Malgorzata Bobrowska-Hägerstrand

Possible role of phospholipid nanotubes in directed transport of membrane vesicles

Abstract We indicate that membrane nanotubes may have an important role in directed transport of membrane vesicles between different membrane-enclosed compartments in cells. We present experimental evidence that small blebs of phospholipid nanotube may travel along the nanotube and act as vehicles for transporting the enclosed solution. We have also observed similar small membrane blebs of a long membrane tube in red blood cells. In both cases the small vesicles seem to be a distended integral part of the membrane tube and not independent vesicles entrapped within the tube.

Amphiphile-induced phosphatidylserine exposure in human erythrocytes.

Nonionic and anionic water-soluble amphiphiles were shown to increase strongly the binding of fluorescein isothiocyanate-conjugated annexin V (FITC-annexin V) in human erythrocytes pretreated with the aminophospholipid translocase (APLT) inhibitor n-ethylmaleimide (NEM). At high sublytic amphiphile-concentrations the binding of FITC-annexin V, monitored in a flow cytometer, was time- and temperature-dependent and occurred heterogeneously in the cell population, with 43-81% of cells being stained above background following incubation for 60 minutes at 37 degrees C. The increased FITC-annexin V binding apparently indicates an increased flop rate of phosphatidylserine (PS) to the outer membrane leaflet. When the NEM-pretreatment was omitted, the FITC-annexin V binding was markedly, but not completely, reduced. In erythrocytes incubated with a zwitter-ionic amphiphile, a small increase in FITC-annexin V binding was detected, while cationic amphiphiles did not induce an increased FITC-annexin V binding. The potency of amphiphiles to induce PS exposure was not related to the type of shape alteration or vesiculation induced. Our results indicate a significant role of the charge status of a membrane intercalated amphiphile for its capability to induce PS exposure.

The lamprey (Lampetra fluviatilis) erythrocyte; morphology, ultrastructure, major plasma membrane proteins and phospholipids, and cytoskeletal organization.

The aim of this study was to characterize the erythrocyte of the lamprey (Lampetra fluviatilis), a primitive vertebrate. The lamprey erythrocyte predominantly has a non-axisymmetric stomatocytelike shape. It has a nucleus and a haemoglobin-filled cytosol with a few organelles and vesicular structures. Surprisingly, there is no marginal band of microtubules. Sodium dodecylsulphate polyacrylamide gel electrophoresis followed by Coomassie blue staining of isolated plasma membranes revealed a single band at the level of the human spectrin doublet. Major bands also occurred at approximately 175 kDa and comigrating with human erythrocyte actin (approximately 45 kDa). The presence of spectrin, actin and vimentin was shown by immunoblotting. Band 3 protein, the anion exchanger in higher vertebrates, seemed to be highly deficient or lacking, as was also the case with ankyrin. Confocal laser scanning microscopy combined with immunocytochemical methods showed spectrin, actin and vimentin mainly to be localized around the nucleus, from where actin- and vimentin-strands extended out into the cytoplasm. Actin also seemed to be present at the plasma membrane. Phospholipid analyses of plasma membrane preparations showed the presence of the same four major phospholipid groups as in the human erythrocyte, although with higher and lower amounts of phosphatidylcholine and sphingomyelin, respectively. The low fluorescein isothiocyanate conjugated annexin V binding, as monitored by flow cytometry, indicated that phosphatidylserine is mainly confined to the inner membrane leaflet in the lamprey erythrocyte plasma membrane.

Membrane skeleton and red blood cell vesiculation at low pH

Shapes of red blood cells at low pH were studied theoretically. It is assumed that the equilibrium shape of the red blood cell corresponds to the minimum of its membrane elastic energy which consists of the bending energy and relative stretching energy of the bilayer, the stretching energy of the skeleton and the interaction energy between the skeleton and the bilayer. It is shown that the aggregation of the skeleton at low pH can cause the red blood cell shape transformation from the stomatocytic shape to the cell shape composed of a spherical parent cell having the bilayer completely underlaid with the skeleton and spherical daughter vesicles without the skeleton.

Torocyte Membrane Endovesicles Induced by Octaethyleneglycol Dodecylether in Human Erythrocytes

Endovesicles induced in human erythrocytes by octaethyleneglycol dodecylether (C12E8) were studied by confocal laser scanning microscopy, using fluorescein isothiocyanate dextran as a nonspecific fluid marker. The endovesicles appeared to consist mainly of a ring-formed toroidal part joined with a central flat membrane segment. The torocyte contour length was several microm. There was usually one torocyte endovesicle per cell. The endovesicles seemed to be located near the cell surface. In sections of C12E8-treated erythrocytes transmission electron microscopy revealed the frequent occurrence of flat membrane structures with a bulby periphery, which apparently are cross sections of torocyte endovesicles. The possible physical mechanisms leading to the observed torocyte endovesicle shape are discussed. The torocyte endovesicles seem to be formed in a process in which an initially stomatocytic invagination loses volume while maintaining a large surface area. Because intercalation of C12E8 in the erythrocyte membrane induces inward membrane bending (stomatocytosis) we assume that C12E8 is preferentially located in the inner lipid layer of the erythrocyte membrane, i.e., in the outer lipid layer of the endovesicle membrane. It is suggested that local disturbances of the lipid molecules in the vicinity of the C12E8 molecules in the outer lipid layer of the endovesicle membrane form membrane inclusions with the effective shape of an inverted truncated cone. If the interaction between the inclusion and the membrane is weak, the membrane of such an endovesicle can be characterized by its negative spontaneous curvature, which may lead to a torocyte endovesicle shape with a small relative volume. Effects of a possible strong interaction between the C12E8-induced membrane inclusions and the membrane on the stability of the torocyte endovesicles are also indicated.

Liposomes composed of a double-chain cationic amphiphile (Vectamidine) induce their own encapsulation into human erythrocytes

Vectamidine is a liposome-forming double-chain cationic amphiphile. The present work was aimed to microscopically study the interactions of Vectamidine liposomes with the human erythrocyte plasma membrane. Vectamidine rapidly induced stomatocytic shapes. Attachment of Vectamidine liposomes to the erythrocyte induced a strong local invagination of the membrane. This frequently resulted in a complete encapsulation of the liposome. Liposomes composed of phosphatidylcholine (neutral) or phosphatidylserine/phosphatidylcholine (anionic) did not perturb the erythrocyte shape. Our results indicate that besides an attraction of Vectamidine liposomes to the plasma membrane, there is a preference of Vectamidine for the inner bilayer leaflet. We suggest that cationic amphiphiles may transfer from membrane-attached liposomes to the plasma membrane and then translocate to the inner bilayer leaflet where they induce a strong local inward bending of the plasma membrane resulting in an encapsulation of the liposome.

A possible physical mechanism of red blood cell vesiculation obtained by incubation at high pH

The membrane of human red blood cells is essentially composed of two parts, the lipid bilayer and the membrane skeleton that interacts with the lipid bilayer. The normal resting shape of the red blood cells at physiological pH 7.4 is the discocyte. However, at alkaline pH approximately equal to 11 the shape of red blood cells is composed of a spherical parent cell and large spherical daughter vesicles. The daughter vesicles may be free or connected to the parent cell by a narrow neck. In this paper we show that the shapes of red blood cells at pH approximately equal to 11 correspond to some of the calculated shapes of a closed lipid bilayer having an extreme area difference between the outer and the inner monolayer. Therefore, it is suggested that the observed shapes of the red blood cells at pH approximately equal to 11 are a consequence of the abolishment of the skeleton bilayer interactions at this pH.

Tethers connecting daughter vesicles and parent red blood cell may be formed due to ordering of anisotropic membrane constituents

Abstract Vesiculation of red blood cells at high alkaline pH was studied experimentally and theoretically. It was observed that the red blood cell daughter vesicles can be connected to the parent cell by thin tubes (tethers). It is shown theoretically that the formation of tethers may be energetically favourable due to the quadrupolar ordering of the anisotropic membrane constituents in the strong curvature field of the tethers.

Modulation of MRP1-like efflux activity in human erythrocytes caused by membrane perturbing agents.

The effect of membrane perturbing agents on the efflux (37°C, 60 min) of the fluorescent probe 2′, 7′-bis-(carboxypropyl)-5(6)-carboxyfluorescein (BCPCF) from human erythrocytes was studied. Several anionic amphiphiles (detergents) markedly inhibited BCPCF efflux (IC50≤⃒40 μM). Most zwitter-ionic amphiphiles were inefficient inhibitors. Non-ionic and cationic amphiphiles had minor effects or increased efflux. Of the aliphatic inhibitors, C12-homologues were the most efficient. Hexanol, ethanol, methyl-β-cyclodextrin (MβCD) and diamide (+ washing) did not influence BCPCF efflux. It is suggested that amphiphiles affect BCPCF efflux by modulating multi-drug resistance protein 1 (MRP1, ABCC1) activity. A negative charge of amphiphiles is essential for the inhibitory effect, while alkyl chain length modulates inhibition. MRP1-mediated BCPCF efflux appears to be relatively insensitive to non-specific plasma membrane modification.

Torocyte shapes of red blood cell daughter vesicles.

The shape of the newly described torocyte red blood cell endovesicles induced by octaethyleneglycol dodecylether (C12E8) is characterized. A possible explanation for the origin and stability of the observed torocyte endovesicles is suggested. Three partly complementary mechanisms are outlined, all originating from the interaction of C12E8 molecules with the membrane. The first is a preferential intercalation of the C12E8 molecule into the inner membrane layer, resulting in a membrane invagination which may finally close, forming an inside-out endovesicle. The second is a preference of the C12E8-induced membrane inclusions (clusters) for small local curvature which would favour torocyte endovesicle shape with large regions of small or even negative membrane mean curvatures, the C12E8 membrane inclusion being defined as a complex composed of the embedded C12E8 molecule and some adjacent phospholipid molecules which are significantly distorted due to the presence of the embedded C12E8 molecule. The preference of the C12E8 inclusions for zero or negative local curvature may also lead to the nonhomogeneous lateral distribution of the C12E8 inclusions resulting in their accumulation in the membrane of torocyte endovesicles. The third possible mechanism is orientational ordering of the C12E8-induced inclusions in the regions of torocyte endovesicles with high local membrane curvature deviator.