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L’imagerie cellulaire et tissulaire connaît un développement considérable résultant à la fois de l’attente des scientifiques et des progrès concertés de nombreuses disciplines. Le développement de la microscopie confocale, de la vidéomicroscopie, de nouveaux fluorochromes, notamment des dérivés de la GFP (green fluorescent protein), et de nouveaux logiciels permettent d’accéder à la visualisation tridimensionnelle des processus biologiques. Leur quantification à l’échelle de la cellule unique est maintenant possible. L’imagerie cellulaire fournit des résultats pertinents sur la stoechiométrie et la dynamique des interactions moléculaires impliquées dans les régulations cellulaires. Des progrès importants ont également été réalisés dans l’optimisation de la résolution temporelle. À l’époque de la post-génomique, une utilisation rationnelle des approches d’imagerie passe par une bonne connaissance de leurs avantages et de leurs limites., Cell and tissue imaging provides scientists with wonderful tools, thanks to a fruitful dialog between chemistry, optical, mechanical, computational sciences and biology. Confocal microscopy, videomicroscopy together with a new generation of fluorochromes (especially those derived from green fluorescent protein, GFP) and image analysis software allow to visualize life in all its dimensions (space and time). Cell imaging also allows to quantify biological processes at the cellular level, to analyse both stoechiometry and dynamics of molecular interactions involved in cell and tissue regulations. Entering the new era of post-genomics requires a better knowledge of advantages and limitations of these new approaches.

In previous work we reported that long term treatment of polarized HT-29 cells by 1-benzyl-2-acetamido-2-deoxy-alpha-d-galactopyranoside (GalNAcalpha-O-bn) induced undersialylation and intracellular distribution of apical glycoproteins such as dipeptidyl peptidase IV (DPP-IV), and we suggested therefore that sialylation could act as an apical targeting signal. In this work, the apical direct biosynthetic route was studied after transfection of polarized enterocyte-like HT-29 5M12 cloned cells with a murine cDNA coding for a soluble form of DPP-IV, which was secreted into the apical medium. A 24-h treatment of transfected cells by GalNAcalpha-O-bn markedly inhibited the apical secretion and the sialylation of this soluble murine DPP-IV, which became blocked inside the cell. A similar short GalNAcalpha-O-bn treatment also induced an intracellular distribution of both endogenous transmembrane DPP-IV and proteins involved in the regulation of the apical trafficking such as the apical t-SNARE syntaxin-3 and the raft-associated protein annexin XIIIb, whereas the basolateral t-SNARE syntaxin-4 kept its normal localization. These apical membrane proteins moved efficiently from trans-Golgi network to apical carrier vesicles but failed to be transported from carrier vesicles to the apical plasma membrane. Isolation of membrane microdomains showed that GalNAcalpha-O-bn induced the formation of abnormal lipid-rich microdomains in comparison to normal rafts, as shown by their lower buoyant density and their depletion in annexin XIIIb. In conclusion, GalNAcalpha-O-bn blocks the anterograde traffic to the apical surface of polarized HT-29 cells at the transport level or docking/fusion level of carrier vesicles.

La polarite est une caracteristique fondamentale de la plupart des cellules eucaryotes. Le caractere primordial de cette polarite est l’individualisation de la membrane plasmique en domaines separes: le domaine apical et le domaine basolateral. Ces deux surfaces exercent des fonctions differentes qui correspondent a une composition tres specifique de chaque domaine en lipides et en proteines. Pour etablir et maintenir leur polarite, les cellules epitheliales doivent trier et envoyer les proteines et les lipides vers la surface cellulaire correcte. Les donnees recentes concernant le transport polarise montrent que le trafic intracellulaire des lipides et des proteines sont deux evenements etroitement lies et qui ne doivent donc pas etre abordes separement. Dans cette revue nous discuterons de quelques mecanismes impliques dans le transport polarise, nous aborderons plus particulierement les donnees recentes concernant le role des microdomaines lipidiques (Rafts) dans le trafic des proteines vers la surface apicale des cellules epitheliales polarisees.

We investigated the signals involved in the apical targeting of dipeptidyl peptidase IV (DPP IV/CD26), an archetypal type II transmembrane glycoprotein. A secretory construct, corresponding to the DPP IV ectodomain, was first stably expressed in both the enterocytic-like cell line Caco-2 and the epithelial kidney MDCK cells. Most of the secretory form of the protein was delivered apically in MDCK cells, whereas secretion was 60% basolateral in Caco-2 cells, indicating that DPP IV ectodomain targeting is cell-type-dependent. A chimera (CTM–GFP) containing only the cytoplasmic and transmembrane domains of mouse DPP IV plus the green fluorescent protein was then studied. In both cell lines, this chimera was preferentially expressed at the apical membrane. By contrast, a secretory form of GFP was randomly secreted, indicating that GFP by itself does not contain cryptic targeting information. Comparison of the sequence of the transmembrane domain of DPP IV and several other apically targeted proteins does not show any consensus, suggesting that the apical targeting signal may be conformational. Neither the DPP IV nor the CTM–GFP chimera was enriched in lipid rafts. Together these results indicate that, besides the well-known raft-dependent apical targeting pathway, the fate of the CTM domain of DPP IV may reveal a new raft-independent apical pathway.

Les phosphodiesterases de type I sont exprimees differemment selon le type cellulaire. Trois membres ont ete identifies, PC-1, B10 et l’autotaxine. Elles ont entre 40 et 50 % d’identite dans leur sequence en acides amines. Les hepatocytes expriment a la fois B10 et PC-1 sur leur membrane plasmique. Cependant, B10 est exclusivement localisee au pole apical tandis que PC-1 est localisee au pole basolateral. Au cours de sa biosynthese dans les hepatocytes, B10 passe transitoirement par le pole basolateral avant d’atteindre le pole apical. L’etape de transcytose entre le pole basolateral et le pole apical fait intervenir un sous-compartiment tubulaire de l’endosome identique a celui du recepteur des IgA polymeriques. Transfectees dans les cellules MDCK d’origine renale et Caco-2 d’origine intestinale, B10 et PC-1 sont exprimees respectivement au pole apical et basolateral, comme dans les hepatocytes. Cependant le transport de B10 a lieu directement vers la surface apicale dans les cellules MDCK; il a lieu en partie directement et en partie par transcytose dans les cellules Caco-2. La troncation du domaine cytoplasmique de PC-1 produit une proteine apicale, ce qui indique que le domaine cytoplasmique porte le signal d’adressage de PC-1 vers la membrane plasmique basolaterale. La recherche du signal d’adressage apical de B10 est en cours.

Le tri des proteines dans les cellules epitheliales represente le processus fondamental de la mise en place et du maintien de la polarite fonctionnelle des cellules epitheliales. Des processus nombreux et interdependants sont mis en oeuvre pour realiser ces etapes specialisees du trafic intracellulaire. Les dernieres donnees de la litterature sur les composantes de ces voies de trafic seront presentees et discutees dans la premiere partie de cet article. Les signaux portes par les proteines pour specifier leur destination representent l’un des elements majeurs de l’adressage. Les signaux basolateraux sont pour l’essentiel connus et relativement univoques, alors que les signaux apicaux sont quant a eux discutes et multiples en terme de nature moleculaire. Le cas des signaux portes par la dipeptidyl peptidase IV (DPP IV/CD26) est particulierement interessant a aborder, car cette proteine rejoint le pole apical des cellules epitheliales selon des voies qui varient avec le type cellulaire considere. Ces voies de trafic variables et complexes qui conduisent a des localisations temporaires de la DPP IV autres que la bordure en brosse apicale sont associees a des fonctions diverses : digestion enzymatique, liaison de proteines circulantes, interaction avec la matrice extracellulaire. Nous avons entrepris l’etude de ces signaux et nous decrirons ici le role potentiel de la glycosylation et celui du domaine transmembranaire dans l’adressage apical de la DPP IV, en insistant sur les capacites des differentes machineries cellulaires a interpreter ces signaux.

Summary Targeting of glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs) in polarized epithelial cells depends on their association with detergent-resistant membrane microdomains called rafts. In MDCK cells, GPI-APs associate with rafts in the trans-Golgi network and are directly delivered to the apical membrane. It has been shown that oligomerization is required for their stabilization in rafts and their apical targeting. In hepatocytes, GPI-APs are first delivered to the basolateral membrane and secondarily reach the apical membrane by transcytosis. We investigated whether oligomerization is required for raft association and apical sorting of GPI-APs in polarized HepG2 cells, and at which step of the pathway oligomerization occurs. Model proteins were wild-type GFP-GPI and a double cysteine GFP-GPI mutant, in which GFP dimerization was impaired. Unlike wild-type GFP-GPI, which was efficiently endocytosed and transcytosed to the apical surface, the double cysteine mutant was basolaterally internalized, but massively accumulated in early endosomes, and reached the bile canaliculi with delayed kinetics. The double cysteine mutant was less resistant to Triton X-100 extraction, and formed fewer high molecular weight complexes. We conclude from these results that, in hepatocytes, oligomerization plays a key role in targeting GPI-APs to the apical membrane, by increasing their affinity for rafts and allowing their transcytosis.

International audience; In polarized hepatocytes, the predominant route for apical resident proteins to reach the apical bile canalicular membrane is transcytosis. Apical proteins are first sorted to the basolateral membrane from which they are internalized and transported to the opposite surface. We have noted previously that transmembrane proteins and GPI-anchored proteins reach the apical bile canaliculi at very different rates. Here, we investigated whether these differences may be explained by the use of distinct endocytic mechanisms. We show that endocytosis of both classes of proteins at the basolateral membrane of polarized hepatic cells is dynamin dependent. However, internalization of transmembrane proteins is clathrin mediated, whereas endocytosis of GPI-anchored proteins does not require clathrin. Further analysis of basolateral endocytosis of GPI-anchored proteins showed that caveolin, as well as the small GTPase cdc42 were dispensable. Alternatively, internalized GPI-anchored proteins colocalized with flotillin-2-positive vesicles, and down-expression of flotillin-2 inhibited endocytosis of GPI-anchored proteins. These results show that basolateral endocytosis of GPI-anchored proteins in hepatic cells occurs via a clathrin-independent flotillin-dependent pathway. The use of distinct endocytic pathways may explain, at least in part, the different rates of transcytosis between transmembrane and GPI-anchored proteins.

Cell and tissue imaging provides scientists with wonderful tools, thanks to a fruitful dialog between chemistry, optical, mechanical, computational sciences and biology. Confocal microscopy, videomicroscopy together with a new generation of fluorochromes (especially those derived from green fluorescent protein, GFP) and image analysis software allow to visualize life in all its dimensions (space and time). Cell imaging also allows to quantify biological processes at the cellular level, to analyse both stoechiometry and dynamics of molecular interactions involved in cell and tissue regulations. Entering the new era of post-genomics requires a better knowledge of advantages and limitations of these new approaches.

The role of glycans in the apical targeting of proteins in epithelial cells remains a debated question. We have expressed the mouse soluble dipeptidyl peptidase IV (DPP IV ectodomain) in kidney (MDCK) and in intestinal (Caco-2) epithelial cell lines, as a model to study the role of glycosylation in apical targeting. The mouse DPP IV ectodomain was secreted mainly into the apical medium by MDCK cells. Exposure of MDCK cells to GalNac-α- O -benzyl, a drug previously described as an inhibitor of mucin O-glycosylation, produced a protein with a lower molecular weight. In addition this treatment resulted in a decreased apical secretion and an increased basolateral secretion of mouse DPP IV ectodomain. When expressed in Caco-2 cells, the mouse DPP IV ectodomain was secreted mainly into the basolateral medium. However, BGN was still able to decrease the amount of apically secreted protein and to increase its basolateral secretion. Neuraminidase digestion showed that the most stricking effect of BGN was a blockade of DPP IV sialylation in both MDCK and Caco-2 cells. These results indicate that a specific glycosylation step, namely, sialylation, plays a key role in the control of the apical targeting of a secreted DPP IV both in MDCK and Caco-2 cells.

Glycosylation was considered the major signal candidate for apical targeting of transmembrane proteins in polarized epithelial cells. However, direct demonstration of the role of glycosylation has proved difficult because non-glycosylated apical transmembrane proteins usually do not reach the cell surface. Here we were able to follow the targeting of the apical transmembrane glycoprotein NPP3 both when glycosylated and non-glycosylated. Transfected in polarized MDCK and Caco-2 cells, NPP3 was exclusively expressed at the apical membrane. The transport kinetics of the protein to the cell surface were studied after metabolic (35)S-labeling and surface immunoprecipitation. The newly synthesized protein was mainly targeted directly to the apical surface in MDCK cells, whereas 50% transited through the basolateral surface in Caco-2 cells. In both cell types, the basolaterally targeted pool was effectively transcytosed to the apical surface. In the presence of tunicamycin, NPP3 was not N-glycosylated. The non-glycosylated protein was partially retained intracellularly but the fraction that reached the cell surface was nevertheless predominantly targeted apically. However, transcytosis of the non-glycosylated protein was partially impaired in MDCK cells. These results provide direct evidence that glycosylation cannot be considered an apical targeting signal for NPP3, although glycosylation is necessary for correct trafficking of the protein to the cell surface.