Rab11 Helps Maintain Apical Crumbs and Adherens Junctions in the Drosophila Embryonic Ectoderm

Background

Tissue morphogenesis and organogenesis require that cells retain stable cell-cell adhesion while changing shape and moving. One mechanism to accommodate this plasticity in cell adhesion involves regulated trafficking of junctional proteins.

Methodology/Principal Findings

Here we explored trafficking of junctional proteins in two well-characterized model epithelia, the Drosophila embryonic ectoderm and amnioserosa. We find that DE-cadherin, the transmembrane protein of adherens junctions, is actively trafficked through putative vesicles, and appears to travel through both Rab5-positive and Rab11-positive structures. We manipulated the functions of Rab11 and Rab5 to examine the effects on junctional stability and morphogenesis. Reducing Rab11 function, either using a dominant negative construct or loss of function alleles, disrupts integrity of the ectoderm and leads to loss of adherens junctions. Strikingly, the apical junctional regulator Crumbs is lost before AJs are destabilized, while the basolateral protein Dlg remains cortical. Altering Rab5 function had less dramatic effects, not disrupting adherens junction integrity but affecting dorsal closure.

Conclusions/Significance

We contrast our results with what others saw when disrupting other trafficking regulators, and when disrupting Rab function in other tissues; together these data suggest distinct mechanisms regulate junctional stability and plasticity in different tissues.     

Drosophila exocyst components Sec5, Sec6, and Sec15 regulate DE-Cadherin trafficking from recycling endosomes to the plasma membrane

The E-Cadherin-catenin complex plays a critical role in epithelial cell-cell adhesion, polarization, and morphogenesis. Here, we have analyzed the mechanism of Drosophila E-Cadherin (DE-Cad) localization. Loss of function of the Drosophila exocyst components sec5, sec6, and sec15 in epithelial cells results in DE-Cad accumulation in an enlarged Rab11 recycling endosomal compartment and inhibits DE-Cad delivery to the membrane. Furthermore, Rab11 and Armadillo interact with the exocyst components Sec15 and Sec10, respectively. Our results support a model whereby the exocyst regulates DE-Cadherin trafficking, from recycling endosomes to sites on the epithelial cell membrane where Armadillo is located.     

Rab11 maintains connections between germline stem cells and niche cells in the Drosophila ovary

All stem cells have the ability to balance their production of self-renewing and differentiating daughter cells. The germline stem cells (GSCs) of the Drosophila ovary maintain such balance through physical attachment to anterior niche cap cells and stereotypic cell division, whereby only one daughter remains attached to the niche. GSCs are attached to cap cells via adherens junctions, which also appear to orient GSC division through capture of the fusome, a germline-specific organizer of mitotic spindles. Here we show that the Rab11 GTPase is required in the ovary to maintain GSC-cap cell junctions and to anchor the fusome to the anterior cortex of the GSC. Thus, rab11-null GSCs detach from niche cap cells, contain displaced fusomes and undergo abnormal cell division, leading to an early arrest of GSC differentiation. Such defects are likely to reflect a role for Rab11 in E-cadherin trafficking as E-cadherin accumulates in Rab11-positive recycling endosomes (REs) and E-cadherin and Armadillo (beta-catenin) are both found in reduced amounts on the surface of rab11-null GSCs. The Rab11-positive REs through which E-cadherin transits are tightly associated with the fusome. We propose that this association polarizes the trafficking by Rab11 of E-cadherin and other cargoes toward the anterior cortex of the GSC, thus simultaneously fortifying GSC-niche junctions, fusome localization and asymmetric cell division. These studies bring into focus the important role of membrane trafficking in stem cell biology.     

A Rab11/Rip11 protein complex regulates apical membrane trafficking via recycling endosomes

Rab11 is a GTPase that regulates endosomal trafficking to apical plasma membrane domains in polarized epithelial cells. We report the identification of a novel Rab11 effector, Rip11. Rip11 is enriched in polarized epithelial cells where, like Rab11, it is localized to subapical recycling endosomes (ARE) and the apical plasma membrane. Using various transport assays, we demonstrate that Rip11 is important for protein trafficking from ARE to the apical plasma membrane. Rip11 is recruited to ARE by binding to Rab11 as well as through a Mg(2+)-dependent interaction of its C2 domain with neutral phospholipids. The association of Rip11 with membranes is regulated by a phosphorylation and dephosphorylation cycle. We propose a model whereby the Rab11/Rip 11 complex regulates vesicle targeting from the ARE.     

Rab11 is required for cell adhesion, maintenance of cell shape and actin-cytoskeleton organization during Drosophila wing development

Intracellular protein trafficking is a key factor in maintaining epithelial cell adhesion and cell shape. Small monomeric Rab GTPases are key players involved in intracellular membrane transport. Rab11, a subfamily of the Ypt/Rab gene family of ubiquitously expressed GTPases, is associated with recycling endosomes, and acts as a master molecule in regulating vesicular trafficking. Wing epithelium of Drosophila has been chosen to address the involvement of Rab11 in trafficking of a cell adhesion molecule, the βPS integrin. Here, we show that Rab11 immunocolocalizes with trans-Golgi network and it is enriched in the centrosomal/recycling endosomal area labeled by γ-Tubulin. Furthermore, Rab11 is required for transcytic and exocytic trafficking of βPS integrin; alterations of Rab11 function by different genetic procedures in wings results in the formation of blisters. We show altered activity of Rab11 affects cell adhesion, cell shape and organization in the actin-cytoskeleton during wing morphogenesis. Finally, using a genetic approach, we demonstrate that Rab11 interacts with the βPS integrin. Collectively, our data suggest that Rab11 regulates cell adhesion, maintenance of cell shape and actin-cytoskeleton organization during Drosophila wing development.     

Drosophila CK1-γ, gilgamesh, controls PCP-mediated morphogenesis through regulation of vesicle trafficking

Cellular morphogenesis, including polarized outgrowth, promotes tissue shape and function. Polarized vesicle trafficking has emerged as a fundamental mechanism by which protein and membrane can be targeted to discrete subcellular domains to promote localized protrusions. Frizzled (Fz)/planar cell polarity (PCP) signaling orchestrates cytoskeletal polarization and drives morphogenetic changes in such contexts as the vertebrate body axis and external Drosophila melanogaster tissues. Although regulation of Fz/PCP signaling via vesicle trafficking has been identified, the interplay between the vesicle trafficking machinery and downstream terminal PCP-directed processes is less established. In this paper, we show that Drosophila CK1-γ/gilgamesh (gish) regulates the PCP-associated process of trichome formation through effects on Rab11-mediated vesicle recycling. Although the core Fz/PCP proteins dictate prehair formation broadly, CK1-γ/gish restricts nucleation to a single site. Moreover, CK1-γ/gish works in parallel with the Fz/PCP effector multiple wing hairs, which restricts prehair formation along the perpendicular axis to Gish. Our findings suggest that polarized Rab11-mediated vesicle trafficking regulated by CK1-γ is required for PCP-directed processes.     

Rab11-FIP2 regulates differentiable steps in transcytosis

Transcytosis through the apical recycling system of polarized cells is regulated by Rab11a and a series of Rab11a-interacting proteins. We have identified a point mutant in Rab11 family interacting protein 2 (Rab11-FIP2) that alters the function of Rab11a-containing trafficking systems. Rab11-FIP2(S229A/R413G) or Rab11-FIP2(R413G) cause the formation of a tubular cisternal structure containing Rab11a and decrease the rate of polymeric IgA transcytosis. The R413G mutation does not alter Rab11-FIP interactions with any known binding partners. Overexpression of Rab11-FIP2(S229A/R413G) alters the localization of a subpopulation of the apical membrane protein GP135. In contrast, Rab11-FIP2(129-512) alters the localization of early endosome protein EEA1. The distributions of both Rab11-FIP2(S229A/R413G) and Rab11-FIP2(129-512) were not dependent on the integrity of the microtubule cytoskeleton. The results indicate that Rab11-FIP2 regulates trafficking at multiple points within the apical recycling system of polarized cells. Rab11a; immunoglobulin A; trafficking; apical recycling; GP135; early endosome; EEA1; Eps15 homology domain     

Rab35 regulates cadherin-mediated adherens junction formation and myoblast fusion

Cadherins are homophilic cell–cell adhesion molecules implicated in many fundamental processes, such as morphogenesis, cell growth, and differentiation. They accumulate at cell–cell contact sites and assemble into large macromolecular complexes named adherens junctions (AJs). Cadherin targeting and function are regulated by various cellular processes, many players of which remain to be uncovered. Here we identify the small GTPase Rab35 as a new regulator of cadherin trafficking and stabilization at cell–cell contacts in C2C12 myoblasts and HeLa cells. We find that Rab35 accumulates at cell–cell contacts in a cadherin-dependent manner. Knockdown of Rab35 or expression of a dominant-negative form of Rab35 impaired N- and M-cadherin recruitment to cell–cell contacts, their stabilization at the plasma membrane, and association with p120 catenin and led to their accumulation in transferrin-, clathrin-, and AP-2–positive intracellular vesicles. We also find that Rab35 function is required for PIP5KIγ accumulation at cell–cell contacts and phosphatidyl inositol 4,5-bisphosphate production, which is involved in cadherin stabilization at contact sites. Finally, we show that Rab35 regulates myoblast fusion, a major cellular process under the control of cadherin-dependent signaling. Taken together, these results reveal that Rab35 regulates cadherin-dependent AJ formation and myoblast fusion.     

Phosphorylation of Rab11-FIP2 regulates polarity in MDCK cells

The Rab11 effector Rab11-family interacting protein 2 (Rab11-FIP2) regulates transcytosis through its interactions with Rab11a and myosin Vb. Previous studies implicated Rab11-FIP2 in the establishment of polarity in Madin-Darby canine kidney (MDCK) cells through phosphorylation of Ser-227 by MARK2. Here we examine the dynamic role of Rab11-FIP2 phosphorylation on MDCK cell polarity. Endogenous Rab11-FIP2 phosphorylated on Ser-227 coalesces on vesicular plaques during the reestablishment of polarity after either monolayer wounding or calcium switch. Whereas expression of the nonphosphorylatable Rab11-FIP2(S227A) elicits a loss in lumen formation in MDCK cell cysts grown in Matrigel, the putative pseudophosphorylated Rab11-FIP2(S227E) mutant induces the formation of cysts with multiple lumens. On permeable filters, Rab11-FIP2(S227E)-expressing cells exhibit alterations in the composition of both the adherens and tight junctions. At the adherens junction, p120 catenin and K-cadherin are retained, whereas the majority of the E-cadherin is lost. Although ZO-1 is retained at the tight junction, occludin is lost and the claudin composition is altered. Of interest, the effects of Rab11-FIP2 on cellular polarity did not involve myosin Vb or Rab11a. These results indicate that Ser-227 phosphorylation of Rab11-FIP2 regulates the composition of both adherens and tight junctions and is intimately involved in the regulation of polarity in epithelial cells.     

Endosomal trafficking of the G protein-coupled receptor somatostatin receptor 3

Intracellular trafficking of G protein-coupled receptors (GPCRs) regulates their surface availability and determines cellular response to agonists. Rab GTPases regulate membrane trafficking and identifying Rab networks controlling GPCR trafficking is essential for understanding GPCR signaling. We used real time imaging to show that somatostatin receptor 3 (SSTR3) traffics through Rab4-, Rab21-, and Rab11-containing endosomes, but largely bypasses Rab5 and Rab7 endosomes. We show that SSTR3 rapidly traffics through Rab4 endosomes but moves slower through Rab21 and Rab11 endosomes. SSTR3 passage through each endosomal compartment is regulated by the cognate Rab since expression of the inactive Rab4/S22N, Rab21/T33N, and Rab11/S25N inhibits SSTR3 trafficking. Thus, Rab4, Rab21, and Rab11 may represent therapeutic targets to modulate surface availability of SSTR3 for agonist binding. Our novel finding that Rab21 regulates SSTR3 trafficking suggests that Rab21 may play a role in trafficking of other GPCRs.     

Myosin Vb is required for trafficking of the cystic fibrosis transmembrane conductance regulator in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretion across fluid-transporting epithelia is regulated, in part, by modulating the number of CFTR Cl(-) channels in the plasma membrane by adjusting CFTR endocytosis and recycling. However, the mechanisms that regulate CFTR recycling in airway epithelial cells remain unknown, at least in part, because the recycling itineraries of CFTR in these cells are incompletely understood. In a previous study, we demonstrated that CFTR undergoes trafficking in Rab11a-specific apical recycling endosomes in human airway epithelial cells. Myosin Vb is a plus-end-directed, actin-based mechanoenzyme that facilitates protein trafficking in Rab11a-specific recycling vesicles in several cell model systems. There are no published studies examining the role of myosin Vb in airway epithelial cells. Thus, the goal of this study was to determine whether myosin Vb facilitates CFTR recycling in polarized human airway epithelial cells. Endogenous CFTR formed a complex with endogenous myosin Vb and Rab11a. Silencing myosin Vb by RNA-mediated interference decreased the expression of wild-type CFTR and DeltaF508-CFTR in the apical membrane and decreased CFTR-mediated Cl(-) secretion across polarized human airway epithelial cells. A recombinant tail domain fragment of myosin Vb attenuated the plasma membrane expression of CFTR by arresting CFTR recycling. The dominant-negative effect was dependent on the ability of the myosin Vb tail fragment to interact with Rab11a. Taken together, these data indicate that myosin Vb is required for CFTR recycling in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells.     

Regulation of apical constriction via microtubule- and Rab11-dependent apical transport during tissue invagination

The formation of an epithelial tube is a fundamental process for organogenesis. During Drosophila embryonic salivary gland (SG) invagination, Folded gastrulation (Fog)-dependent Rho-associated kinase (Rok) promotes contractile apical myosin formation to drive apical constriction. Microtubules (MTs) are also crucial for this process and are required for forming and maintaining apicomedial myosin. However, the underlying mechanism that coordinates actomyosin and MT networks still remains elusive. Here, we show that MT-dependent intracellular trafficking regulates apical constriction during SG invagination. Key components involved in protein trafficking, such as Rab11 and Nuclear fallout (Nuf), are apically enriched near the SG invagination pit in a MT-dependent manner. Disruption of the MT networks or knockdown of Rab11 impairs apicomedial myosin formation and apical constriction. We show that MTs and Rab11 are required for apical enrichment of the Fog ligand and the continuous distribution of the apical determinant protein Crumbs (Crb) and the key adherens junction protein E-Cadherin (E-Cad) along junctions. Targeted knockdown of crb or E-Cad in the SG disrupts apical myosin networks and results in apical constriction defects. Our data suggest a role of MT- and Rab11-dependent intracellular trafficking in regulating actomyosin networks and cell junctions to coordinate cell behaviors during tubular organ formation.     

Recycling and resensitization of the neurokinin 1 receptor. Influence of agonist concentration and Rab GTPases

Substance P (SP) induces endocytosis and recycling of the neurokinin 1 receptor (NK1R) in endothelial cells and spinal neurons at sites of inflammation and pain, and it is thus important to understand the mechanism and function of receptor trafficking. We investigated how the SP concentration affects NK1R trafficking and determined the role of Rab GTPases in trafficking. NK1R trafficking was markedly influenced by the SP concentration. High SP (10 nM) induced translocation of the NK1R and beta-arrestin 1 to perinuclear sorting endosomes containing Rab5a, where NK1R remained for >60 min. Low SP (1 nM) induced translocation of the NK1R to early endosomes located immediately beneath the plasma membrane that also contained Rab5a and beta-arrestin 1, followed by rapid recycling of the NK1R. Overexpression of Rab5a promoted NK1R translocation to perinuclear sorting endosomes, whereas the GTP binding-deficient mutant Rab5aS34N caused retention of the NK1R in superficial early endosomes. NK1R translocated from superficial early endosomes to recycling endosomes containing Rab4a and Rab11a, and Rab11aS25N inhibited NK1R recycling. Rapid NK1R recycling coincided with resensitization of SP-induced Ca2+ mobilization and with the return of surface SP binding sites. Resensitization was minimally affected by inhibition of vacuolar H(+)-ATPase and phosphatases but was markedly suppressed by disruption of Rab4a and Rab11a. Thus, whereas beta-arrestins mediate NK1R endocytosis, Rab5a regulates translocation between early and sorting endosomes, and Rab4a and Rab11a regulate trafficking through recycling endosomes. We have thus identified a new function of Rab5a as a control protein for directing concentration-dependent trafficking of the NK1R into different intracellular compartments and obtained evidence that Rab4a and Rab11a contribute to G-protein-coupled receptor recycling from early endosomes.     

Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane

The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane.     

Rho1 regulates adherens junction remodeling by promoting recycling endosome formation through activation of myosin II

Once adherens junctions (AJs) are formed between polarized epithelial cells they must be maintained because AJs are constantly remodeled in dynamic epithelia. AJ maintenance involves endocytosis and subsequent recycling of E-cadherin to a precise location along the basolateral membrane. In the Drosophila pupal eye epithelium, Rho1 GTPase regulates AJ remodeling through Drosophila E-cadherin (DE-cadherin) endocytosis by limiting Cdc42/Par6/aPKC complex activity. We demonstrate that Rho1 also influences AJ remodeling by regulating the formation of DE-cadherin–containing, Rab11-positive recycling endosomes in Drosophila postmitotic pupal eye epithelia. This effect of Rho1 is mediated through Rok-dependent, but not MLCK-dependent, stimulation of myosin II activity yet independent of its effects upon actin remodeling. Both Rho1 and pMLC localize on endosomal vesicles, suggesting that Rho1 might regulate the formation of recycling endosomes through localized myosin II activation. This work identifies spatially distinct functions for Rho1 in the regulation of DE-cadherin–containing vesicular trafficking during AJ remodeling in live epithelia.     

Rab11 family interacting protein 2 associates with Myosin Vb and regulates plasma membrane recycling

Plasma membrane recycling is an important process necessary for maintaining membrane composition. The motor protein myosin Vb regulates plasma membrane recycling through its association with Rab11a. Overexpression of the tail of myosin Vb disrupts trafficking out of plasma membrane recycling systems and leads to the accumulation of Rab11a in both polarized and non-polarized cells. We have investigated the association of Rab11 family interacting protein 2 (Rab11-FIP2) with myosin Vb as an adapter protein between Rab11a and myosin Vb. Immunofluorescence studies indicated a colocalization of endogenous Rab11-FIP2 with green fluorescent protein-myosin Vb tail overexpressed in Madin-Darby canine kidney (MDCK) cells. Yeast two hybrid assays showed that amino acids 129-356 of Rab11-FIP2 were important for binding to myosin Vb tail. In vitro association assays and co-transfection experiments in both MDCK and HeLa cells confirmed this result but further refined the binding site to amino acids 129-290 of Rab11-FIP2. Like myosin Vb, functional studies indicated that Rab11-FIP2 is also important for normal plasma membrane recycling. Green fluorescent protein-Rab11-FIP2 (129-512), which lacks its amino-terminal C2 domain, functioned as a dominant negative acting truncation that caused accumulation of Rab11a and disrupted IgA trafficking in MDCK cells and transferrin trafficking in HeLa cells. The ternary association of myosin Vb and Rab11-FIP2 with Rab11a suggests that a multimeric protein complex is involved in vesicle trafficking through plasma membrane recycling systems.     

Rab13 mediates the continuous endocytic recycling of occludin to the cell surface

During epithelial morphogenesis, adherens junctions (AJs) and tight junctions (TJs) undergo dynamic reorganization, whereas epithelial polarity is transiently lost and reestablished. Although ARF6-mediated endocytic recycling of E-cadherin has been characterized and implicated in the rapid remodeling of AJs, the molecular basis for the dynamic rearrangement of TJs remains elusive. Occludin and claudins are integral membrane proteins comprising TJ strands and are thought to be responsible for establishing and maintaining epithelial polarity. Here we investigated the intracellular transport of occludin and claudins to and from the cell surface. Using cell surface biotinylation and immunofluorescence, we found that a pool of occludin was continuously endocytosed and recycled back to the cell surface in both fibroblastic baby hamster kidney cells and epithelial MTD-1A cells. Biochemical endocytosis and recycling assays revealed that a Rab13 dominant active mutant (Rab13 Q67L) inhibited the postendocytic recycling of occludin, but not that of transferrin receptor and polymeric immunoglobulin receptor in MTD-1A cells. Double immunolabelings showed that a fraction of endocytosed occludin was colocalized with Rab13 in MTD-1A cells. These results suggest that Rab13 specifically mediates the continuous endocytic recycling of occludin to the cell surface in both fibroblastic and epithelial cells.     

Making and breaking contacts: the cellular biology of cadherin regulation

Cadherin-mediated cell-cell interactions are dynamic processes, and cadherin function is tightly regulated in response to cellular context and signaling. Ultimately, cadherin regulation is likely to reflect the interplay between a range of fundamental cellular processes, including surface organization of receptors, cytoskeletal organization and cell trafficking, that are coordinated by signaling events. In this review we focus on recent advances in understanding how interplay with membrane trafficking and other cell-cell junctions can control cadherin function. The endocytosis of cadherins, and their post-internalization fate, influences surface expression and metabolic stability of these adhesion receptors. Similarly, at the surface, components of tight junctions provide a mode of cross-talk that regulates assembly of adherens junctions.     

Dennd3 Functions as a Guanine Nucleotide Exchange Factor for Small GTPase Rab12 in Mouse Embryonic Fibroblasts

Small GTPase Rab12 regulates mTORC1 (mammalian target of rapamycin complex 1) activity and autophagy through controlling PAT4 (proton/amino acid transporter 4) trafficking from recycling endosomes to lysosomes, where PAT4 is degraded. However, the precise regulatory mechanism of the Rab12-mediated membrane trafficking pathway remained to be determined because a physiological Rab12-GEF (guanine nucleotide exchange factor) had yet to be identified. In this study we performed functional analyses of Dennd3, which has recently been shown to possess a GEF activity toward Rab12 in vitro. The results showed that knockdown of Dennd3 in mouse embryonic fibroblast cells caused an increase in the amount of PAT4 protein, the same as Rab12 knockdown did, and knockdown of Dennd3 and overexpression of Dennd3 were found to result in an increase and a decrease, respectively, in the intracellular amino acid concentration. Dennd3 overexpression was also found to reduce mTORC1 activity and promoted autophagy in a Rab12-dependent manner. Unexpectedly, however, Dennd3 knockdown had no effect on mTORC1 activity or autophagy despite increasing the intracellular amino acid concentration. Further study showed that Dennd3 knockdown reduced Akt activity, and the reduction in Akt activity is likely to have canceled out amino acid-induced mTORC1 activation through PAT4. These findings indicated that Dennd3 not only functions as a Rab12-GEF but also modulates Akt signaling in mouse embryonic fibroblast cells.     

Rab14 and its exchange factor FAM116 link endocytic recycling and adherens junction stability in migrating cells

Rab GTPases define the vesicle trafficking pathways underpinning cell polarization and migration. Here, we find that Rab4, Rab11, and Rab14 and the candidate Rab GDP-GTP exchange factors (GEFs) FAM116A and AVL9 are required for cell migration. Rab14 and its GEF FAM116A localize to and act on an intermediate compartment of the transferrin-recycling pathway prior to Rab11 and after Rab5 and Rab4. This Rab14 intermediate recycling compartment has specific functions in migrating cells discrete from early and recycling endosomes. Rab14-depleted cells show increased N-cadherin levels at junctional complexes and cannot resolve cell-cell junctions. This is due to decreased shedding of cell-surface N-cadherin by the ADAM family protease ADAM10/Kuzbanian. In FAM116A- and Rab14-depleted cells, ADAM10 accumulates in a transferrin-positive endocytic compartment, and the cell-surface level of ADAM10 is correspondingly reduced. FAM116 and Rab14 therefore define an endocytic recycling pathway needed for ADAM protease trafficking and regulation of cell-cell junctions.