Bernadette Chailley

The effects of membrane lipid order and cholesterol on the internal and external cationic sites of the Na+−K+ pump in erythrocytes

cholesterol depletion alters the apparent affinity of the internal cationic sites and the maximal translocation rate but not the affinity of the external cationic sites of the Na+-K+ pump in human erythrocytes. To test whether these effects were mediated by a direct cholesterol-internal site interaction or by a change in membrane lipid order, the effects of five fluidizing amphiphiles (chlorpromazine, imipramine, benzyl alcohol, sodium oleate and sodium benzenesulphonate) on the kinetic parameters of the Na+-K+ pump were determined. The cholesterol removal and all the agents used induced dose-response decreases in membrane lipid order as measured by fluorescence polarization or ESR. Positive and neutral amphiphiles mimicked the effects of cholesterol removal on the affinity of the internal sites of the pump and to a lesser extent on the maximal translocation rate. Anionic amphiphiles had no effect on internal sites, probably because they distributed preferentially within the outer leaflet on the membrane. These results indicate that cholesterol controls the affinity of the internal sites of the Na+-K+ pump by altering the membrane lipid order. In contrast, neither cholesterol depletion nor the agents used altered the affinity of the external sites of the Na+-K+ pump. This difference in sensitivity to membrane lipids order suggests that internal and external cationic sites, although borne by the same protein, are in different lipid environments.

Development and functions of the cytoskeleton during ciliogenesis in metazoa

Summary— The different steps of ciliogenesis occurring in quail oviduct were compared to the ciliogenesis pattern described in other metazoan species.

Organization and functions of cytoskeleton in metazoan ciliated cells

Summary— Ciliated cells are characterized by a highly organized cytoskeleton which is connected with the ciliary apparatus. The organization of microtubules, microfilaments, and cytokeratin filaments is described and the relationships of each network with the ciliary apparatus are emphasized. Possible functions of such a complex cytoskeleton are discussed.

Detection of plasma membrane cholesterol by filipin during microvillogenesis and ciliogenesis in quail oviduct.

Using filipin as a probe for the presence of membrane cholesterol, the evolution of cholesterol distribution in the apical plasma membrane was studied during estrogen-induced ciliogenesis in quail oviduct and compared with the distribution of intramembrane particles (IMPs). Ciliary growth is preceded by the first step of microvillus differentiation. Microvilli emerge in membrane domains rich in IMPs and devoid of filipin-cholesterol (f-c) complexes. However growing microvillus membrane shows f-c complexes. During ciliary growth, microvilli lengthen from 0.5 to 2 microns, indicating that the microvillar membrane is not a membrane reservoir for ciliogenesis. During ciliary growth, the characteristic ciliary necklace IMP rows appear progressively at the base of cilia. The first IMP row is organized in a membrane circlet lacking of f-c complexes, whereas the new shaft membrane in the middle of the circlet exhibits numerous complexes. These two different domains of the cilia keep their specificity during ciliary growth. Only the ciliary tip shows fewer complexes than the shaft membrane. The apical membrane of differentiated ciliated cells is thus composed of various domains, the ciliary shaft full of f-c complexes and poor in IMPs, the ciliary necklace is devoid of f-c complexes and rich in IMPs, the microvilli membrane is rich in both IMPs and f-c complexes, and the interciliary membrane is poor in both f-c complexes and IMPs, whereas the undifferentiated cells exhibit an apical membrane in which f-c complexes and IMPs are distributed homogeneously.

Organization of actin microfilaments in the apical border of oviduct ciliated cells

Summary— Actin microfilaments were localized in quail oviduct ciliated cells using decoration with myosin subfragment S1 and immunogold labeling. These polarized epithelial cells show a well developed cytoskeleton due to the prosence of numerous cilia and microvilli at their apical pole. Most S1‐decorated microfilaments extend from the microvilli downward towards the upper part of the ciliary striated rootlets with which they are connected. From the microvillous roots, a few microfilaments connect the proximal part of the basal body or the basal foot associated with the basal body. Microfilament polarity is shown by S1 arrowheads pointing away from the microvillous tip to the cell body. Furthermore, short microfilaments are attached to the plasma membrane at the anchoring sites of basal bodies and run along the basal body. The polarity of these short microfilaments is directed from the basal body anchoring fibers downward to the cytoplasm. At the cell periphery, microfilaments from microvillous roots and ciliary apparatus are connected with those of the circumferential actin belt which is associated with the apical zonula adhaerens.

Changes in morphology and in polyphosphoinositide turnover of human erythrocytes after cholesterol depletion.

Human erythrocytes were cholesterol-depleted (5-25%) by incubation with phosphatidylcholine vesicles in media containing Ca2+ at different concentrations (0, 28 nM, 5 microM or 1 mM). After removal of the vesicles, the cells were reincubated with [32P]phosphate in the same media. Control (incubated in buffer alone) and cholesterol-maintained erythrocytes (incubated with cholesterol/phosphatidylcholine vesicles) were treated similarly. Cholesterol depletion induced the conversion of the cells into stomatocytes III and spherostomatocytes and decreased the turnover rate of phosphatidylinositol phosphate and of phosphatidylinositol bisphosphate. None of these effects were observed in cholesterol-maintained cells. In cholesterol-depleted cells, they occurred without changes in the ATP specific activity or in the polyphosphoinositide concentrations. Moreover, these modifications of shape and of lipid metabolism were proportional to the extent of the cholesterol depletion and were independent of the external Ca2+ concentration. In contrast, other effects of cholesterol depletion, a decrease in the turnover rate of phosphatidic acid, a decrease in diacylglycerol and in phosphatidic acid concentrations were dependent on the external Ca2+ concentration. Thus it appears that the shape change was not correlated with a change in the concentrations of these phospholipids or of diacylglycerol and therefore cannot be explained by a bilayer couple mechanism involving these phospholipids. However, the spherostomatocytic transformation was correlated with the decrease in the turnover rate of the polyphosphoinositides, but not with the turnover rate of phosphatidic acid, suggesting a role for the turnover of the polyphosphoinositides in the maintenance of the erythrocyte shape.

Immunodetection of annexins 1 and 2 in ciliated cells from quail oviduct.

Summary— Annexins I and 2 are Ca2+‐binding proteins related to the cytoskeletal proteins which have been reported to bind in a calcium‐dependent manner to F‐actin and phospholipids in vitro. Proteins immunologically related to the brain 37‐kDa annexin I and 36‐kDa annexin 2 were characterized by immunoblotting epithelial ciliated cells from quail oviduct. They were detected by immunofluorescence in ciliated as well as glandular cells, using antisera and purified antibodies directed against pig brain annexins. The pattern of labeling was found in the apical part of both cell types, with close membrane association. However, a wider distribution was observed in mature ciliated cells: annexins were localized in the well developed cytoskeletal meshwork in which the ciliary apparatus is tightly anchored. After immunogold labeling, annexins 1 and 2 were located in the same area as spectrin 240/235 and at the connection sites of F‐actin; both these cytoskeletals proteins were associated with the appendages of the basal body. In contrast, annexins were not detected in immature epithelial cells, while actin and spectrin were present. During ciliogenesis, the staining gradually appeared associated with the lateral and apical membranes. In this cellular model, the annexins may function during exocytosis in gland epithelial cells, where a close cytoskeleton‐membrane association is observed; moreover, in ciliated cells, a relationship between cytoskeletal elements of the terminal web and annexins may exist.

Structure and Assembly of the Oviduct Ciliary Membrane

For species whose fertilization is internal, the oviduct is a part of the female genital tract which is the site for a number of different steps required for reproduction, including gamete transport, fertilization, and early embryonic development.

Formation of protein polymers in erythrocyte ghosts incubated with sonicated lipid vesicles. Effects on spectrin extractibility, permeability of ghosts to vesicles, intramembrane particle distribution and bleb formation.

Abstract The incubation of human erythrocyte white ghosts with phosphatidylcholine (PC) vesicles or cholesterol/phosphatidylcholine (C/PC) vesicles under hypotonic or isotonic conditions generated membrane protein cross-links. The latter appeared in the form of a high molecular weight polymer after SDS-polyacrylamide gel electrophoresis. The polymer started to develop within a few minutes of incubation, arising largely from spectrin, and required 24 h or more for completion. It occurred regardless of cholesterol depletion undergone by the ghosts in the presence of PC vesicles. It was not reversed upon further incubation in a hypotonic, vesicle-free medium. When initial incubation was carried out under hypotonic conditions, a number of other alterations were recorded: (i) spectrin extractibility was abolished; (ii) ghosts became gradually impermeable to vesicles within a few hours, a process referred to as slow ‘resealing’ and generating an irreversible sequestration of the vesicles; (iii) intramembrane particles aggregated and blebs free of intramembrane particles pinched off inward or outward. When initial incubation was conducted under isotonic conditions, the following was observed: (i) spectrin was unextractible, as could be expected; (ii) vesicles did not enter the ghosts, a fact indicating an immediate and complete impermeabilization of ghosts to vesicles, a process referred to as fast ‘resealing’; (iii) intramembrane particle aggregation and blebs free of intramembrane particles were also present. When initial incubation was performed under isotonic conditions, but in the absence of vesicles, the polymer failed to be associated with spectrin inextractibility. These data support the view that lipid vesicles generate a high molecular weight polymer-associated, slow resealing of erythrocyte ghosts that differs, at least in part, from the polymer-free, fast resealing induced by a vesicle-free isotonic medium. Resistance to β-mercaptoethanol of the polymer makes unlikely the sole participation of disulfide bonds. Absence of added Ca 2+ in the medium is inconsistent with the transglutaminase-catalyzed formation of amide linkages. When ghosts were separated from the vesicles by a cellophane membrane upon hypotonic incubation, spectrin remained extractible and no polymer developed. Sonication of the vesicles under nitrogen and in the presence of butylated hydroxytoluene did not prevent the formation of the polymer.