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Amphiphilic aminoglycoside derivatives are potential new antimicrobial agents mostly developed to fight resistant bacteria. The mechanism of action of the 3',6-dinonyl neamine, one of the most promising derivative, has been investigated on Gram-negative bacteria, including P. aeruginosa. In this study, we have assessed its mechanism of action against Gram-positive bacteria, S. aureus and B. subtilis. By conducting time killing experiments, we assessed the bactericidal effect induced by 3',6-dinonyl neamine on S. aureus MSSA and MRSA. By measuring the displacement of BODIPY™-TR cadaverine bound to lipoteichoic acids (LTA), we showed that 3',6-dinonyl neamine interacts with these bacterial surface components. We also highlighted the ability of 3',6-dinonyl neamine to enhance membrane depolarization and induce membrane permeability, by using fluorescent probes, DiSC3C(5) and propidium iodide, respectively. These effects are observed for both MSSA and MRSA S. aureus as well as for B. subtilis. By electronic microscopy, we imaged the disruption of membrane integrity of the bacterial cell wall and by fluorescence microscopy, we demonstrated changes in the localization of lipids from the enriched-septum region and the impairment of the formation of septum. At a glance, we demonstrated that 3',6-dinonyl neamine interferes with multiple targets suggesting a low ability of bacteria to acquire resistance to this agent. In turn, the amphiphilic neamine derivatives are promising candidates for development as novel multitarget therapeutic antibiotics.

We recently reported that trace insertion of exogenous fluorescent (green BODIPY) analogs of sphingomyelin into living red blood cells partially spread onto coverslips labels submicrometric domains, visible by confocal microscopy. We here extend this feature to endogenous sphingomyelin, upon binding of a sphingomyelin-specific non-toxic fragment of the earthworm toxin, lysenin, fused to the red monomeric fluorescent protein, mCherry (construct named lysenin*). Specificity of lysenin* binding was verified with composition-defined liposomes and by loss of 125I-lysenin* binding to erythrocytes upon sphingomyelin depletion by sphingomyelinase. 125I-lysenin* binding isotherm indicated saturation at 3.5x10^6molecules/red blood cell, i.e. ~3% of sphingomyelin coverage. Non-saturating lysenin* concentration also labeled submicrometric domains on the plasma membrane of partially spread erythrocytes, co-localizing with inserted green BODIPY-sphingomyelin, and abrogated by sphingomyelinase. Lysenin*-labeled domains were stable in time and space and were regulated by temperature and cholesterol. The abundance, size, positioning and segregation of lysenin*-labeled domains from other lipids (BODIPY-phosphatidylcholine or -glycosphingolipids) depended on membrane tension. Similar lysenin*-labeled domains were evidenced in red blood cells gently suspended in three-dimensional gel. Taken together, these data demonstrate submicrometric compartmentation of endogenous sphingomyelin at the membrane of a living cell in vitro, and suggest it may be a genuine feature of erythrocytes in vivo.

The thyroid is a highly vascularized endocrine gland, displaying a characteristic epithelial organization in closed spheres, called follicles. Here we investigate how endothelial cells are recruited into the developing thyroid and if they control glandular organization as well as thyrocytes and C-cells differentiation. We show that endothelial cells closely surround, and then invade the expanding thyroid epithelial cell mass to become closely associated with nascent polarized follicles. This close and sustained endothelial:epithelial interaction depends on epithelial production of the angiogenic factor, Vascular Endothelial Growth Factor-A (VEGF-A), as its thyroid-specific genetic inactivation reduced the endothelial cell pool of the thyroid by >90%. Vegfa KO also displayed decreased C-cells differentiation and impaired organization of the epithelial cell mass into follicles. We developed an ex vivo model of thyroid explants that faithfully mimicks bilobation of the thyroid anlagen, endothelial and C-cells invasion, folliculogenesis and differentiation. Treatment of thyroid explants at e12.5 with a VEGFR2 inhibitor ablated the endothelial pool and reproduced ex vivo folliculogenesis defects observed in conditional Vegfa KO. In the absence of any blood supply, rescue by embryonic endothelial progenitor cells restored folliculogenesis, accelerated lumen expansion and stimulated calcitonin expression by C-cells. In conclusion, our data demonstrate that, in developing mouse thyroid, epithelial production of VEGF-A is necessary for endothelial cells recruitment and expansion. In turn, endothelial cells control epithelial reorganization in follicles and C-cells differentiation.

Infection of Spodoptera frugiperda (Sf9) cells by baculovirus (BV) is well established for transgene expression of soluble proteins, but few correctly folded transmembrane proteins have been so produced. We here report the use of the BV/Sf9 (BVES) method for the expression and transfer, via microvesicles, of the exclusive lysosomal exporters for cystine and sialic acid, human cystinosin and sialin. These proteins and their mRNA are released into the culture medium as very low-density microvesicles (~1.05 g/ml), which do not label for lysobisphosphatidic acid. The presence of the human transgene proteins in the vesicles was confirmed by western blotting and confirmed and quantified by mass spectrometry. Addition of vesicles to cultures of human fibroblast lines deficient in either cystinosin or sialin produced a progressive depletion of stored lysosomal cystine or sialic acid, respectively. The depletion effect was slow (T1/2 ~48 h), saturable (down to ~40% of initial after 4 days) and stable (one week). Surprisingly, BV infection of Spodoptera appeared to induce expression and release into microvesicles of the insect orthologue of cystinosin, but not of sialin. We conclude that BVES is an effective method to express and transfer functional transmembrane proteins so as to study their properties in mammalian cells, and has a generic potential for transport protein replacement therapy.

Micrometric membrane lipid segregation is controversial. We addressed this issue in attached erythrocytes and found that fluorescent boron dipyrromethene (BODIPY) analogs of glycosphingolipids (GSLs) [glucosylceramide (BODIPY-GlcCer) and monosialotetrahexosylganglioside (GM1BODIPY)], sphingomyelin (BODIPY-SM), and phosphatidylcholine (BODIPY-PC inserted into the plasma membrane spontaneously gathered into distinct submicrometric domains. GM1BODIPY domains colocalized with endogenous GM1 labeled by cholera toxin. All BODIPY-lipid domains disappeared upon erythrocyte stretching, indicating control by membrane tension. Minor cholesterol depletion suppressed BODIPY-SM and BODIPY-PC but preserved BODIPY-GlcCer domains. Each type of domain exchanged constituents but assumed fixed positions, suggesting self-clustering and anchorage to spectrin. Domains showed differential association with 4.1R versus ankyrin complexes upon antibody patching. BODIPY-lipid domains also responded differentially to uncoupling at 4.1R complexes [protein kinase C (PKC) activation] and ankyrin complexes (in spherocytosis, a membrane fragility disease). These data point to micrometric compartmentation of polar BODIPY-lipids modulated by membrane tension, cholesterol, and differential association to the two nonredundant membrane:spectrin anchorage complexes. Micrometric compartmentation might play a role in erythrocyte membrane deformability and fragility.

Micrometric lipid compartmentation at the plasma membrane is disputed. Using live confocal imaging, we found that three unrelated fluorescent sphingomyelin (SM) analogs spontaneously clustered at the outer leaflet into micrometric domains, contrasting with homogeneous labelling by DiIC18 and TMA-DPH. In erythrocytes, these domains were round, randomly distributed, and reversibly coalesced under hypotonicity. BODIPY-SM and -glucosylceramide showed distinct temperature-dependence, in the same ranking as Tm for corresponding natural lipids, indicating phase behaviour. Scanning electron microscopy excluded micrometric surface structural features. In CHO cells, similar surface micrometric patches were produced by either direct BODIPY-SM insertion or intracellular processing from BODIPY-ceramide, ruling out aggregation artefacts. BODIPY-SM surface micrometric patches were refractory to endocytosis block or actin depolymerization and clustered upon cholesterol deprivation, indicating self-clustering at the plasma membrane. BODIPY-SM excimers further suggested clustering in ordered domains. Segregation of BODIPY-SM and -lactosylceramide micrometric domains showed coexistence of distinct phases. Consistent with micrometric domain boundaries, fluorescence recovery after photobleaching (FRAP) revealed restriction of BODIPY-SM lateral diffusion over long-range, but not short-range, contrasting with comparable high mobile fraction of BODIPY-lactosylceramide in both ranges. Controlled perturbations of endogenous SM pool similarly affected BODIPY-SM domain size by confocal imaging and its mobile fraction by FRAP. The latter evidence supports the hypothesis that, as shown for BODIPY-SM, endogenous SM spontaneously clusters at the plasmalemma outer leaflet of living cells into ordered micrometric domains, defined in shape by liquid-phase coexistence and in size by membrane tension and cholesterol. This proposal remains speculative and calls for further investigations.

SummaryFor several days after antigenic stimulation, human cytolytic T lymphocyte (CTL) clones exhibit a decrease in their effector activity and in their binding to human leukocyte antigen (HLA)-peptide tetramers. We observed that, when in this state, CTLs lose the colocalization of the T cell receptor (TCR) and CD8. Effector function and TCR-CD8 colocalization were restored with galectin disaccharide ligands, suggesting that the binding of TCR to galectin plays a role in the distancing of TCR from CD8. These findings appear to be applicable in vivo, as TCR was observed to be distant from CD8 on human tumor-infiltrating lymphocytes, which were anergic. These lymphocytes recovered effector functions and TCR-CD8 colocalization after ex vivo treatment with galectin disaccharide ligands. The separation of TCR and CD8 molecules could be one major mechanism of anergy in tumors and other chronic stimulation conditions.

Gentamicin accumulates in lysosomes and induces apoptosis in kidney proximal tubules and renal cell lines. Using LLC-PK1 cells, we have examined the concentration- and time-dependency of the effects exerted by gentamicin (1-3 mM; 0-3 days) on (i) lysosomal stability; (ii) activation of mitochondrial pathway; (iii) occurrence of apoptosis (concentrations larger than 3 mM caused extensive necrosis as assessed by the measurement of lactate dehydrogenase release). Within 2 h, gentamicin induced a partial relocalization [from lysosomes to cytosol] of the weak organic base acridine orange. We thereafter observed (a) a loss of mitochondrial membrane potential (as from 10 h, based on spectrophotometric and confocal microscopy using JC1 probe) and (b) the release of cytochrome c from granules to cytosol, and the activation of caspase-9 (as from 12 h; evidenced by Western blot analysis). Increase in caspase-3 activity (assayed with Ac-DEVD-AFC in the presence of z-VAD-fmk]) and appearance of fragmented nuclei (DAPI staining) was then detected as from 16 to 24 h together with nuclear fragmentation. Gentamicin produces a fast (within 4 h) release of calcein from negatively-charged liposomes at pH 5.4, which was slowed down by raising the pH to 7.4, or when phosphatidylinositol was replaced by cardiolipin (to mimic the inner mitochondrial membrane). The present data provide temporal evidence that gentamicin causes apoptosis in LLC-PK1 with successive alteration of the permeability of lysosomes, triggering of the mitochondrial pathway, and activation of caspase-3.

Endocytosis of the somatostatin analogue, octreotide, by the proximal tubule-derived opossum kidney (OK) cell line.BackgroundNephrotoxicity of cancer therapy using radiolabeled somatostatin analogues such as octreotide is due to ultrafiltration and reuptake by proximal tubular cells (PTCs). The mechanism of uptake is unknown. It could occur either by receptor-mediated endocytosis via a somatostatin receptor or, alternatively, the multiligand megalin/cubilin tandem receptor, or by fluid-phase endocytosis. To define the mechanisms of internalization and to identify potential receptors, we have studied the uptake and processing of octreotide by the PTC-derived opossum kidney (OK) cell line.MethodsWe compared the kinetics of uptake and fate of 111In-diethylenetriamine pentaacetic acid (DTPA)-D-Phe1-octreotide and 125I-human serum albumin (125I-HSA). To determine the contribution of receptor-mediated endocytosis, we tested competition for uptake by octreotide and somatostatin and by various megalin/cubilin ligands [receptor-associated protein (RAP), albumin, transferrin, insulin, polymixin B] or basic amino acids. The subcellular localization of fluorescein isothiocyanate (FITC)-D-Phe1-octreotide was studied by confocal microscopy.ResultsKinetics of uptake of 111In-DTPA-D-Phe1-octreotide and 125I-HSA by OK cells were comparable, but only the somatostatin analogue was significantly retained intact. All megalin/cubilin ligands and basic amino acids strongly inhibited 125I-HSA uptake, but these could not compete for >50% of 111In-DTPA-D-Phe1-octreotide uptake. The same was found for somatostatin and octreotide. The noncompetable uptake of 111In-DTPA-D-Phe1-octreotide was comparable to the clearance of Lucifer Yellow, a marker of fluid-phase endocytosis. By confocal microscopy, FITC-D-Phe1-octreotide colocalized with transferrin in endosomes, then accumulated in lysosomes.ConclusionReceptor-mediated endocytosis via megalin/cubilin and fluid-phase endocytosis contribute about equally to the uptake of radiolabeled somatostatin analogues by OK cells.

Altered polarity and expression of H + -ATPase without ultrastructural changes in kidneys of Dent's disease patients. Background Dent's disease is a proximal tubule (PT) disorder characterized by low-molecular-weight proteinuria (LWMP) that may be associated with hypercalciuria, nephrocalcinosis, and renal failure. It is caused by inactivating mutations of the renal chloride channel ClC-5, which colocalizes with the vacuolar H + -ATPase in PT cells and α-type intercalated cells. Examinations of knockout mice have established the role of ClC-5 in PT endocytosis, but the consequences of ClC-5 mutations on the polarity of H + -ATPase and other plasma membrane proteins remain unknown. Methods We have studied renal biopsies from eight patients with Dent's disease, due to inactivating ClC-5 mutations, by light and electron microscopy, and by immunohistochemical staining. All patients exhibited LMWP, and renal function ranged from normal to end-stage renal failure. Results Light microscopy revealed either normal renal architecture or glomerulosclerosis, tubular dedifferentiation and atrophy, and mild interstitial fibrosis. Focal, hyaline casts, sometimes calcified, were identified at all stages. Electron microscopy did not reveal any ultrastructural abnormalities in PT cells, and the endocytic apparatus was apparently normal. However, immunohistochemical studies demonstrated a consistent inversion of H + -ATPase polarity in PT cells to a basolateral distribution contrasting with its apical location in the normal kidney. This inversion of polarity was specific for H + -ATPase and did not affect distribution of aminopeptidase, megalin, and Na + /K + -ATPase. Furthermore, apical H + -ATPase expression was absent in α-type intercalated cells. Conclusion ClC-5 mutations are associated with modifications in the polarity and expression of H + -ATPase, but not ultrastructural alterations in PT cells. These findings help further understanding of the role of ClC-5 and the pathophysiology of Dent's disease.

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.

Cystinosis is a multi-systemic lysosomal storage disease caused by defective transmembrane cystine transporter, cystinosin (CTNS gene). In the mouse model of cystinosis, Ctns−/− mice, it has already been shown that hematopoietic stem and progenitor cell (HSC) transplantation provides long-term protection of kidneys and eyes, which are affected early on in cystinosis. Tissue repair involves transfer of cystinosin-bearing lysosomes from HSPCs differentiated as macrophages into deficient adjacent cells, via tunnelling nanotubes (TNTs). Hypothyroidism is the most frequent and earliest endocrine complication in cystinotic patients and was recently shown as a complication in the Ctns−/− mice too. Here, we are evaluating the benefit of HSPC transplantation in the thyroid of Ctns−/− mice. Abundant engraftment of bone marrow-derived cells in Ctns−/− thyroid correlated with drastic decreased of cystine content, normalization of TSH level and correction of the structure of a large fraction of thyrocytes. In the thyroid microenvironment, HSPCs differentiated into a distinct, mixed macrophage/dendritic cell lineage expressing CD45 and MHCII, but not-detectably CD11b and F4/80. Like in cystinotic kidneys, HSPC-derived cells produced TNT-like extensions capable of crossing the follicular basement laminae. Interestingly, HSPCs themselves further squeezed into follicles, allowing extensive contact with thyrocytes, but did not transdifferentiate into Nkx2.1-expressing cells, the hallmark of thyrocytes. This is the first report demonstrating the potential of HSPC transplantation to correct thyroid disease, and supports a major multisystemic benefit of stem cells therapy for cystinosis.