Donatienne Tyteca

Lack of cadherins Celsr2 and Celsr3 impairs ependymal ciliogenesis, leading to fatal hydrocephalus

Ependymal cells form the epithelial lining of cerebral ventricles. Their apical surface is covered by cilia that beat in a coordinated fashion to facilitate circulation of the cerebrospinal fluid (CSF). The genetic factors that govern the development and function of ependymal cilia remain poorly understood. We found that the planar cell polarity cadherins Celsr2 and Celsr3 control these processes. In Celsr2-deficient mice, the development and planar organization of ependymal cilia are compromised, leading to defective CSF dynamics and hydrocephalus. In Celsr2 and Celsr3 double mutant ependyma, ciliogenesis is markedly impaired, resulting in lethal hydrocephalus. The membrane distribution of Vangl2 and Fzd3, two key planar cell polarity proteins, was disturbed in Celsr2 mutants, and even more so in Celsr2 and Celsr3 double mutants. Our findings suggest that planar cell polarity signaling is involved in ependymal cilia development and in the pathophysiology of hydrocephalus, with possible implications in other ciliopathies.

The subapical compartment: a traffic center in membrane polarity development.

Spatially separated apical and basolateral plasma membrane domains that have distinct functions and molecular compositions are a characteristic feature of epithelial cell polarity. The subapical compartment (SAC), also known as the common endosome (CE), where endocytic pathways from both surfaces merge, plays a crucial role in the maintenance and probably the biogenesis of these distinct membrane domains. Although differences in morphology are apparent, the same principal features of a SAC can be distinguished in different types of epithelial cells. As polarity develops, the compartment acquires several distinct machineries that, in conjunction with the cytoskeleton, are necessary for polarized trafficking. Disrupting trafficking via the SAC and hence bypassing its sorting machinery, as occurs upon actin depolymerization, leads to mis-sorting of apical and basolateral molecules, thereby compromising the development of polarity. The structural and functional integrity of the compartment in part depends on microtubules. Moreover, the acquisition of a particular set of Rab proteins, including Rab11 and Rab3, appears to be crucial in regulating molecular sorting and vesicular transport relevant both to recycling to either plasma membrane domain and to de novo assembly of the apical domain. Furthermore, subcompartmentalization of the SAC appears to be key to its various functions.

Molecular identification of aspartate N-acetyltransferase and its mutation in hypoacetylaspartia

The brain-specific compound NAA (N-acetylaspartate) occurs almost exclusively in neurons, where its concentration reaches approx. 20 mM. Its abundance is determined in patients by MRS (magnetic resonance spectroscopy) to assess neuronal density and health. The molecular identity of the NAT (N-acetyltransferase) that catalyses NAA synthesis has remained unknown, because the enzyme is membrane-bound and difficult to purify. Database searches indicated that among putative NATs (i.e. proteins homologous with known NATs, but with uncharacterized catalytic activity) encoded by the human and mouse genomes two were almost exclusively expressed in brain, NAT8L and NAT14. Transfection studies in HEK-293T [human embryonic kidney-293 cells expressing the large T-antigen of SV40 (simian virus 40)] indicated that NAT8L, but not NAT14, catalysed the synthesis of NAA from L-aspartate and acetyl-CoA. The specificity of NAT8L, its Km for aspartate and its sensitivity to detergents are similar to those described for brain Asp-NAT. Confocal microscopy analysis of CHO (Chinese-hamster ovary) cells and neurons expressing recombinant NAT8L indicates that it is associated with the ER (endoplasmic reticulum), but not with mitochondria. A mutation search in the NAT8L gene of the only patient known to be deficient in NAA disclosed the presence of a homozygous 19 bp deletion, resulting in a change in reading frame and the absence of production of a functional protein. We conclude that NAT8L, a neuron-specific protein, is responsible for NAA synthesis and is mutated in primary NAA deficiency (hypoacetylaspartia). The molecular identification of this enzyme will lead to new perspectives in the clarification of the function of this most abundant amino acid derivative in neurons and for the diagnosis of hypoacetylaspartia in other patients.

Src triggers circular ruffling and macropinocytosis at the apical surface of polarized MDCK cells.

We addressed the role of Src on cortical actin dynamics and polarized endocytosis in MDCK cells harboring a thermosensitive v‐src mutant. Shifting monolayers established at 40 °C (non‐permissive temperature) to 34 °C (permissive temperature) rapidly reactivated v‐Src kinase, but tight junctions and cell polarity resisted for >6 h. At this interval, activated v‐src was recruited on apical vesicles, induced cortactin‐associated apical circular ruffles productive of macropinosomes, thereby accelerating apical pinocytosis by approximately fivefold. Ruffling and macropinosome formation were selectively abrogated by inhibitors of actin polymerization, phosphoinositide 3‐kinase, phospholipase C, and phospholipase D, which all returned apical pinocytosis to the level observed at 40 °C, underscoring the distinct control of apical micropinocytosis and macropinocytosis. Src promoted microtubule‐dependent fusion of macropinosomes to the apical recycling endosome (ARE), causing its strong vacuolation. However, preservation of tubulation and apical polarity indicated that its function was not affected. The ARE was labeled for v‐src, Rab11, and rabankyrin‐5 but not early endosome antigen 1, thus distinguishing two separate Rab5‐dependent apical pathways. The mechanisms of Src‐induced apical ruffling and macropinocytosis could shed light on the triggered apical enteroinvasive pathogens entry and on the apical differentiation of osteoclasts.

Cellular Pharmacokinetics and Pharmacodynamics of the Glycopeptide Antibiotic Oritavancin (LY333328) in a Model of J774 Mouse Macrophages

ABSTRACT The intracellular pharmacokinetics and pharmacodynamics of oritavancin (LY333328) were studied in cultured cells. Oritavancin was avidly accumulated by J774 and THP-1 macrophages and rat fibroblasts and to a lesser extent by LLC-PK1 and Caco-2 cells. In J774 macrophages, the level of accumulation reached a plateau (at 370-fold the extracellular concentration) within 24 h and was partly defeated by a rise in serum protein levels. Efflux was incomplete (with a plateau at two-thirds of the original level at 6 h). In short-term kinetic studies, oritavancin uptake was linear for up to 4 h (as was the case for horseradish peroxidase and small latex beads, used as markers of the fluid phase and adsorptive endocytosis, respectively), which was in contrast to azithromycin and chloroquine uptake (which accumulate in cells by diffusion and segregation). The rates of clearance of oritavancin and latex beads were comparable (150 and 120 μl × mg of protein−1 × h−1, respectively) and were approximately 200 times higher than that of horseradish peroxidase. Oritavancin accumulation was partially reduced by monensin but was unaffected by acidic pH (these conditions abolished chloroquine accumulation). Cell-associated oritavancin was found in lysosomal fractions after homogenization of J774 macrophages and fractionation by isopycnic centrifugation. Oritavancin was bactericidal against intracellular Staphylococcus aureus (phagolysosomal infection) but was unable to control the intracellular growth of Listeria monocytogenes (cytosolic infection), even though its cellular concentration largely exceeded the MIC (0.02 mg/liter) and minimal bactericidal concentration (2 mg/liter). We conclude that oritavancin enters cells by adsorptive endocytosis (favored by its lipophilic side chain and/or the presence of three protonatable amines), which drives it to lysosomes, where it exerts antibiotic activity.

Recent progress on lipid lateral heterogeneity in plasma membranes: From rafts to submicrometric domains

The concept of transient nanometric domains known as lipid rafts has brought interest to reassess the validity of the Singer-Nicolson model of a fluid bilayer for cell membranes. However, this new view is still insufficient to explain the cellular control of surface lipid diversity or membrane deformability. During the past decades, the hypothesis that some lipids form large (submicrometric/mesoscale vs nanometric rafts) and stable (>min vs s) membrane domains has emerged, largely based on indirect methods. Morphological evidence for stable submicrometric lipid domains, well-accepted for artificial and highly specialized biological membranes, was further reported for a variety of living cells from prokaryot es to yeast and mammalian cells. However, results remained questioned based on limitations of available fluorescent tools, use of poor lipid fixatives, and imaging artifacts due to non-resolved membrane projections. In this review, we will discuss recent evidence generated using powerful and innovative approaches such as lipid-specific toxin fragments that support the existence of submicrometric domains. We will integrate documented mechanisms involved in the formation and maintenance of these domains, and provide a perspective on their relevance on membrane deformability and regulation of membrane protein distribution.

Amyloid precursor protein controls cholesterol turnover needed for neuronal activity.

Perturbation of lipid metabolism favours progression of Alzheimer disease, in which processing of Amyloid Precursor Protein (APP) has important implications. APP cleavage is tightly regulated by cholesterol and APP fragments regulate lipid homeostasis. Here, we investigated whether up or down regulation of full‐length APP expression affected neuronal lipid metabolism. Expression of APP decreased HMG‐CoA reductase (HMGCR)‐mediated cholesterol biosynthesis and SREBP mRNA levels, while its down regulation had opposite effects. APP and SREBP1 co‐immunoprecipitated and co‐localized in the Golgi. This interaction prevented Site‐2 protease‐mediated processing of SREBP1, leading to inhibition of transcription of its target genes. A GXXXG motif in APP sequence was critical for regulation of HMGCR expression. In astrocytes, APP and SREBP1 did not interact nor did APP affect cholesterol biosynthesis. Neuronal expression of APP decreased both HMGCR and cholesterol 24‐hydroxylase mRNA levels and consequently cholesterol turnover, leading to inhibition of neuronal activity, which was rescued by geranylgeraniol, generated in the mevalonate pathway, in both APP expressing and mevastatin treated neurons. We conclude that APP controls cholesterol turnover needed for neuronal activity.

Role of oxidative stress in lysosomal membrane permeabilization and apoptosis induced by gentamicin, an aminoglycoside antibiotic

Gentamicin, an aminoglycoside antibiotic used to treat severe bacterial infections, may cause acute renal failure. At therapeutic concentrations, gentamicin accumulates in lysosomes and induces apoptosis in kidney proximal tubular cells. In gentamicin-treated renal LLC-PK1 cells, acridine orange release from lysosomes, previously interpreted as lysosomal membrane permeabilization, precedes the apoptotic cascade that develops during incubation with gentamicin. However, the link between gentamicin lysosomal accumulation and apoptosis remains unclear. We here examined if reactive oxygen species (ROS) production could account for gentamicin-induced acridine orange release and apoptosis, and the implication of iron in these events. We found that gentamicin induced ROS production prior to, and at lower drug concentrations than required for, acridine orange release and apoptosis. ROS antioxidant or scavenger, catalase, and N-acetylcysteine largely prevented these events. Vital confocal imaging revealed that gentamicin-induced ROS production occurs in lysosomes. Deferoxamine, an iron chelator, which is endocytosed and accumulates in lysosomes, largely prevented gentamicin-induced ROS production as well as apoptosis. Direct evidence for gentamicin-induced permeabilization of lysosomal membrane was provided by showing the release into the cytosol of Lucifer yellow, a membrane-impermeant endocytic tracer with a comparable molecular weight as gentamicin. Altogether, our data demonstrate a key role of lysosomal iron and early ROS production in gentamicin-induced lysosomal membrane permeabilization and apoptosis.

Three unrelated sphingomyelin analogs spontaneously cluster into plasma membrane micrometric domains

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.

The macrolide antibiotic azithromycin interacts with lipids and affects membrane organization and fluidity: studies on Langmuir-Blodgett monolayers, liposomes and J774 macrophages

The macrolide antibiotic azithromycin was shown to markedly inhibit endocytosis. Here we investigate the interaction of azithromycin with biomembranes and its effects on membrane biophysics in relation to endocytosis. Equilibrium dialysis and 31P NMR revealed that azithromycin binds to lipidic model membranes and decreases the mobility of phospholipid phosphate heads. In contrast, azithromycin had no effect deeper in the bilayer, based on fluorescence polarization of TMA-DPH and DPH, compounds that, respectively, explore the interfacial and hydrophobic domains of bilayers, and it did not induce membrane fusion, a key event of vesicular trafficking. Atomic force microscopy showed that azithromycin perturbed lateral phase separation in Langmuir-Blodgett monolayers, indicating a perturbation of membrane organization in lateral domains. The consequence of azithromycin/phospholipid interaction on membrane endocytosis was next evaluated in J774 macrophages by using three tracers with different insertion preferences inside the biological membranes and intracellular trafficking: C6-NBD-SM, TMA-DPH and N-Rh-PE. Azithromycin differentially altered their insertion into the plasma membrane, slowed down membrane trafficking towards lysosomes, as evaluated by the rate of N-Rh-PE self-quenching relief, but did not affect bulk membrane internalization of C6-NBD-SM and TMA-DPH. Azithromycin also decreased plasma membrane fluidity, as shown by TMA-DPH fluorescence polarization and confocal microscopy after labeling by fluorescent concanavalin A. We conclude that azithromycin directly interacts with phospholipids, modifies biophysical properties of membrane and affects membrane dynamics in living cells. This antibiotic may therefore help to elucidate the physico-chemical properties underlying endocytosis.