The kinesin KIF16B mediates apical transcytosis of transferrin receptor in AP-1B-deficient epithelia.

Polarized epithelial cells take up nutrients from the blood through receptors that are endocytosed and recycle back to the basolateral plasma membrane (PM) utilizing the epithelial-specific clathrin adaptor AP-1B. Some native epithelia lack AP-1B and therefore recycle cognate basolateral receptors to the apical PM, where they carry out important functions for the host organ. Here, we report a novel transcytotic pathway employed by AP-1B-deficient epithelia to relocate AP-1B cargo, such as transferrin receptor (TfR), to the apical PM. Lack of AP-1B inhibited basolateral recycling of TfR from common recycling endosomes (CRE), the site of function of AP-1B, and promoted its transfer to apical recycling endosomes (ARE) mediated by the plus-end kinesin KIF16B and non-centrosomal microtubules, and its delivery to the apical membrane mediated by the small GTPase rab11a. Hence, our experiments suggest that the apical recycling pathway of epithelial cells is functionally equivalent to the rab11a-dependent TfR recycling pathway of non-polarized cells. They define a transcytotic pathway important for the physiology of native AP-1B-deficient epithelia and report the first microtubule motor involved in transcytosis.

Clathrin is a key regulator of basolateral polarity.

Clathrin-coated vesicles are vehicles for intracellular trafficking in all nucleated cells, from yeasts to humans. Many studies have demonstrated their essential roles in endocytosis and cellular signalling processes at the plasma membrane. By contrast, very few of their non-endocytic trafficking roles are known, the best characterized being the transport of hydrolases from the Golgi complex to the lysosome. Here we show that clathrin is required for polarity of the basolateral plasma membrane proteins in the epithelial cell line MDCK. Clathrin knockdown depolarized most basolateral proteins, by interfering with their biosynthetic delivery and recycling, but did not affect the polarity of apical proteins. Quantitative live imaging showed that chronic and acute clathrin knockdown selectively slowed down the exit of basolateral proteins from the Golgi complex, and promoted their mis-sorting into apical carrier vesicles. Our results demonstrate a broad requirement for clathrin in basolateral protein trafficking in epithelial cells.

Modulation of E-cadherin monomer folding by cooperative binding of calcium ions.

Classical cadherins are transmembrane glycoproteins involved in calcium-dependent cell-cell adhesion. Calcium ions are coordinated at the interface between successive modules of the cadherin ectodomain and are thought to regulate the adhesive interactions of cadherins when present at millimolar concentrations. It is widely accepted that calcium plays a critical role in cadherin-mediated cell-cell adhesion, but the nature of cadherin-calcium binding remains a matter of debate. We investigated the parameters of noncovalent cadherin-calcium binding, using the two N-terminal modules of E-cadherin (E/EC12) with a native N-terminal end and nondenaturing electrospray ionization mass spectrometry. By directly visualizing the molecular complexes, we demonstrated that E/EC12 binds three calcium ions, with an average KD of 20 +/- 0.7 microM. These calcium ions bound cooperatively to E/EC12 in its monomeric state, and these properties were not modified by an N-terminal extension consisting of a single methionine residue. This binding induced specific structural changes, as shown by assessments of protease sensitivity, circular dichroism, and mass spectrometry. Furthermore, the D103A mutation (a residue involved in E-cadherin adhesive function) modified calcium binding and led to a loss of cooperativity and the absence of structural changes, despite calcium binding. As the amino acids involved in calcium binding are found within the cadherin consensus motif, our findings may be relevant to other members of the cadherin family.

AP1B sorts basolateral proteins in recycling and biosynthetic routes of MDCK cells.

The epithelial-specific adaptor AP1B sorts basolateral proteins, but the trafficking routes where it performs its sorting role remain controversial. Here, we used an RNAi approach to knock down the medium subunit of AP1B (mu1B) in the prototype epithelial cell line Madin-Darby canine kidney (MDCK). Mu1B-knocked down MDCK cells displayed loss of polarity of several endogenous and exogenous basolateral markers transduced via adenovirus vectors, but exhibited normal polarity of apical markers. We chose two well characterized basolateral protein markers, the transferrin receptor (TfR) and the vesicular stomatitis virus G protein, to study the sorting role of AP1B. A surface-capture assay introduced here showed that mu1B-knocked down MDCK cells plated on filters at confluency and cultured for 4.5 d, sorted TfR correctly in the biosynthetic route but incorrectly in the recycling route. In contrast, these same cells missorted vesicular stomatitis virus G apically in the biosynthetic route. Strikingly, recently confluent MDCK cells (1-3 d) displayed AP1B-dependence in the biosynthetic route of TfR, which decreased with additional days in culture. Sucrose density gradient analysis detected AP1B predominantly in TfR-rich endosomal fractions in MDCK cells confluent for 1 and 4 d. Our results are consistent with the following model: AP1B sorts basolateral proteins in both biosynthetic and recycling routes of MDCK cells, as a result of its predominant functional localization in recycling endosomes, which constitute a post-Golgi station in the biosynthetic route of some plasma membrane proteins. TfR utilizes a direct route from Golgi to basolateral membrane that is established as the epithelial monolayer matures.

Evolving endosomes: how many varieties and why?

The cell biologist's insight into endosomal diversity, in terms of both form and function, has increased dramatically in the past few years. This understanding has been promoted by the availability of powerful new techniques that allow imaging of both cargo and machinery in the endocytic process in real time, and by our ability to inhibit components of this machinery by RNA interference. The emerging picture from these studies is of a highly complex, dynamic and adaptable endosomal system that interacts at various points with the secretory system of the cell.

[Molecular aspects of cadherin-mediated cell adhesion: the first moments of the interaction]. / Aspects moléculaires de l'adherence cellulaire cadhérine-dépendante: les premiers moments de l'interaction.

Cadherins play a major role in the development and maintenance of all solid tissues. These transmembrane glycoproteins are responsible for calcium-dependent homophilic cell interactions. Recently, many different experimental approaches have been used to untangle the molecular basis of cadherin-mediated adherence. Various models have been suggested, particularly from high-resolution structures. Whilst the adherence mechanism is still under controversy, it is widely accepted that the specificity of the adherent interaction is localized to the N-terminal domain. New biophysical techniques together with biological approaches will allow a better understanding of how cadherins regulate cell-cell adherence. Integrating kinetics properties of cadherin interaction at the single molecule level has led to a greater understanding of cadherin molecular regulations.

Fast dissociation kinetics between individual E-cadherin fragments revealed by flow chamber analysis.

E-cadherin is the predominant adhesion molecule of epithelia. The interaction between extracellular segments of E-cadherin in the membrane of opposing cells is homophilic and calcium dependent. Whereas it is widely accepted that the specificity of the adhesive interaction is localized to the N-terminal domain, the kinetics of the recognition process are unknown. We report the first quantitative data describing the dissociation kinetics of individual E-cadherin interactions. Aggregation assays indicate that the two outermost domains of E-cadherin (E/EC1-2) retain biological activity when chemically immobilized on glass beads. Cadherin fragment trans-interaction was analysed using a flow chamber technique. Transient tethers had first-order kinetics, suggesting a unimolecular interaction. The unstressed lifetime of individual E-cadherin interactions was as brief as 2 s. A fast off rate and the low tensile strength of the E-cadherin bond may be necessary to support the high selectivity and plasticity of epithelial cell interactions.