Shafaq-zadah, Massi et al. (2011) International Worm Meeting "AP-1 controls an apical trafficking pathway required for PAR-6 localisation and epithelial polarity."

Brocard, Lysiane, Michaux, Gregoire, Shafaq-zadah, Massi

[International Worm Meeting, 2011]

The establishment and the maintenance of epithelial polarity are essential for animal growth and development and the PAR proteins play an essential role in that process. However little is known about the mechanisms required for their localisation in epithelial cells. We have uncovered an apical trafficking pathway dependent on clathrin/CHC-1, dynamin/DYN-1, RAB-5, RAB-11 and the clathrin adaptor complex AP-1 which are all required for the maintenance of PAR-6 apical localisation in intestinal epithelial cells. Because AP-1 was found to be implicated in basolateral targeting in mammalian epithelial cells we decided to focus on this new and essential function of AP-1 at the apical cortex during embryonic morphogenesis. We found that AP-1 depletion does not affect epithelial polarity establishment and the initial formation of the intestinal lumen. However it induces a complete loss of apico-basal polarity of PAR-6 localisation later during embryonic elongation; while other polarity determinants are unaffected PAR-6 becomes homogenously distributed at the apical and basolateral cortex. This loss of PAR-6 asymmetric localisation triggers de novo formation of ectopic intestinal lumens along the lateral membranes of adjacent cells as shown by light and electron microscopy. In addition we found that AP-1 sorts apical transmembrane cargos, is required for the maintenance of RAB-11 apical recycling endosomes and acts upstream of RAB-11. We also identified a function for RAB-11 itself in the maintenance of a single intestinal lumen. Finally we found that this AP-1 function is dependent on the mu1-II subunit APM-1 while the mu1-I subunit UNC-101 is not implicated. Altogether our results demonstrate an essential function for an AP-1 dependent apical trafficking pathway which is required for the maintenance of PAR-6 localisation and epithelial polarity.

shafaq-zadah, massiullah et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "The AP-1 adaptor complex is required for the maintenance of epithelial junction integrity during C.elegans embryonic development"

Michaux, Gregoire, shafaq-zadah, massiullah, Brocard, Lysiane, Wissler, Frederic, Labouesse, Michel

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Epithelial polarity is acquired and maintained by various mechanisms. To identify new genes involved in this process we performed an RNAi screen and looked for epithelial polarity defects. The transmembrane protein CHE-14, which accumulates at the apical pole, and the junction protein DLG-1 were used as markers. Consistent with predictions, loss of proteins known to be required for epithelial polarity affects CHE-14 and/or DLG-1 localisation. We found that several genes implicated in membrane traffic display a similar CHE-14 mislocalisation phenotype, including the adaptor complex AP-1 and the ESCRT-III member VPS-32. We decided to focus on AP-1 and show that RNAi ag ainst two different subunits both induces an arrest at late stage of elongation and an accumulation of CHE-14 at the lateral plasma membrane. All tested junction proteins were present and look normal during early elongation. However, at the 3-fold stage EM analysis of AP-1(RNAi) embryos revealed that junctions were extended and misplaced towards the baso-lateral pole, leading to a dramatic expansion of the apical pole. We also found that the apical PAR-6 protein is correctly localised at the onset of elongation but progressively loses its apical concentration at the late 3-fold stage. Furthermore, to test how the loss of AP-1 might affect junction dynamics, FRAP experiments were performed on a GFP-tagged E-cadherin expressing worm, showing its recovery was affected. Together, these results suggest an unexpected role for AP-1 in the maintenance of epithelial junctions, possibly through the localisation of apical PAR proteins and E-cadherin recycling/trafficking pathways.