Patrick Van Der Smissen

Loss of chloride channel ClC-5 impairs endocytosis by defective trafficking of megalin and cubilin in kidney proximal tubules

Loss of the renal endosome-associated chloride channel, ClC-5, in Dents disease and knockout (KO) mice strongly inhibits endocytosis of filtered proteins by kidney proximal tubular cells (PTC). The underlying mechanism remains unknown. We therefore tested whether this endocytic failure could primarily reflect a loss of reabsorption by the multiligand receptors, megalin, and cubilin, caused by a trafficking defect. Impaired protein endocytosis in PTC of ClC-5 KO mice was demonstrated by (i) a major decreased uptake of injected125I-β2-microglobulin, but not of the fluid-phase tracer, FITC-dextran, (ii) reduced labeling of endosomes by injected peroxidase and for the endogenous megalin/cubilin ligands, vitamin D- and retinol-binding proteins, and (iii) urinary appearance of low-molecular-weight proteins and the selective cubilin ligand, transferrin. Contrasting with preserved mRNA levels, megalin and cubilin abundance was significantly decreased in kidney extracts of KO mice. Percoll gradients resolving early and late endosomes (Rab5a, Rab7), brush border (villin, aminopeptidase M), and a dense peak comprising lysosomes (acid hydrolases) showed a disappearance of the brush border component for megalin and cubilin in KO mice. Quantitative ultrastructural immunogold labeling confirmed the overall decrease of megalin and cubilin in PTC and their selective loss at the brush border. In contrast, total contents of the rate-limiting endocytic catalysts, Rab5a and Rab7, were unaffected. Thus, impaired protein endocytosis caused by invalidation of ClC-5 primarily reflects a trafficking defect of megalin and cubilin in PTC.

Restoring the Association of the T Cell Receptor with CD8 Reverses Anergy in Human Tumor-Infiltrating Lymphocytes

For 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.

A Galectin-3 Ligand Corrects the Impaired Function of Human CD4 and CD8 Tumor-Infiltrating Lymphocytes and Favors Tumor Rejection in Mice

Human CD8(+) tumor-infiltrating T lymphocytes (TIL), in contrast with CD8(+) blood cells, show impaired IFN-γ secretion on ex vivo restimulation. We have attributed the impaired IFN-γ secretion to a decreased mobility of T-cell receptors on trapping in a lattice of glycoproteins clustered by extracellular galectin-3. Indeed, we have previously shown that treatment with N-acetyllactosamine, a galectin ligand, restored this secretion. We strengthened this hypothesis here by showing that CD8(+) TIL treated with an anti-galectin-3 antibody had an increased IFN-γ secretion. Moreover, we found that GCS-100, a polysaccharide in clinical development, detached galectin-3 from TIL and boosted cytotoxicity and secretion of different cytokines. Importantly, we observed that not only CD8(+) TIL but also CD4(+) TIL treated with GCS-100 secreted more IFN-γ on ex vivo restimulation. In tumor-bearing mice vaccinated with a tumor antigen, injections of GCS-100 led to tumor rejection in half of the mice, whereas all control mice died. In nonvaccinated mice, GCS-100 had no effect by itself. These results suggest that a combination of galectin-3 ligands and therapeutic vaccination may induce more tumor regressions in cancer patients than vaccination alone.

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.

ZONAB Promotes Proliferation and Represses Differentiation of Proximal Tubule Epithelial Cells

Epithelial polarization modulates gene expression. The transcription factor zonula occludens 1 (ZO-1)-associated nucleic acid binding protein (ZONAB) can shuttle between tight junctions and nuclei, promoting cell proliferation and expression of cyclin D1 and proliferating cell nuclear antigen (PCNA), but whether it also represses epithelial differentiation is unknown. Here, during mouse kidney ontogeny and polarization of proximal tubular cells (OK cells), ZONAB and PCNA levels decreased in parallel and inversely correlated with increasing apical differentiation, reflected by expression of megalin/cubilin, maturation of the brush border, and extension of the primary cilium. Conversely, ZONAB reexpression and loss of apical differentiation markers provided a signature for renal clear cell carcinoma. In confluent OK cells, ZONAB overexpression increased proliferation and PCNA while repressing megalin/cubilin expression and impairing differentiation of the brush border and primary cilium. Reporter and chromatin immunoprecipitation assays demonstrated that megalin and cubilin are ZONAB target genes. Sparsely plated OK cells formed small islands composed of distinct populations: Cells on the periphery, which lacked external tight junctions, strongly expressed nuclear ZONAB, proliferated, and failed to differentiate; central cells, surrounded by continuous junctions, lost nuclear ZONAB, stopped proliferating, and engaged in apical differentiation. Taken together, these data suggest that ZONAB is an important component of the mechanisms that sense epithelial density and participates in the complex transcriptional networks that regulate the switch between proliferation and differentiation.

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.

Membrane destabilization induced by beta-amyloid peptide 29-42: importance of the amino-terminus

Increasing evidence implicates interactions between Abeta-peptides and membrane lipids in Alzheimers disease. To gain insight into the potential role of the free amino group of the N-terminus of Abeta29-42 fragment in these processes, we have investigated the ability of Abeta29-42 unprotected and Abeta29-42 N-protected to interact with negatively-charged liposomes and have calculated the interaction with membrane lipids by conformational analysis. Using vesicles mimicking the composition of neuronal membranes, we show that both peptides have a similar capacity to induce membrane fusion and permeabilization. The fusogenic effect is related to the appearance of non-bilayer structures where isotropic motions occur as shown by 31P and 2H NMR studies. The molecular modeling calculations confirm the experimental observations and suggest that lipid destabilization could be due to the ability of both peptides to adopt metastable positions in the presence of lipids. In conclusion, the presence of a free or protected (acetylated) amino group in the N-terminus of Abeta29-42 is therefore probably not crucial for destabilizing properties of the C-terminal fragment of Abeta peptides.

Time Course of Pathogenic and Adaptation Mechanisms in Cystinotic Mouse Kidneys

Cystinosis, a main cause of Fanconi syndrome, is reproduced in congenic C57BL/6 cystinosin knockout (KO) mice. To identify the sequence of pathogenic and adaptation mechanisms of nephropathic cystinosis, we defined the onset of Fanconi syndrome in KO mice between 3 and 6 months of age and analyzed the correlation with structural and functional changes in proximal tubular cells (PTCs), with focus on endocytosis of ultrafiltrated disulfide-rich proteins as a key source of cystine. Despite considerable variation between mice at the same age, typical event sequences were delineated. At the cellular level, amorphous lysosomal inclusions preceded cystine crystals and eventual atrophy without crystals. At the nephron level, lesions started at the glomerulotubular junction and then extended distally. In situ hybridization and immunofluorescence revealed progressive loss of expression of megalin, cubilin, sodium-glucose cotransporter 2, and type IIa sodium-dependent phosphate cotransporter, suggesting apical dedifferentiation accounting for Fanconi syndrome before atrophy. Injection of labeled proteins revealed that defective endocytosis in S1 PTCs led to partial compensatory uptake by S3 PTCs, suggesting displacement of endocytic load and injury by disulfide-rich cargo. Increased PTC apoptosis allowed luminal shedding of cystine crystals and was partially compensated for by tubular proliferation. We conclude that lysosomal storage triggered by soluble cystine accumulation induces apical PTC dedifferentiation, which causes transfer of the harmful load of disulfide-rich proteins to more distal cells, possibly explaining longitudinal progression of swan-neck lesions. Furthermore, our results suggest that subsequent adaptation mechanisms include lysosomal clearance of free and crystalline cystine into urine and ongoing tissue repair.

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

Abstract 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 μM] against caspase-3 and aurin [1 μM] 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.

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.