Clathrin and AP1 are required for apical sorting of glycosyl phosphatidyl inositol-anchored proteins in biosynthetic and recycling routes in Madin-Darby canine kidney cells.

Recently, studies in animal models demonstrate potential roles for clathrin and AP1 in apical protein sorting in epithelial tissue. However, the precise functions of these proteins in apical protein transport remain unclear. Here, we reveal mistargeting of endogenous glycosyl phosphatidyl inositol-anchored proteins (GPI-APs) and soluble secretory proteins in Madin-Darby canine kidney (MDCK) cells upon clathrin heavy chain or AP1 subunit knockdown (KD). Using a novel directional endocytosis and recycling assay, we found that these KD cells are not only affected for apical sorting of GPI-APs in biosynthetic pathway but also for their apical recycling and basal-to-apical transcytosis routes. The apical distribution of the t-SNARE syntaxin 3, which is known to be responsible for selective targeting of various apical-destined cargo proteins in both biosynthetic and endocytic routes, is compromised suggesting a molecular explanation for the phenotype in KD cells. Our results demonstrate the importance of biosynthetic and endocytic routes for establishment and maintenance of apical localization of GPI-APs in polarized MDCK cells.

Localization of NPFxD motif-containing proteins in Aspergillus nidulans.

During growth, filamentous fungi produce polarized cells called hyphae. It is generally presumed that polarization of hyphae is dependent upon secretion through the Spitzenkörper, as well as a mechanism called apical recycling, which maintains a balance between the tightly coupled processes of endocytosis and exocytosis. Endocytosis predominates in an annular domain called the sub-apical endocytic collar, which is located in the region of plasma membrane 1-5 µm distal to the Spitzenkörper. It has previously been proposed that one function of the sub-apical endocytic collar is to maintain the apical localization of polarization proteins. These proteins mark areas of polarization at the apices of hyphae. However, as hyphae grow, these proteins are displaced along the membrane and some must then be removed at the sub-apical endocytic collar in order to maintain the hyphoid shape. While endocytosis is fairly well characterized in yeast, comparatively little is known about the process in filamentous fungi. Here, a bioinformatics approach was utilized to identify 39 Aspergillus nidulans proteins that are predicted to be cargo of endocytosis based on the presence of an NPFxD peptide motif. This motif is a necessary endocytic signal sequence first established in Saccharomyces cerevisiae, where it marks proteins for endocytosis through an interaction with the adapter protein Sla1p. It is hypothesized that some proteins that contain this NPFxD peptide sequence in A. nidulans will be potential targets for endocytosis, and therefore will localize either to the endocytic collar or to more proximal polarized regions of the cell, e.g. the apical dome or the Spitzenkörper. To test this, a subset of the motif-containing proteins in A. nidulans was tagged with GFP and the dynamic localization was evaluated. The documented localization patterns support the hypothesis that the motif marks proteins for localization to the polarized cell apex in growing hyphae.

Myosin 5b loss of function leads to defects in polarized signaling: implication for microvillus inclusion disease pathogenesis and treatment.

Microvillus inclusion disease (MVID) is an autosomal recessive condition resulting in intractable secretory diarrhea in newborns due to loss-of-function mutations in myosin Vb (Myo5b). Previous work suggested that the apical recycling endosomal (ARE) compartment is the primary location for phosphoinositide-dependent protein kinase 1 (PDK1) signaling. Because the ARE is disrupted in MVID, we tested the hypothesis that polarized signaling is affected by Myo5b dysfunction. Subcellular distribution of PDK1 was analyzed in human enterocytes from MVID/control patients by immunocytochemistry. Using Myo5b knockdown (kd) in Caco-2BBe cells, we studied phosphorylated kinases downstream of PDK1, electrophysiological parameters, and net water flux. PDK1 was aberrantly localized in human MVID enterocytes and Myo5b-deficient Caco-2BBe cells. Two PDK1 target kinases were differentially affected: phosphorylated atypical protein kinase C (aPKC) increased fivefold and phosohoprotein kinase B slightly decreased compared with control. PDK1 redistributed to a soluble (cytosolic) fraction and copurified with basolateral endosomes in Myo5b kd. Myo5b kd cells showed a decrease in net water absorption that could be reverted with PDK1 inhibitors. We conclude that, in addition to altered apical expression of ion transporters, depolarization of PDK1 in MVID enterocytes may lead to aberrant activation of downstream kinases such as aPKC. The findings in this work suggest that PDK1-dependent signaling may provide a therapeutic target for treating MVID.

Gb3-binding lectins as potential carriers for transcellular drug delivery.

OBJECTIVES: Epithelial cell layers as well as endothelia forming the blood-brain barrier can drastically reduce the efficiency of drug targeting. Our goal was to investigate lectins recognizing the glycosphingolipid globotriaosylceramide (Gb3) for their potential as carriers for transcytotic drug delivery. METHODS: We utilized an in vitro model based on Madin-Darby canine kidney cells transfected with Gb3 synthase to characterize transcytosis of the Gb3-binding lectins LecA from Pseudomonas aeruginosa and the B-subunit of Shiga toxin (StxB). RESULTS: Both lectins were rapidly transcytosed from the apical to the basolateral plasma membrane and vice versa. Whereas StxB proceeded on retrograde and transcytotic routes, LecA avoided retrograde transport. This differential trafficking could be explained by our observation that LecA and StxB segregated into different domains during endocytosis. Furthermore, inhibiting the small GTPase Rab11a, which organizes trafficking through apical recycling endosomes, blocked basolateral to apical transcytosis of both lectins. CONCLUSIONS: Gb3-binding lectins are promising candidates for transcytotic drug delivery. Our findings highlight that LecA and StxB, which both bind Gb3 but exhibit dissimilar valence and molecular structures of their carbohydrate binding sites and can take divergent intracellular trafficking routes. This opens up the possibility of developing tailor-made glycosphingolipid-binding carrier lectins, which take optimized trafficking pathways.