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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.

Polarity is a fundamental characteristic of most eukaryotic cells. The plasma membrane of such cells consists in two structurally and functionally different domains, i.e., the basolateral and the apical membrane, separated by tight junctions. The generation of the distinct molecular identity of both domains and its maintenance in spite of the dynamics of lipids and proteins at either surface requires sophisticated sorting and trafficking mechanisms. Recent progress in the field of polarized trafficking reveals that, for a detailed understanding of its mechanism and regulation, an integrated approach that includes the flow of both lipids and proteins is imperative. In this review, some recent progress in understanding mechanisms involved in protein sorting and trafficking is discussed. We focus on the role of lipid microdomains (Rafts) in trafficking of proteins to the apical surface of polarized cells.

International audience; The human colon cancer cell line HT-29 remains totally undifferentiated when glucose is present in the culture medium (HT-29 Glc+), while the same cells may undergo typical enterocytic differentiation after reaching confluence when grown in glucose-deprived medium (HT-29 Glc-). Recently, we demonstrated a deficiency in the overall N-glycan processing in confluent undifferentiated cells, whereas differentiated cells follow a classical pattern of N-glycosylation. The main changes in N-glycosylation observed in confluent undifferentiated cells may be summarised as follows: 1) the conversion of high mannose into complex glycopeptides is greatly decreased; 2) this decreased conversion could be a consequence of an accumulation of Man9-8-GlcNAc2-Asn high mannose species. Whether these changes in N-glycan processing appear progressively during cell culture or are already present from the beginning of the culture was investigated in this study by comparing the actual status of N-glycan processing in exponentially growing HT-29 Glc- and HT-29 Glc+ cells. Under these conditions, HT-29 Glc- cells do not exhibit any characteristics of differentiation. The conversion of high mannose into complex glycoproteins is severely reduced in HT-29 Glc+ cells, regardless of the growth phase studied. In contrast, HT-29 Glc- cells display a normal pattern of N-glycan processing in both growth phases. We therefore conclude that N-glycan processing may be used as an early biochemical marker of the enterocytic differentiation process of HT-29 cells.

Rotavirus is the major causative agent of severe gastroenteritis in young children worldwide. After assembly steps in the Endoplasmic Reticulum (ER), virions are released without any cellular lysis at the apical side of enterocytes, following an atypical trafficking pathway that bypasses the Golgi apparatus. This work is aimed at understanding the mechanisms of ER exit of rotavirus particles as well as their unconventional trafficking. Components of the protein folding and quality control machinery in the ER were found associated with viral assembly complexes. Consistent with this observation, viral infection induced ER stress, which activates a specific cellular response named the Unfold Protein Response (UPR). Rotavirus infection modulated differently the three UPR signaling pathways and only the IRE1 and PERK pathways were required for viral morphogenesis. Autophagy, besides being a degradative process, has recently been shown to be potentially involved in unconventional secretion pathways. We showed that the differentiation process of human intestinal epithelial Caco-2 cells into enterocyte-like phenotype was marked by the increase in expression of autophagic markers and the reduction of autophagic flux. Rotavirus infection blocked both the initiation and late steps of autophagy, in both undifferentiated and differentiated Caco-2 cells. Surprisingly, only in undifferentiated Caco-2 cells, rotavirus infection induced a lipidation of LC3 that was not associated with autophagy but correlated with a cleavage of ATG3, a protein directly involved in the lipidation process. Neither autophagy nor the lipidation of LC3 were required for rotavirus morphogenesis in Caco-2 cells.; Le rotavirus est le principal agent étiologique des gastro-entérites infantiles. Après l'assemblage dans le réticulum endoplasmique (RE), les virions sont relargués au niveau du pôle apical des entérocytes selon une voie de trafic atypique ne passant pas par l'appareil de Golgi. Ce travail a pour but d'étudier les mécanismes de sortie du RE et le trafic atypique du rotavirus. Des acteurs de la machinerie réticulaire de repliement et de contrôle qualité des protéines ont été retrouvés associés aux intermédiaires d'assemblage du rotavirus. En corrélation avec cela, l'infection provoque un stress du RE et active une réponse cellulaire spécifique, l'Unfolded Protein Response (UPR). Le rotavirus module de manière différentielle les trois voies de signalisation de l'UPR et seules les voies IRE1 et PERK sont requises pour la morphogénèse virale. L'autophagie, en dehors de son rôle dans la dégradation du matériel cellulaire, a été récemment impliquée dans des voies de sécrétion non conventionnelles. Dans les cellules épithéliales intestinales Caco-2, la différenciation cellulaire se manifeste par l'augmentation de l'expression des marqueurs autophagiques et la diminution du flux autophagique. Quelque soit l'état de différenciation des cellules, l'infection à rotavirus bloque à la fois la formation et la maturation des autophagosomes. Uniquement dans les cellules Caco-2 non différenciées, l'infection induit une lipidation de LC3 qui n'est pas associée à l'autophagie, mais qui corrèle avec un clivage de la protéine ATG3 impliquée dans le processus de lipidation. Ni l'autophagie, ni la lipidation de LC3 ne sont requises pour la morphogénèse du rotavirus dans les cellules Caco-2.

Rotavirus is the major causative agent of severe gastroenteritis in young children worldwide. After assembly steps in the Endoplasmic Reticulum (ER), virions are released without any cellular lysis at the apical side of enterocytes, following an atypical trafficking pathway that bypasses the Golgi apparatus. This work is aimed at understanding the mechanisms of ER exit of rotavirus particles as well as their unconventional trafficking. Components of the protein folding and quality control machinery in the ER were found associated with viral assembly complexes. Consistent with this observation, viral infection induced ER stress, which activates a specific cellular response named the Unfold Protein Response (UPR). Rotavirus infection modulated differently the three UPR signaling pathways and only the IRE1 and PERK pathways were required for viral morphogenesis. Autophagy, besides being a degradative process, has recently been shown to be potentially involved in unconventional secretion pathways. We showed that the differentiation process of human intestinal epithelial Caco-2 cells into enterocyte-like phenotype was marked by the increase in expression of autophagic markers and the reduction of autophagic flux. Rotavirus infection blocked both the initiation and late steps of autophagy, in both undifferentiated and differentiated Caco-2 cells. Surprisingly, only in undifferentiated Caco-2 cells, rotavirus infection induced a lipidation of LC3 that was not associated with autophagy but correlated with a cleavage of ATG3, a protein directly involved in the lipidation process. Neither autophagy nor the lipidation of LC3 were required for rotavirus morphogenesis in Caco-2 cells., Le rotavirus est le principal agent étiologique des gastro-entérites infantiles. Après l'assemblage dans le réticulum endoplasmique (RE), les virions sont relargués au niveau du pôle apical des entérocytes selon une voie de trafic atypique ne passant pas par l'appareil de Golgi. Ce travail a pour but d'étudier les mécanismes de sortie du RE et le trafic atypique du rotavirus. Des acteurs de la machinerie réticulaire de repliement et de contrôle qualité des protéines ont été retrouvés associés aux intermédiaires d'assemblage du rotavirus. En corrélation avec cela, l'infection provoque un stress du RE et active une réponse cellulaire spécifique, l'Unfolded Protein Response (UPR). Le rotavirus module de manière différentielle les trois voies de signalisation de l'UPR et seules les voies IRE1 et PERK sont requises pour la morphogénèse virale. L'autophagie, en dehors de son rôle dans la dégradation du matériel cellulaire, a été récemment impliquée dans des voies de sécrétion non conventionnelles. Dans les cellules épithéliales intestinales Caco-2, la différenciation cellulaire se manifeste par l'augmentation de l'expression des marqueurs autophagiques et la diminution du flux autophagique. Quelque soit l'état de différenciation des cellules, l'infection à rotavirus bloque à la fois la formation et la maturation des autophagosomes. Uniquement dans les cellules Caco-2 non différenciées, l'infection induit une lipidation de LC3 qui n'est pas associée à l'autophagie, mais qui corrèle avec un clivage de la protéine ATG3 impliquée dans le processus de lipidation. Ni l'autophagie, ni la lipidation de LC3 ne sont requises pour la morphogénèse du rotavirus dans les cellules Caco-2.