Contribution of plasma membrane lipid domains to red blood cell (re)shaping.

Although lipid domains have been evidenced in several living cell plasma membranes, their roles remain largely unclear. We here investigated whether they could contribute to function-associated cell (re)shaping. To address this question, we used erythrocytes as cellular model since they (i) exhibit a specific biconcave shape, allowing for reversible deformation in blood circulation, which is lost by membrane vesiculation upon aging; and (ii) display at their outer plasma membrane leaflet two types of submicrometric domains differently enriched in cholesterol and sphingomyelin. We here reveal the specific association of cholesterol- and sphingomyelin-enriched domains with distinct curvature areas of the erythrocyte biconcave membrane. Upon erythrocyte deformation, cholesterol-enriched domains gathered in high curvature areas. In contrast, sphingomyelin-enriched domains increased in abundance upon calcium efflux during shape restoration. Upon erythrocyte storage at 4 °C (to mimick aging), lipid domains appeared as specific vesiculation sites. Altogether, our data indicate that lipid domains could contribute to erythrocyte function-associated (re)shaping.

Hematopoietic Stem Cells Transplantation Can Normalize Thyroid Function in a Cystinosis Mouse Model.

Hypothyroidism is the most frequent and earliest endocrine complication in cystinosis, a multisystemic lysosomal storage disease caused by defective transmembrane cystine transporter, cystinosin (CTNS gene). We recently demonstrated in Ctns(-/-) mice that altered thyroglobulin biosynthesis associated with endoplasmic reticulum stress, combined with defective lysosomal processing, caused hypothyroidism. In Ctns(-/-) kidney, hematopoietic stem cell (HSC) transplantation provides long-term functional and structural protection. Tissue repair involves transfer of cystinosin-bearing lysosomes from HSCs differentiated as F4/80 macrophages into deficient kidney tubular cells, via tunneling nanotubes that cross basement laminae. Here we evaluated the benefit of HSC transplantation for cystinotic thyroid and investigated the underlying mechanisms. HSC engraftment in Ctns(-/-) thyroid drastically decreased cystine accumulation, normalized the TSH level, and corrected the structure of a large fraction of thyrocytes. In the thyroid microenvironment, HSCs differentiated into a distinct, mixed macrophage/dendritic cell lineage expressing CD45 and major histocompatibility complex II but low CD11b and F4/80. Grafted HSCs closely apposed to follicles and produced tunneling nanotube-like extensions that crossed follicular basement laminae. HSCs themselves further squeezed into follicles, allowing extensive contact with thyrocytes, but did not transdifferentiate into Nkx2.1-expressing cells. Our observations revealed significant differences of basement lamina porosity between the thyroid and kidney and/or intrinsic macrophage invasive properties once in the thyroid microenvironment. The contrast between extensive thyrocyte protection and low HSC abundance at steady state suggests multiple sequential encounters and/or remanent impact. This is the first report demonstrating the potential of HSC transplantation to correct thyroid disease and supports a major multisystemic benefit of stem cell therapy for cystinosis.

A mouse model suggests two mechanisms for thyroid alterations in infantile cystinosis: decreased thyroglobulin synthesis due to endoplasmic reticulum stress/unfolded protein response and impaired lysosomal processing.

Thyroid hormones are released from thyroglobulin (Tg) in lysosomes, which are impaired in infantile/nephropathic cystinosis. Cystinosis is a lysosomal cystine storage disease due to defective cystine exporter, cystinosin. Cystinotic children develop subclinical and then overt hypothyroidism. Why hypothyroidism is the most frequent and earliest endocrine complication of cystinosis is unknown. We here defined early alterations in Ctns(-/-) mice thyroid and identified subcellular and molecular mechanisms. At 9 months, T4 and T3 plasma levels were normal and TSH was moderately increased (∼4-fold). By histology, hyperplasia and hypertrophy of most follicles preceded colloid exhaustion. Increased immunolabeling for thyrocyte proliferation and apoptotic shedding indicated accelerated cell turnover. Electron microscopy revealed endoplasmic reticulum (ER) dilation, apical lamellipodia indicating macropinocytic colloid uptake, and lysosomal cystine crystals. Tg accumulation in dilated ER contrasted with mRNA down-regulation. Increased expression of ER chaperones, glucose-regulated protein of 78 kDa and protein disulfide isomerase, associated with alternative X-box binding protein-1 splicing, revealed unfolded protein response (UPR) activation by ER stress. Decreased Tg mRNA and ER stress suggested reduced Tg synthesis. Coordinated increase of UPR markers, activating transcription factor-4 and C/EBP homologous protein, linked ER stress to apoptosis. Hormonogenic cathepsins were not altered, but lysosome-associated membrane protein-1 immunolabeling disclosed enlarged vesicles containing iodo-Tg and impaired lysosomal fusion. Isopycnic fractionation showed iodo-Tg accumulation in denser lysosomes, suggesting defective lysosomal processing and hormone release. In conclusion, Ctns(-/-) mice showed the following alterations: 1) compensated primary hypothyroidism and accelerated thyrocyte turnover; 2) impaired Tg production linked to ER stress/UPR response; and 3) altered endolysosomal trafficking and iodo-Tg processing. The Ctns(-/-) thyroid is useful to study disease progression and evaluate novel therapies.

Intravital multi-photon microscopy reveals several levels of heterogeneity in endocytic uptake by mouse renal proximal tubules.

Understanding renal function requires one to integrate the structural complexity of kidney nephrons and the dynamic nature of their cellular processes. Multi-photon fluorescence microscopy is a state-of-the-art imaging technique for in vivo analysis of kidney tubules structure and function in real time. This study presents visual evidence for several levels of heterogeneity of proximal tubular endocytic uptake in the superficial renal mouse cortex and illustrates the potential of multi-photon microscopy for providing a comprehensive and dynamic portrayal of renal function.

Decrease of elastic moduli of DOPC bilayers induced by a macrolide antibiotic, azithromycin.

The elastic properties of membrane bilayers are key parameters that control its deformation and can be affected by pharmacological agents. Our previous atomic force microscopy studies revealed that the macrolide antibiotic, azithromycin, leads to erosion of DPPC domains in a fluid DOPC matrix [A. Berquand, M. P. Mingeot-Leclercq, Y. F. Dufrene, Real-time imaging of drug-membrane interactions by atomic force microscopy, Biochim. Biophys. Acta 1664 (2004) 198-205.]. Since this observation could be due to an effect on DOPC cohesion, we investigated the effect of azithromycin on elastic properties of DOPC giant unilamellar vesicles (GUVs). Microcinematographic and morphometric analyses revealed that azithromycin addition enhanced lipid membranes fluctuations, leading to eventual disruption of the largest GUVs. These effects were related to change of elastic moduli of DOPC, quantified by the micropipette aspiration technique. Azithromycin decreased both the bending modulus (k(c), from 23.1+/-3.5 to 10.6+/-4.5 k(B)T) and the apparent area compressibility modulus (K(app), from 176+/-35 to 113+/-25 mN/m). These data suggested that insertion of azithromycin into the DOPC bilayer reduced the requirement level of both the energy for thermal fluctuations and the stress to stretch the bilayer. Computer modeling of azithromycin interaction with DOPC bilayer, based on minimal energy, independently predicted that azithromycin (i) inserts at the interface of phospholipid bilayers, (ii) decreases the energy of interaction between DOPC molecules, and (iii) increases the mean surface occupied by each phospholipid molecule. We conclude that azithromycin inserts into the DOPC lipid bilayer, so as to decrease its cohesion and to facilitate the merging of DPPC into the DOPC fluid matrix, as observed by atomic force microscopy. These investigations, based on three complementary approaches, provide the first biophysical evidence for the ability of an amphiphilic antibiotic to alter lipid elastic moduli. This may be an important determinant for drug: lipid interactions and cellular pharmacology.

Azithromycin, a lysosomotropic antibiotic, impairs fluid-phase pinocytosis in cultured fibroblasts.

The dicationic macrolide antibiotic azithromycin inhibits the uptake of horseradish peroxidase (HRP) by fluid-phase pinocytosis in fibroblasts in a time- and concentration-dependent fashion without affecting its decay (regurgitation and/or degradation). The azithromycin effect is additive to that of nocodazole, known to impair endocytic uptake and transport of solutes along the endocytic pathway. Cytochemistry (light and electron microscopy) shows a major reduction by azithromycin in the number of HRP-labeled endocytic vesicles at 5 min (endosomes) and 2 h (lysosomes). Within 3 h of exposure, azithromycin also causes the appearance of large and light-lucentlelectron-lucent vacuoles, most of which can be labeled by lucifer yellow when this tracer is added to culture prior to azithromycin exposure. Three days of treatment with azithromycin result in the accumulation of very large vesicles filled with pleiomorphic content, consistent with phospholipidosis. These vesicles are accessible to fluorescein-labeled bovine serum albumin (FITC-BSA) and intensively stained with filipin, indicating a mixed storage with cholesterol. The impairment of HRP pinocytosis directly correlates with the amount of azithromycin accumulated by the cells, but not with the phospholipidosis induced by the drug. The proton ionophore monensin, which completely suppresses azithromycin accumulation, also prevents inhibition of HRP uptake. Erythromycylamine, another dicationic macrolide, also inhibits HRP pinocytosis in direct correlation with its cellular accumulation and is as potent as azithromycin at equimolar cellular concentrations. We suggest that dicationic macrolides inhibit fluid-phase pinocytosis by impairing the formation of pinocytic vacuoles and endosomes.

Topical gene transfer into rat skin using electroporation.

PURPOSE: To investigate whether electroporation can be used for topical gene delivery and for DNA expression in rat keratinocytes. METHODS: The localization of a fluorescent-labelled plasmid and the expression of a reporter gene (pEGFP-N1) coding for Green Fluorescent Protein (GFP) in stripped skin were assessed by Confocal Laser Scanning Microscopy (CLSM). RESULTS: The plasmid penetrated into the epidermis within minutes after electroporation and entered the keratinocyte cytoplasm within hours. A localized expression of GFP was observed for at least 7 days in the epidermis. Skin viability was not compromised by electroporation. CONCLUSIONS: Electroporation enhances the delivery, and hence the expression, of topically applied plasmid DNA on the skin. It could be a promising alternative method to administer DNA, particularly for DNA vaccines, in the skin in vivo.

Identification of caspase-3 and caspase-activated deoxyribonuclease in rat blastocysts and their implication in the induction of chromatin degradation (but not nuclear fragmentation) by high glucose.

Previous investigations have shown that maternal diabetes impairs rodent embryo development during the earliest phase of gestation. Exposure to high concentrations of glucose before implantation results in a decrease in the number of cells per embryo and in a concomitant increase in two nuclear markers of apoptosis, chromatin degradation and nuclear fragmentation. In the present study, we show that two intracellular effectors of apoptosis, caspase-3 and caspase-activated deoxyribonuclease (CAD), are involved in the embryotoxicity of high glucose. Using reverse transcription-polymerase chain reaction and immunocytochemistry, we first demonstrated that these two effectors were expressed in rat blastocysts. The two effectors were detected in all the cells of the blastocysts and the immuno-signals were excluded from the nuclei. Rat blastocysts were incubated for 24 h in either 6 mM or 28 mM glucose in the presence or absence of specific inhibitors (DEVD-CHO [10 microM] against caspase-3 and aurin [1 microM] against CAD). After incubation, blastocysts were examined for the proportion of nuclei showing signs of chromatin degradation or nuclear fragmentation. Addition of DEVD-CHO or aurin was found to inhibit the increase in chromatin degradation induced by high glucose. None of these two inhibitors prevented the increase in nuclear fragmentation triggered by excess glucose. Our data indicate that chromatin degradation and nuclear fragmentation are two nuclear damages that are induced separately by high glucose in rat blastocysts. Chromatin degradation is apparently mediated by the activation of caspase-3 and CAD.

Expression and role of Bcl-2 in rat blastocysts exposed to high D-glucose.

Bcl-2 mRNA expression was detected in rat blastocysts by in situ hybridization. The distribution of mRNA expression was rather heterogenous, with approximately 2% of high-expressing cells. In vitro exposure to 28 mmol/l D-glucose for 24 h resulted in a significant increase in the proportion of these cells compared with control embryos in either 6 mmol/l D-glucose or 28 mmol/l D+L-glucose. Heterogeneity in the expression of Bcl-2 was also observed at the protein level by immunocytochemistry. Exposure to 28 mmol/l D-glucose significantly increased the incidence of chromatin degradation (karyolysis) and nuclear fragmentation (karyorhexis), two nuclear markers of apoptosis in rat blastocysts. When two different antisense oligodeoxynucleotides designed to block Bcl-2 expression were added to 28 mmol/1 D-glucose, the incidence of karyolysis (but not karyorhexis) was increased compared with embryos in 28 mmol/l D-glucose alone. These data suggest that Bcl-2 is involved in the protective response against the induction of karyolysis in blastocysts on their exposure to high concentrations of D-glucose in vitro, whereas karyorhexis appears to result from the activation of an intracellular pathway that is independent of Bcl-2.

Constitutive macropinocytosis in oncogene-transformed fibroblasts depends on sequential permanent activation of phosphoinositide 3-kinase and phospholipase C.

Macropinocytosis results from the closure of lamellipodia generated by membrane ruffling, thereby reflecting cortical actin dynamics. Both transformation of Rat-1 fibroblasts by v-Src or K-Ras and stable transfection for expression of dominant-positive, wild-type phosphoinositide 3-kinase (PI3K) regulatory subunit p85 alpha constitutively led to stress fiber disruption, cortical actin recruitment, extensive ruffling, and macropinosome formation, as measured by a selective acceleration of fluid-phase endocytosis. These alterations closely correlated with activation of PI3K and phosphatidylinositol-specific phospholipase C (PI-PLC), as assayed by 3-phosphoinositide synthesis in situ and in vitro and inositol 1, 4,5 trisphosphate steady-state levels, respectively; they were abolished by stable transfection of v-Src-transformed cells for dominant-negative truncated p85 alpha expression and by pharmacological inhibitors of PI3K and PI-PLC, indicating a requirement for both enzymes. Whereas PI3K activation resisted PI-PLC inhibition, PI-PLC activation was abolished by a PI3K inhibitor and dominant-negative transfection, thus placing PI-PLC downstream of PI3K. Together, these data suggest that permanent sequential activation of both PI3K and PI-PLC is necessary for the dramatic reorganization of the actin cytoskeleton in oncogene-transformed fibroblasts, resulting in constitutive ruffling and macropinocytosis.

Regulation of macropinocytosis in v-Src-transformed fibroblasts: cyclic AMP selectively promotes regurgitation of macropinosomes.

Stable transformation of Rat-1 fibroblasts by the v-Src oncoprotein results into the constitutive formation of macropinosomes. In the present report, we found that macropinosomes do not fuse with transferrin-containing endosomes and investigated the effects of cyclic AMP as a regulator of macropinocytosis in this cell system. The permeant analogs dibutyryl cyclic AMP and 8-bromo-cyclic AMP, as well as the pharmacological activator of adenylate cyclase forskolin, similarly decreased by about 35% the net endocytic accumulation of the fluid-phase tracer horseradish peroxidase at intervals >5 minutes in v-Src-transformed cells but not in the non-transformed parental Rat-1 cell line. However, and in contrast to the phospholipase C inhibitor 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate or the phosphatidylinositol 3-kinase inhibitor wortmannin, dibutyryl cyclic AMP neither returned the peroxidase accumulation rate of v-Src-transformed cells to that of parental Rat-1/control cells, nor prevented macropinosome formation, as shown by confocal microscopy. Detailed analysis of the kinetics of tracer entry and efflux in transformed cells revealed that dibutyryl cyclic AMP inhibited peroxidase accumulation only after intervals >5 minutes, due to accelerated peroxidase regurgitation, but did not alter the rate of transferrin recycling. Taken together, these data indicate that, in v-Src-transformed fibroblasts, macropinocytosis and micropinocytosis serve different pathways and that cyclic AMP affects neither micropinocytosis nor the formation of macropinosomes, but selectively promotes regurgitation therefrom.

Yersinia enterocolitica induces apoptosis in macrophages by a process requiring functional type III secretion and translocation mechanisms and involving YopP, presumably acting as an effector protein.

Yersiniae, causative agents of plague and gastrointestinal diseases, secrete and translocate Yop effector proteins into the cytosol of macrophages, leading to disruption of host defense mechanisms. It is shown in this report that Yersinia enterocolitica induces apoptosis in macrophages and that this effect depends on YopP. Functional secretion and translocation mechanisms are required for YopP to act, strongly suggesting that this protein exerts its effect intracellularly, after translocation into the macrophages. YopP shows a high level of sequence similarity with AvrRxv, an avirulence protein from Xanthomonas campestris, a plant pathogen that induces programmed cell death in plant cells. This indicates possible similarities between the strategies used by pathogenic bacteria to elicit programmed cell death in both plant and animal hosts.

Quantitative analysis of clustering on biological membranes: methodology and application to ligand-induced asialoglycoprotein receptor redistribution on rat hepatocytes.

Ligand-induced receptor clustering is the first step in receptor mediated endocytosis of asialoglycoproteins by rat hepatocytes. This well-characterized receptor was used as a model system to set up a general method for the quantitative analysis and the visualization of molecular clustering on surface replicas, using a two-step approach. In the first step, aiming at the quantitation of clustering, gold-labeled asialoglycoprotein receptors on the cell surface were assumed to reflect two populations, one of clustered and one of not-clustered receptors. The experimental distribution of nearest neighbor distances of labeled receptors was adjusted by least square fitting to the sum of two functions, each corresponding to the theoretical nearest neighbor distance distribution of randomly distributed points, corresponding to non-clustered and clustered particle concentrations, respectively. The resolution of the experimental nearest neighbor distribution into these two components yielded an objective estimate of the number of labeled receptors in clusters and of the total surface of clusters. The second step, aiming at the visualization of clusters, rested on the fact that the distance of a point to its neighbors is shorter in a cluster than outside the cluster. Accordingly, each particle was ordered according to the sum of the distances to its nearest neighbors. Clustered particles with the lowest cumulative distances were extracted, in proportion of the extent of clustering determined in the first step, and displayed on the computer screen. This translated the two components into the two corresponding populations of identified particles. This method demonstrated that, if rat hepatocytes were incubated at 4 degrees C with either asialofetuin-gold complexes before fixation, or with asialofetuin prior to fixation and immunogold labeling of the ASGP-R, up to 65% of the receptors became clustered on the dorsal cell surface in areas where receptors could be concentrated up to 20-fold, as compared with randomly distributed molecules. In ultrathin sections, clusters essentially corresponded to clathrin-coated pits. In principle, this method is generic and can be applied to other biological systems, especially when statistical analysis is needed for studying the dynamics of the clustering process.

Structure and dynamics of the fenestrae-associated cytoskeleton of rat liver sinusoidal endothelial cells.

This article describes the cytoskeleton associated with fenestrae and sieve plates of rat liver sinusoidal endothelial cells. Fenestrae control the exchange between the blood and parenchymal cells. We present evidence indicating that several agents that change the fenestrae and sieve plates also cause changes in the cytoskeleton. Cultured liver endothelial cells (LECs) were slightly fixed and treated with cytoskeleton extraction buffer. Detergent-extracted whole mounts of cultured cells were prepared for either scanning electron microscopy (SEM) or transmission electron microscopy (TEM). Extracted cells show an integral intricate cytoskeleton; sieve plates and fenestrae are delineated by cytoskeleton elements. Fenestrae are surrounded by a filamentous, fenestrae-associated cytoskeleton with a mean filament thickness of 16 nm. Sieve plates are surrounded and delineated by microtubuli, which form a network together with additional branching cytoskeletal elements. The addition of ethanol to cultured cells enlarged the diameter for these fenestrae-associated cytoskeleton rings by 5%, whereas serotonin treatment reduced the diameter by 20%. These observations indicate that the fenestrae-associated cytoskeleton probably changes the size of fenestrae after different treatments. After treatment with cytochalasin B the number of fenestrae increased. However, cytochalasin B did not change the structure of the fenestrae-associated cytoskeleton ring, but disperses the microtubuli. In conclusion, LECs have a cytoskeleton that defines and supports sieve plates and fenestrae. Fenestrae-associated cytoskeleton is a dynamic structure and plays a role in maintaining and regulating the size of fenestrae after different treatments. Therefore, the fenestrae-associated cytoskeleton controls the important hepatic function of endothelial filtration.

Ligand-induced clustering of asialoglycoprotein receptors on rat hepatocytes at 4 degrees C.

The platinum-carbon replica technique was applied to rat hepatocyte monolayers to visualize the distribution of the receptor responsible for the clearance of asialoglycoproteins. In a first series of experiments, hepatocytes were mildly fixed, either immediately after transfer to 4 degrees C, or after up to 24 h of incubation at 4 degrees C in the presence or absence of soluble asialofetuin (ASF). The asialoglycoprotein receptors were then immunolabeled by a rabbit antiserum followed by protein A-gold complexes. Average labeling was 41 +/- 2 particles x microns -2 (mean +/- SEM, n = 50), corresponding to an efficiency of about 25% as compared to radioligand binding data. Nearest neighbor analysis of gold particles showed an almost random distribution on cells incubated without asialofetuin, but significant clustering after exposure to the ligand. In a second series of experiments, hepatocytes were incubated at 4 degrees C for 2 or 24 h with asialofetuin adsorbed onto 15-nm gold particles (2 x 10(12) particles x ml-1, 32 molecules x particle-1), then fixed. On average, 20 +/- 2 (n = 14) and 38 +/- 6 (n = 19) particles x microns -2 were observed after 2 and 24 h, respectively. By comparison to control preparations, clustering of particles was found at both time intervals, although no significant difference could be detected between 2 and 24 h using the Kolmogorov-Smirnov test when the abundance of particles was taken into consideration. Summarizing, (1) in absence of ligand, the asialoglycoprotein receptor is almost randomly distributed on rat hepatocytes, (2) both soluble ASF and ASF-gold complexes induce receptor clustering, (3) even at 4 degrees C, the plasma membrane remains sufficiently fluid for receptor clustering.