Mannock, Langmuir, 1997, n13, 265). We have measured the limiting hydration of the nFd3m phase in a number of lipid systems. We have n found that electrical conductivity measurements can offer a simple n way of distinguishing bicontinuous from discontinuous (inverse n micellar) phases. We have also discovered a new n optically-isotropic phase in a few lipid systems, at lower n hydrations than the Fd3m cubic phase. The n X-ray diffraction pattern does not appear to index as cubic, n and we assume that this phase consists of a non-cubic complex n 3-D packing of inverse micelles. It gives rise to three n different fracture planes by FFEM, one of which is identical to the n [111] fracture plane of the Fd3m cubic n phase, with two different sub-domains with alternating aspects n along the [111] direction being present. This implies that n the two inverse micellar phases may be related by a restacking n transition of planes normal to the cubic [111] direction, n analogous to the fcc–hcp restacking transition of hard spheres.

Polymorphic phase behavior of perfluoroalkylated phosphatidylcholines

The polymorphic phase behavior of the F-alkyl modified phosphatidylcholines FnCmPC with Fn = CnF2n + 1 and Cm = -(CH2)m- and the physicochemical properties of their aqueous dispersions have been investigated. We show that the supramolecular assemblies formed by F4C4PC, F6C4PC, F8C4PC and F4C10PC dispersed in water consist of liposomes. F6C10PC forms, as does F8C10PC, a ribbon-like phase (two-dimensional centered rectangular lattice) at 25 degrees C, but on heating, it forms a lamellar phase. Upon cooling, the lamellar gel phase is metastable and converts slowly back into the ribbon-like phase. Analyses of the dispersions before and after heat sterilization and upon storage at 25 degrees C reveal an exceptional stability of the FnCmPC-based liposomes which contrasts strongly with that of DPPC vesicles. This enhanced stability most likely arises from the increased hydrophobic character resulting from the presence of the perfluoroalkyl tails. The gel to fluid phase transition temperature of the FnCmPCs is found to be related to the total length of the hydrophobic chain and more markedly to the length of the perfluoroalkyl tail. This phase transition is first induced by the melting of the fluorocarbon chain. Each portion of the Fn tail and of the hydrocarbon spacer experiences intrinsic changes of molecular motion with temperature. The partitioning of a lipophilic/hydrophilic paramagnetic probe between the aqueous and lipidic phases present in the FnCmPC dispersions shows that an increase in fluorophilic character results in a lower solubility of the probe in the membrane, thus reflecting a dramatic decrease of the membranes lipophilicity.

Redistribution of intramembrane particles related to acetylcholine release by cholinergic synaptosomes

Acetylcholine release was measured on suspensions of pure cholinergic synaptosomes, isolated from torpedo electric organ. Transmitter release was triggered by two different methods: KCl depolarization, or action of a venom extracted from a polychaete annelid Glycera convoluta . This venom was known to increase considerably the miniature endplate potential frequency at neuromuscular junctions. Ultrarapid freezing of synaptosomes in suspension in the absence of fixation, followed by freeze fracture, permitted us to show: (1) That the venom does not trigger the appearance of endo-exocytotic pits in the presynaptic membrane, in contrast to KCl depolarization. (2) That both KCl depolarization and venom action lead to a decrease in the number of small P-face intramembrane particles and to an increase in the number of medium-sized E-face particles. In addition, the venom increased the number of medium-sized P-face particles. The redistribution of the intramembrane particles is discussed in relation to the release of transmitter which has been measured in parallel.

Structure of serum low-density lipoprotein: II. A freeze-etching electron microscopy study

Normal human and diet-induced hyperlipidemic Rhesus monkey serum low-density lipoprotein structure was investigated by freeze-etching electron microscopy employing a novel rapid freezing technique. Using turnip yellow mosaic virus as a standard, the technique was shown to be capable of providing information regarding the general architecture of particles in solution. Human and monkey serum low-density lipoprotein particle morphology appeared to deviate markedly from that of a perfect sphere. Instead, the outer layer of the particles appeared to consist of a small number of globules. The number and dimensions of these globules as well as their arrangement are in remarkable agreement with the tetrahedral model proposed by Luzzati et al. (1979) in the preceding paper for the low temperature form of the Rhesus monkey low-density lipoprotein.

[27] Studies of lipoproteins by freeze-fracture and etching electron microscopy

Publisher Summary Freeze-drying, freeze-fracturing and etching, and low-temperature electron microscopy of frozen hydrated specimens are the methods used for studying cryofixed material. This chapter describes a simple and inexpensive procedure for the study of plasma lipoproteins using a Balzers freeze-etching unit. The technique consists of four essential steps: cryofixation of the sample solution, either in the presence or absence of antifreeze agents, fracture of the cryofixed sample solution followed by etching of the fractured surface, and finally heavy metal replication of the fractured and etched surface. Freeze-fracture electron microscopy has become the preeminent technique for the structural study of cell membranes and lipid phases following subsequent improvements, and has been applied to the study of biological macromolecules in solution. Although the freeze-fracture/etching technique may provide detailed information on lipoprotein morphology, the chemical nature of the various components observed on the replica cannot be determined by the freeze-fracture/etching technique alone. Preliminary studies suggest that this technique may be very useful for the study of serum lipoproteins.

Ultrastructural changes and transmitter release induced by depolarization of cholinergic synaptosomes: A freeze-fracture study of a synaptosomal fraction from torpedo electric organ

A Ca-dependent transmitter release was triggered by K+ depolarization in pure cholinergic synaptosomes isolated from Torpedo electric organ. The synaptosomes were freeze fractured after ultrarapid freezing in the absence of any fixation. A quantitative analysis of freeze-fracture micrographs shows a Ca-dependent increase of the number of pits in the presynaptic membrane upon K+ depolarization. We were able to distinguish two populations of pits after K+ depolarization with or without 4-aminopyridine. This drug reduced the number of the large (20 to 150 nm) pits and increased the number of the small (10 nm) pits while ACh release was unaffected or enhanced. The large pits might represent endoexocytotic events. The small pits, found on the P face without symmetric holes on the E face, could represent either “synaptopores” related to synaptic vesicles or holes left in the P face by specialized particles detached with the E face. These micropits might well be the site of ACh release.

Freeze-fracture study of cardiotoxin action on axonal membrane and axonal membrane lipid vesicles

Freeze-fracture electron microscopy was used to follow morphological changes induced by Naja mossambica mossambica venom cardiotoxins on crab axonal membranes and thier lipids. It was shown that the extent of morphological changes depended drastically on the purity of cardiotoxin preparations and on their nature. Highly purified cardiotoxin induced mainly fusion of membrane or lipid vesicles. The extent of fusion and other morphological changes depended on the nature of cardiotoxin used: VII4 cardiotoxin induced only fusion while VII1 led to further modifications of membranes and liposomes. The most spectacular morphological changes were observed with axonal membranes treated with cardiotoxin containing traces of venom phospholipase A2. At low cardiotoxin concentration (10(-7)-10(-5) M) important intramembrane particle aggregation was observed and at higher concentrations (more than 10(-4) M) intramembrane particles disappeared from the membrane and were found in solution. The membrane vesicles, devoid of intramembrane particles, were observed to fuse rapidly into liposome-like aggregates. These morphological changes are interpreted as being due to the removal of intrinsic membrane proteins from the membrane by the combined action of cardiotoxin and phospholipase A2.

Calcium‐induced acetylcholine release and intramembrane particle occurrence in proteoliposomes equipped with mediatophore

Summary— Proteoliposomes obtained from the mediatophore, a purified Torpedo electric organ nerve terminals protein, and endogenous lipids were used for a study of calcium‐induced release of acetylcholine and freeze‐fracture electron microscopy. Large intramembrane particles were induced by the influx of calcium into proteoliposomes, as previously observed for synaptosomes or stimulated electric organ nerve terminals. The involvement of mediatophore in a calcium dependent acetylcholine translocation seems therefore to be related to the occurrence of a category of intramembrane particles in the course of the release process.

The egg-shell of Drosophila melanogaster VI, structural analysis of the wax layer in laid eggs

Utilizing freeze-fracturing conventional electron microscopy and scanning electron microscopy methods, a wax layer was identified, sealing the oocyte of Drosophila melanogaster. In mature egg-shells wax forms a hydrophobic layer surrounding the oocyte and lying between, and in very close contact with the vitelline membrane (interiorly) and the crystalline intermediate chorionic layer (exteriorly). In cross-fractured views it is less than 50 A thick whereas in longitudinal fracturing it reveals smooth fracture faces of a multilayered material in the form of hydrophobic areas or plaques (0.5-1 microns in diameter) which are partially overlapping and highly compressed between the vitelline membrane and the innermost chorionic layer. The evidence for this layer being a wax are the facts that a) it is not preserved in conventional fat-extracting electron microscopy methods, b) it directs laterally the fracture planes during freeze-fracturing and reveals smooth fracture faces. Analysis of the structural features of wax in mature egg-shell in various species of Drosophilidae have shown that the wax layer exhibits indistinguishable (among the species) hydrophobic plaques, which have the same size and thickness with Drosophila melanogaster. These data provide structural evidence explaining the physiological resistance of the insect eggs studied, against water loss or water uptake, whenever they are laid on substrates with extreme environmental conditions. In addition, the data demonstrate how an extracellular substance can be organized to perform that function.

The eggshell of Drosophila melanogaster. VIII. Morphogenesis of the wax layer during oogenesis

Utilizing freeze-fracturing and conventional electron microscopy methods, we have studied the details of morphogenesis and construction of the wax layer envelope from Oregon R and mutants of Drosophila melanogaster eggs during oogenesis. The wax layer is synthesized and secreted by the follicular cells in the form of lipid vesicles during stage 10b. During secretion (stages 10b, 11 and 12) the lipid vesicles are accumulated on the vitelline membrane surface and become flat. At the late stages of choriogenesis (stages 13, 14) the lipid vesicles are compressed tightly between the vitelline membrane and the other already constructed eggshell layers, so the wax layer becomes very thin and is hardly seen in cross-fractured views.