The cellular vesicle transport machinery delivers E-cadherin and other transmembrane proteins from sites of protein synthesis in the endoplasmic reticulum to specific destinations at the cell surface [10
]. Vesicle trafficking can also remove E-cadherin from the plasma membrane by endocytosis [7
]. The internalization of surface E-cadherin into early endosomes requires the vesicle scission factor dynamin and the Rab5 GTPase. E-cadherin in the early endosomes is then either routed to the lysosome for degradation or to recycling endosomes in a process that requires the Rab11 GTPase. E-cadherin in recycling endosomes is returned to the cell surface by fusion of exocytic vesicles with the plasma membrane ().
Molecular mechanisms of E-cadherin trafficking
Several lines of evidence demonstrate that E-cadherin is dynamically trafficked to and from the plasma membrane, even in the absence of obvious changes in cell shape or cell interactions. When E-cadherin is selectively labeled at the surface of epithelial cells in culture, labeled protein becomes detectable inside the cell, indicating that E-cadherin protein is internalized from the cell surface [11
]. After removal of the surface label and further incubation, labeled E-cadherin is detected on the cell surface, demonstrating that internalized protein is recycled back to the plasma membrane. These results have been extended in vivo
, where imaging experiments in epithelial cells of the Drosophila
pupal notum demonstrate that labeled E-cadherin at the basolateral cell surface is internalized and recycled to the apical membrane () [12
]. Disruption of E-cadherin trafficking results in an irregular distribution of adherens junctions and ultimately leads to a loss of epithelial adhesion [12
]. Together, these results indicate that E-cadherin turnover is required to maintain a dynamic and functional population of E-cadherin at the plasma membrane.
What are the mechanisms that direct E-cadherin trafficking? Two classes of spatial inputs have been shown to affect this process. First, the subcellular distribution of vesicular compartments correlates with sites of active protein turnover in some tissues () [13
], suggesting that the localization of vesicular compartments can direct protein delivery to specific regions at the cell surface. Second, localized molecules can target endocytosis and exocytosis to specific surface domains (). For example, the conserved multi-protein exocyst complex is thought to provide a spatial landmark on the plasma membrane for the fusion of exocytic vesicles from the Golgi and the recycling endosome [21
]. Exocyst proteins mark sites of active membrane and protein delivery, including the growing bud tip in S. cerevisiae
], intercellular junctions in mammalian epithelial cells [25
] and the apical rhabdomere in Drosophila
]. In Drosophila
mutants defective for specific subunits of the exocyst complex, E-cadherin becomes trapped in recycling endosomes in the pupal notum [12
] and embryo [13
], suggesting that the exocyst is required for the delivery of Ecadherin to the cell surface (). E-cadherin's cytoplasmic binding partner β-catenin/Armadillo directly interacts with Sec10 [12
], the exocyst subunit which, together with Sec15, mediates the tethering of exocytic vesicles to the exocyst complex [22
]. This interaction could promote the trafficking of E-cadherin by linking vesicles containing E-cadherin and β-catenin to the exocyst to promote their fusion with the plasma membrane. Alternatively, this interaction could recruit the exocyst to sites of β-catenin localization at the cell surface, targeting membrane trafficking more generally to the region of the adherens junctions.
E-cadherin trafficking in Drosophila
In addition to the spatial regulation of E-cadherin exocytosis, endocytosis is also spatially regulated by the PDZ domain protein Par-6 and the atypical protein kinase C (aPKC), two evolutionarily conserved regulators of cell polarity. Par-6 and aPKC assemble into a complex and are recruited to the apical cell cortex through an interaction between Par-6 and the active GTP-bound form of the Cdc42 GTPase [28
]. Apical aPKC phosphorylates and inactivates basolateral proteins, which contributes to the segregation of the plasma membrane into complementary apical and basolateral domains [29
]. In addition to this role, recent studies demonstrate that the Par-6–aPKC–Cdc42 complex regulates E-cadherin endocytosis in the Drosophila
pupal epithelium () [14
]. Disruption of endocytosis in mutants for shibire
, the Drosophila
dynamin homolog [31
], prevents the release of endocytic vesicles into the cytoplasm. Live tracking of the fate of surface E-cadherin in dynamin mutant cells reveals that E-cadherin is trapped in aborted endocytic intermediates [14
]. Mutants for par-6
, or cdc42
phenocopy these defects in E-cadherin endocytosis, and direct trafficking assays demonstrate that endocytosis originates from the apical junctions [14
]. Endocytosis can also occur at the basal surface in these cells () [12
], indicating that there are other spatial inputs into this process.
Cdc42 is known to bind and activate WASp, which promotes actin nucleation by the Arp2/3 complex [32
], and the Arp2/3 complex has been implicated in endocytosis [33
]. Consistent with these interactions, Wasp and Arp3 are required for E-cadherin endocytosis in the Drosophila
pupal notum [14
]. The Cdc42-interacting protein Cip4 [35
] associates with WASp and dynamin and is also required for E-cadherin endocytosis [15
]. Taken together, these data suggest a model in which Cdc42 recruits dynamin, Wasp and the Arp2/3 complex to adherens junctions to promote E-cadherin endocytosis.
Although these studies identify several key players in E-cadherin trafficking, gaps in our understanding remain. For example, Cdc42 is required for the apical localization of Par-6 and aPKC [14
], raising the question of how Cdc42 activity is spatially regulated to target endocytosis to the apical cell surface. In the early C. elegans
embryo, the Cdc42 GTPase activating protein PAC-1 negatively regulates Cdc42 at contacting cell surfaces, restricting the Par-6–aPKC–Cdc42 complex to the outer, non-contacting cell surfaces [36
]. Another issue is that expression of dominant-negative Cdc42 stimulates endocytosis in the Drosophila
embryo, as judged from an elevated rate of uptake of a fluorescent dye [16
]. This result stands in contrast to the cdc42
mutant phenotype in pupae, and raises the possibility of tissue-specific differences in the role of the Par-6–aPKC–Cdc42 complex in endocytosis.
Mutations in exocyst proteins or the Par-6–aPKC–Cdc42 complex cause a breakdown of adherens junctions, which is at least in part attributable to a disruption of E-cadherin trafficking [12
]. However, it is important to consider that many transmembrane proteins in addition to Ecadherin are transported through the vesicle trafficking machinery, some of which could stabilize adherens junctions indirectly, such as the apical Crumbs protein [13
]. In addition, aPKC phosphorylates and inhibits the endocytic protein Numb [38
], which regulates cadherin trafficking and adhesion in neuroepithelial cells [40
]. Moreover, aPKC also regulates adherens junctions by phosphorylating Crumbs and the basolateral proteins Lethal giant larvae (Lgl) and Par-1 [30
]. Unlike Numb, these substrates have not been implicated in cadherin trafficking, suggesting that the Par-6-aPKC complex may regulate adherens junctions through trafficking-dependent and independent mechanisms.