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After mammalian cell invasion, the parasite Toxoplasma multiplies in a self-made membrane-bound compartment, the parasitophorous vacuole (PV). We previously showed that Toxoplasma interacts with many host cell organelles, especially from recycling pathways, and sequestrates Rab11A and Rab11B vesicles into the PV. Here, we examine the specificity of host Rab11 vesicle interaction with the PV by focusing on the recruitment of subpopulations of Rab11 vesicles characterized by different effectors, for example, Rab11-family interacting roteins (FIPs) or Arf6. Our quantitative microscopic analysis illustrates the presence of intra-PV vesicles with FIPs from class I (FIP1C, FIP2, FIP5) and class II (FIP3, FIP4) but to various degrees. The intra-PV delivery of vesicles with class I, but not class II, FIPs is dependent on Rab11 binding. Cell depletion of Rab11A results in a significant decrease in intra-PV FIP5, but not FIP3 vesicles. Class II FIPs also bind to Arf6, and we observe vesicles associated with FIP3-Rab11A or FIP3-Arf6 complexes concomitantly within the PV. Abolishing FIP3 binding to both Rab11 and Arf6 reduces the number of intra-PV FIP3 vesicles. These data point to a selective process of mammalian Rab11 vesicle recognition and scavenging mediated by Toxoplasma , suggesting that specific parasite PV proteins may be involved in these processes.

Macropinocytosis is an actin-driven form of clathrin-independent endocytosis that generates an enlarged structure, the macropinosome. Although many studies focus on signaling molecules and phosphoinositides involved in initiating macropinocytosis, the commitment to forming a macropinosome and the handling of that membrane have not been studied in detail. Here we show in HT1080 cells, a human fibrosarcoma cell line, a requirement for microtubules, dynein, the JIP3 microtubule motor scaffold protein, and Arf6, a JIP3 interacting protein, for the formation and inward movement of the macropinosome. While actin and myosin II also play critical roles in the formation of ruffling membrane, microtubules provide an important tract for initiation, sealing, and transport of the macropinosome through the actin- and myosin-rich lamellar region.

Hemidesmosomes (HDs) are epithelial-specific cell-matrix adhesions that stably anchor the intracellular keratin network to the extracellular matrix. Although their main role is to protect the epithelial sheet from external mechanical strain, how HDs respond to mechanical stress remains poorly understood. Here we identify a pathway essential for HD remodeling and outline its role with respect to α6β4 integrin recycling. We find that α6β4 integrin chains localize to the plasma membrane, caveolae, and ADP-ribosylation factor-6+ (Arf6+) endocytic compartments. Based on fluorescence recovery after photobleaching and endocytosis assays, integrin recycling between both sites requires the small GTPase Arf6 but neither caveolin1 (Cav1) nor Cavin1. Strikingly, when keratinocytes are stretched or hypo-osmotically shocked, α6β4 integrin accumulates at cell edges, whereas Cav1 disappears from it. This process, which is isotropic relative to the orientation of stretch, depends on Arf6, Cav1, and Cavin1. We propose that mechanically induced HD growth involves the isotropic flattening of caveolae (known for their mechanical buffering role) associated with integrin diffusion and turnover., (© 2018 Osmani et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Although TrkA postendocytic sorting significantly influences neuronal cell survival and differentiation, the molecular mechanism underlying TrkA receptor sorting in the recycling or degradation pathways remains poorly understood. Here we demonstrate that Golgi-localized, γ adaptin-ear-containing ADP ribosylation factor-binding protein 3 (GGA3) interacts directly with the TrkA cytoplasmic tail through an internal DXXLL motif and mediates the functional recycling of TrkA to the plasma membrane. We find that GGA3 depletion by siRNA delays TrkA recycling, accelerates TrkA degradation, attenuates sustained NGF-induced Akt activation, and reduces cell survival. We also show that GGA3's effect on TrkA recycling is dependent on the activation of Arf6. This work identifies GGA3 as a key player in a novel DXXLL-mediated endosomal sorting machinery that targets TrkA to the plasma membrane, where it prolongs the activation of Akt signaling and survival responses., (© 2015 Li et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Oligodendrocyte precursor cells differentiate to produce myelin sheaths that insulate axons to ensure fast propagation of action potentials. Many aspects of differentiation are regulated by multiple extracellular signals. However, their intracellular signalings remain elusive. We show that Rab35 and its effector, ACAP2, a GTPase-activating protein that switches off Arf6 activity, negatively regulate oligodendrocyte morphological differentiation. Knockdown of Rab35 or ACAP2 with their respective small interfering RNAs promotes differentiation. As differentiation initiates, the activities of Rab35 and ACAP2 are down-regulated. The activity of Arf6, in contrast, is up-regulated. Arf6 knockdown inhibits differentiation, indicating that Rab35 and ACAP2 negatively regulate differentiation by down-regulating Arf6. Importantly, as differentiation proceeds, the activity of cytohesin-2, a guanine nucleotide exchange factor that switches on Arf6 activity, is up-regulated. Pharmacological inhibition of cytohesin-2 inhibits differentiation, suggesting that cytohesin-2 promotes differentiation by activating Arf6. Furthermore, using oligodendrocyte-neuronal cocultures, we find that knockdown of Rab35 or ACAP2 promotes myelination, whereas inhibition of cytohesin-2 or knockdown of Arf6 inhibits myelination. Thus Rab35/ACAP2 and cytohesin-2 antagonistically control oligodendrocyte differentiation and myelination through Arf6 regulation, presenting a unique small GTPase on/off switching mechanism.

Epithelial cysts comprise the structural units of the glandular epithelium. Although glandular inversion in epithelial tumors is thought to be a potential mechanism for the establishment of metastatic disease, little is known about the morphogenic cues and signaling pathways that govern glandular polarity and organization. Using organotypic cultures of Madin-Darby canine kidney cells in reconstituted basement membrane, we show that cellular depletion of the small GTP-binding protein ARF6 promotes the formation of inverted cysts, wherein the apical cell membrane faces the cyst exterior, and the basal domain faces the central lumen, while individual cell polarity is maintained. These cysts are also defective in interactions with laminin at the cyst-matrix interface. This inversion of glandular orientation is accompanied by Rac1 inactivation during early cystogenesis, and temporal activation of Rac1 is sufficient to recover the normal cyst phenotype. In an unnatural collagen I microenvironment, ARF6-depleted, inverted epithelial cysts exhibit some loss of cell polarity, a marked increase in Rho activation and Rac1 inactivation, and striking rearrangement of the surrounding collagen I matrix. These studies demonstrate the importance of ARF6 as a critical determinant of glandular orientation and the matrix environment in dictating structural organization of epithelial cysts.

Cell function requires the integration of cytoskeletal organization and membrane trafficking. Small GTP-binding proteins are key regulators of these processes. We find that EPI64, an apical microvillar protein with a Tre-2/Bub2/Cdc16 (TBC) domain that stabilizes active Arf6 and has RabGAP activity, regulates Arf6-dependent membrane trafficking. Expression of EPI64 in HeLa cells induces the accumulation of actin-coated vacuoles, a distinctive phenotype seen in cells expressing constitutively active Arf6. Expression of EPI64 with defective RabGAP activity does not induce vacuole formation. Coexpression of Rab8a suppresses the vacuole phenotype induced by EPI64, and EPI64 expression lowers the level of Rab8-GTP in cells, strongly suggesting that EPI64 has GAP activity toward Rab8a. JFC1, an effector for Rab8a, colocalizes with and binds directly to a C-terminal region of EPI64. Together this region and the N-terminal TBC domain of EPI64 are required for the accumulation of vacuoles. Through analysis of mutants that uncouple JFC1 from either EPI64 or from Rab8-GTP, our data suggest a model in which EPI64 binds JFC1 to recruit Rab8a-GTP for deactivation by the RabGAP activity of EPI64. We propose that EPI64 regulates membrane trafficking both by stabilizing Arf6-GTP and by inhibiting the recycling of membrane through the tubular endosome by decreasing Rab8a-GTP levels.

As the initial barrier to viral entry, the plasma membrane along with the membrane trafficking machinery and cytoskeleton are of fundamental importance in the viral cycle. However, little is known about the contribution of plasma membrane dynamics during early human immunodeficiency virus type 1 (HIV-1) infection. Considering that ADP ribosylation factor 6 (Arf6) regulates cellular invasion via several microorganisms by coordinating membrane trafficking, our aim was to study the function of Arf6-mediated membrane dynamics on HIV-1 entry and infection of T lymphocytes. We observed that an alteration of the Arf6-guanosine 5'-diphosphate/guanosine 5'-triphosphate (GTP/GDP) cycle, by GDP-bound or GTP-bound inactive mutants or by specific Arf6 silencing, inhibited HIV-1 envelope-induced membrane fusion, entry, and infection of T lymphocytes and permissive cells, regardless of viral tropism. Furthermore, cell-to-cell HIV-1 transmission of primary human CD4(+) T lymphocytes was inhibited by Arf6 knockdown. Total internal reflection fluorescence microscopy showed that Arf6 mutants provoked the accumulation of phosphatidylinositol-(4,5)-biphosphate-associated structures on the plasma membrane of permissive cells, without affecting CD4-viral attachment but impeding CD4-dependent HIV-1 entry. Arf6 silencing or its mutants did not affect fusion, entry, and infection of vesicular stomatitis virus G-pseudotyped viruses or ligand-induced CXCR4 or CCR5 endocytosis, both clathrin-dependent processes. Therefore we propose that efficient early HIV-1 infection of CD4(+) T lymphocytes requires Arf6-coordinated plasma membrane dynamics that promote viral fusion and entry.

Myoblast fusion is an essential step during myoblast differentiation that remains poorly understood. M-cadherin-dependent pathways that signal through Rac1 GTPase activation via the Rho-guanine nucleotide exchange factor (GEF) Trio are important for myoblast fusion. The ADP-ribosylation factor (ARF)6 GTPase has been shown to bind to Trio and to regulate Rac1 activity. Moreover, Loner/GEP(100)/BRAG2, a GEF of ARF6, has been involved in mammalian and Drosophila myoblast fusion, but the specific role of ARF6 has been not fully analyzed. Here, we show that ARF6 activity is increased at the time of myoblast fusion and is required for its implementation in mouse C2C12 myoblasts. Specifically, at the onset of myoblast fusion, ARF6 is associated with the multiproteic complex that contains M-cadherin, Trio, and Rac1 and accumulates at sites of myoblast fusion. ARF6 silencing inhibits the association of Trio and Rac1 with M-cadherin. Moreover, we demonstrate that ARF6 regulates myoblast fusion through phospholipase D (PLD) activation and phosphatidylinositol 4,5-bis-phosphate production. Together, these data indicate that ARF6 is a critical regulator of C2C12 myoblast fusion and participates in the regulation of PLD activities that trigger both phospholipids production and actin cytoskeleton reorganization at fusion sites.

Caveolin-1 and caveolae are often lost in cancer. We found that levels of caveolin-1 and polymerase I and transcript release factor (PTRF)/cavin-1 correlated closely in a panel of cancer and normal cells. Caveolin-1 reexpression in cancer cells lacking both proteins induced formation of long membrane tubules rarely seen in normal cells. PTRF/cavin-1 inhibited tubule formation when coexpressed with caveolin-1 in these cells, whereas suppression of PTRF/cavin-1 expression in cells that normally expressed both genes stimulated tubule formation by endogenous caveolin-1. Caveolin-1 tubules shared several features with previously described Rab8 tubules. Coexpressed Rab8 and caveolin-1 labeled the same tubules (as did EHD proteins), and synergized to promote tubule formation, whereas a dominant-interfering Rab8 mutant inhibited caveolin-1 tubule formation. Both overexpression and inhibition of dynamin-2 reduced the abundance of caveolin-1 tubules. Caveolin-1 reexpression in SK-BR-3 breast cancer cells also induced formation of short membrane tubules close to cortical actin filaments, which required actin filaments but not microtubules. Actomyosin-induced tension destabilized both long and short tubules; they often snapped and resolved to small vesicles. Actin filament depolymerization or myosin II inhibition reduced tension and stabilized tubules. These data demonstrate a new function for PTRF/cavin-1, a new functional interaction between caveolin-1 and Rab8 and that actomyosin interactions can induce tension on caveolin-1-containing membranes.

Tumor development in glandular tissues is associated with structural alterations in the hollow ducts and spherical structures that comprise such tissues. We describe a signaling axis involving sustained activation of the GTP-binding protein, ARF6, that provokes dramatic changes in the organization of epithelial cysts, reminiscent of tumorigenic glandular phenotypes. In reconstituted basement membrane cultures of renal epithelial cysts, enhanced ARF6 activation induces the formation of cell-filled glandular structures with multiple lumens and disassembled cadherin-based cell-cell contacts. All of these alterations are accompanied by growth factor receptor internalization into signaling endosomes and reversed by blocking ARF6 activation or receptor endocytosis. Receptor localization in signaling endosomes results in hyperactive extracellular signal-regulated kinase signaling leading to Bcl-2 stabilization and aberrant cysts. Similarly, formation of hyperproliferative and disorganized mammary acini induced by chronic stimulation of colony-stimulating factor 1 receptor is coupled to endogenous ARF6 activation and constitutive receptor internalization and is reversed by ARF6 inhibition. These findings identify a previously unrecognized link between ARF6-regulated receptor internalization and events that drive dramatic alterations in cyst morphogenesis providing new mechanistic insight into the molecular processes that can promote epithelial glandular disruption.

ADP-ribosylation factor (Arf) 6 activity is crucially involved in the regulation of E-cadherin-based cell-cell adhesions. Erythropoietin-producing hepatocellular carcinoma (Eph)-family receptors recognize ligands, namely, ephrins, anchored to the membrane of apposing cells, and they mediate cell-cell contact-dependent events. Here, we found that Arf6 activity is down-regulated in Madin-Darby canine kidney cells, which is dependent on cell density and calcium ion concentration, and we provide evidence of a novel signaling pathway by which ligand-activated EphA2 suppresses Arf6 activity. This EphA2-mediated suppression of Arf6 activity was linked to the induction of cell compaction and polarization, but it was independent of the down-regulation of extracellular signal-regulated kinase 1/2 kinase activity. We show that G protein-coupled receptor kinase-interacting protein (Git) 1 and noncatalytic region of tyrosine kinase (Nck) 1 are involved in this pathway, in which ligand-activated EphA2, via its phosphorylated Tyr594, binds to the Src homology 2 domain of Nck1, and then via its Src homology 3 domain binds to the synaptic localizing domain of Git1 to suppress Arf6 activity. We propose a positive feedback loop in which E-cadherin-based cell-cell contacts enhance EphA-ephrinA signaling, which in turn down-regulates Arf6 activity to enhance E-cadherin-based cell-cell contacts as well as the apical-basal polarization of epithelial cells.

The generation of novel genes and proteins throughout evolution has been proposed to occur as a result of whole genome and gene duplications, exon shuffling, and retrotransposition events. The analysis of such genes might thus shed light into the functional complexity associated with highly evolved species. One such case is represented by TBC1D3, a primate-specific gene, harboring a TBC domain. Because TBC domains encode Rab-specific GAP activities, TBC-containing proteins are predicted to play a major role in endocytosis and intracellular traffic. Here, we show that the TBC1D3 gene originated late in evolution, likely through a duplication of the RNTRE locus, and underwent gene amplification during primate speciation. Despite possessing a TBC domain, TBC1D3 is apparently devoid of Rab-GAP activity. However, TBC1D3 regulates the optimal rate of epidermal growth factor-mediated macropinocytosis by participating in a novel pathway involving ARF6 and RAB5. In addition, TBC1D3 binds and colocalize to GGA3, an ARF6-effector, in an ARF6-dependent manner, and synergize with it in promoting macropinocytosis, suggesting that the two proteins act together in this process. Accordingly, GGA3 siRNA-mediated ablation impaired TBC1D3-induced macropinocytosis. We thus uncover a novel signaling pathway that appeared after primate speciation. Within this pathway, a TBC1D3:GGA3 complex contributes to optimal propagation of signals, ultimately facilitating the macropinocytic process.

Trafficking of H-Ras was examined to determine whether it can enter cells through clathrin-independent endocytosis (CIE). H-Ras colocalized with the CIE cargo protein, class I major histocompatibility complex, and it was sequestered in vacuoles that formed upon expression of an active mutant of Arf6, Q67L. Activation of Ras, either through epidermal growth factor stimulation or the expression of an active mutant of Ras, G12V, induced plasma membrane ruffling and macropinocytosis, a stimulated form of CIE. Live imaging of cells expressing H-RasG12V and fluorescent protein chimeras with pleckstrin homology domains that recognize specific phosphoinositides showed that incoming macropinosomes contained phosphatidylinositol 4,5-bisphosphate (PIP(2)) and phosphatiylinositol 3,4,5-trisphosphate (PIP(3)). PIP(2) loss from the macropinosome was followed by the recruitment of Rab5, a downstream target of Ras, and then PIP(3) loss. Our studies support a model whereby Ras can signal on macropinosomes that pass through three distinct stages: PIP(2)/PIP(3), PIP(3)/Rab5, and Rab5. Vacuoles that form in cells expressing Arf6Q67L trap Ras signaling in the first stage, recruiting the active form of the Ras effectors extracellular signal-regulated kinase and protein kinase B (Akt) but not Rab5. Arf6 stimulation of macropinocytosis also involves passage through the distinct lipid phases, but recruitment of Akt is not observed.

The small GTP-binding protein Arf6 regulates membrane remodeling at cell peripheries and plays crucial roles in higher orders of cellular functions including tumor invasion. Here we show that Fbx8, an F-box protein bearing the Sec7 domain, mediates ubiquitination of Arf6. This ubiquitination did not appear to be linked to immediate proteasomal degradation of Arf6, whereas Fbx8 knockdown caused hyperactivation of Arf6. Expression of Fbx8 protein was substantially lost in several breast tumor cell lines, in which Arf6 activity is pivotal for their invasion. Forced expression of Fbx8 in these cells suppressed their Arf6 activities and invasive activities, in which the F-box and Sec7 domains of Fbx8 are required. Together with the possible mechanism as to how Fbx8-mediated ubiquitination interferes with the functions of Arf6, we propose that Fbx8 provides a novel suppressive control of Arf6 activity through noncanonical ubiquitination. Our results indicate that dysfunction of Fbx8 expression may contribute to the invasiveness of some breast cancer cells.

ARNO is a soluble guanine nucleotide exchange factor (GEF) for the Arf family of GTPases. Although in biochemical assays ARNO prefers Arf1 over Arf6 as a substrate, its localization in cells at the plasma membrane (PM) suggests an interaction with Arf6. In this study, we found that ARNO activated Arf1 in HeLa and COS-7 cells resulting in the recruitment of Arf1 on to dynamic PM ruffles. By contrast, Arf6 was activated less by ARNO than EFA6, a canonical Arf6 GEF. Remarkably, Arf6 in its GTP-bound form recruited ARNO to the PM and the two proteins could be immunoprecipitated. ARNO binding to Arf6 was not mediated through the catalytic Sec7 domain, but via the pleckstrin homology (PH) domain. Active Arf6 also bound the PH domain of Grp1, another ARNO family member. This interaction was direct and required both inositol phospholipids and GTP. We propose a model of sequential Arf activation at the PM whereby Arf6-GTP recruits ARNO family GEFs for further activation of other Arf isoforms.

The ADP ribosylation factor (Arf)1 orthologue in the divergent eukaryote Trypanosoma brucei (Tb) shares characteristics with both Arf1 and Arf6 and has a vital role in intracellular protein trafficking. TbARF1 is Golgi localized in trypanosomes but associates with the plasma membrane when expressed in human cells. Depletion of TbARF1 by RNA interference causes a major decrease in endocytosis, which correlates with Rab5 dissociation from early endosomes. Although the Golgi remains intact, parasites display enlarged flagellar pockets and intracellular flagella. An increase in active GTP-bound TbARF1 in bloodstream parasites is rapidly lethal, correlating with a defect in Golgi-to-lysosome transport. We conclude that the essential Golgi-localizing T. brucei ARF1 has a primary role in the maintenance of both post-Golgi transport and endocytosis and that it is significantly divergent from other characterized ARFs.

ARF6 and Rac1 are small GTPases known to regulate remodelling of the actin cytoskeleton. Here, we demonstrate that these monomeric G proteins are sequentially activated when HEK 293 cells expressing the angiotensin type 1 receptor (AT(1)R) are stimulated with angiotensin II (Ang II). After receptor activation, ARF6 and Rac1 transiently form a complex. Their association is, at least in part, direct and dependent on the nature of the nucleotide bound to both small G proteins. ARF6-GTP preferentially interacts with Rac1-GDP. AT(1)R expressing HEK293 cells ruffle, form membrane protrusions, and migrate in response to agonist treatment. ARF6, but not ARF1, depletion using small interfering RNAs recapitulates the ruffling and migratory phenotype observed after Ang II treatment. These results suggest that ARF6 depletion or Ang II treatment are functionally equivalent and point to a role for endogenous ARF6 as an inhibitor of Rac1 activity. Taken together, our findings reveal a novel function of endogenously expressed ARF6 and demonstrate that by interacting with Rac1, this small GTPase is a central regulator of the signaling pathways leading to actin remodeling.

In the process of internalization of molecules from the extracellular milieu, a cell uses multiple endocytic pathways, consequently generating different endocytic vesicles. These primary endocytic vesicles are targeted to specific destinations inside the cell. Here, we show that GPI-anchored proteins are internalized by an Arf6-independent mechanism into GPI-anchored protein-enriched early endosomal compartments (GEECs). Internalized GPI-anchored proteins and the fluid phase are first visualized in GEECs that are acidic, primary endocytic structures, negative for early endosomal markers, Rab4, Rab5, and early endosome antigen (EEA)1. They subsequently acquire Rab5 and EEA1 before homotypic fusion with other GEECs, and heterotypic fusion with endosomes containing cargo from the clathrin-dependent endocytic pathway. Although, the formation of GEECs is unaffected by inhibition of Rab5 GTPase and phosphatidylinositol-3'-kinase (PI3K) activity, their fusion with sorting endosomes is dependent on both activities. Overexpression of Rab5 reverts PI3K inhibition of fusion, providing evidence that Rab5 effectors play important roles in heterotypic fusion between the dynamin-independent GEECs and clathrin- and dynamin-dependent sorting endosomes.

Extracellular signal-regulated kinase (Erk) is widely recognized for its central role in cell proliferation and motility. Although previous work has shown that Erk is localized at endosomal compartments, no role for Erk in regulating endosomal trafficking has been demonstrated. Here, we report that Erk signaling regulates trafficking through the clathrin-independent, ADP-ribosylation factor 6 (Arf6) GTPase-regulated endosomal pathway. Inactivation of Erk induced by a variety of methods leads to a dramatic expansion of the Arf6 endosomal recycling compartment, and intracellular accumulation of cargo, such as class I major histocompatibility complex, within the expanded endosome. Treatment of cells with the mitogen-activated protein kinase kinase (MEK) inhibitor U0126 reduces surface expression of MHCI without affecting its rate of endocytosis, suggesting that inactivation of Erk perturbs recycling. Furthermore, under conditions where Erk activity is inhibited, a large cohort of Erk, MEK, and the Erk scaffold kinase suppressor of Ras 1 accumulates at the Arf6 recycling compartment. The requirement for Erk was highly specific for this endocytic pathway, because its inhibition had no effect on trafficking of cargo of the classical clathrin-dependent pathway. These studies reveal a previously unappreciated link of Erk signaling to organelle dynamics and endosomal trafficking.

RNA interference-mediated depletion of phospholipase D2 (PLD2), but not PLD1, inhibited recycling of transferrin receptors in HeLa cells, whereas the internalization rate was unaffected by depletion of either PLD. Although reduction of both PLD isoforms inhibits PLD activity stimulated by phorbol 12-myristic 13-acetate, only depletion of PLD2 decreased nonstimulated activity. Cells with reduced PLD2 accumulated a greater fraction of transferrin receptors in a perinuclear compartment that was positive for Rab11, a marker of recycling endosomes. EFA6, an exchange factor for Arf6, has been proposed to stimulate the recycling of transferrin receptors. Thus, one consequence of EFA6 overexpression would be a reduction of the internal pool of receptors. We confirmed this observation in control HeLa cells; however, overexpression of EFA6 failed to decrease the internal pool of transferrin receptors that accumulate in cells previously depleted of PLD2. These observations suggest that either PLD2 is required for a constitutive Arf6-mediated recycling pathway or in the absence of PLD2 transferrin receptors accumulate in recycling endosomes that are not responsive to overexpression of EFA6.

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins mediate cellular membrane fusion events and provide a level of specificity to donor-acceptor membrane interactions. However, the trafficking pathways by which individual SNARE proteins are targeted to specific membrane compartments are not well understood. In neuroendocrine cells, synaptosome-associated protein of 25 kDa (SNAP25) is localized to the plasma membrane where it functions in regulated secretory vesicle exocytosis, but it is also found on intracellular membranes. We identified a dynamic recycling pathway for SNAP25 in PC12 cells through which plasma membrane SNAP25 recycles in approximately 3 h. Approximately 20% of the SNAP25 resides in a perinuclear recycling endosome-trans-Golgi network (TGN) compartment from which it recycles back to the plasma membrane. SNAP25 internalization occurs by constitutive, dynamin-independent endocytosis that is distinct from the dynamin-dependent endocytosis that retrieves secretory vesicle constituents after exocytosis. Endocytosis of SNAP25 is regulated by ADP-ribosylation factor (ARF)6 (through phosphatidylinositol bisphosphate synthesis) and is dependent upon F-actin. SNAP25 endosomes, which exclude the plasma membrane SNARE syntaxin 1A, merge with those derived from clathrin-dependent endocytosis containing endosomal syntaxin 13. Our results characterize a robust ARF6-dependent internalization mechanism that maintains an intracellular pool of SNAP25, which is compatible with possible intracellular roles for SNAP25 in neuroendocrine cells.

In epithelial cell lines, apical but not basolateral clathrin-mediated endocytosis has been shown to be affected by actin-disrupting drugs. Using electron and fluorescence microscopy, as well as biochemical assays, we show that the amount of actin dedicated to endocytosis is limiting at the apical surface of epithelia. In part, this contributes to the low basal rate of clathrin-dependent endocytosis observed at this epithelial surface. ARF6 in its GTP-bound state triggers the recruitment of actin from the cell cortex to the clathrin-coated pit to enable dynamin-dependent endocytosis. In addition, we show that perturbation of the apical endocytic system by expression of a clathrin heavy-chain mutant results in the collapse of microvilli. This phenotype was completely reversed by the expression of an ARF6-GTP-locked mutant. These observations indicate that concomitant to actin recruitment, the apical clathrin endocytic system is deeply involved in the morphology of the apical plasma membrane.

In this study, we investigated the role of phospholipase D (PLD) in mediating Arf6 function in cells. Expression of Arf6 mutants that are defective in activating PLD, Arf6N48R and Arf6N48I, inhibited membrane recycling to the plasma membrane (PM), resulting in an accumulation of tubular endosomal membranes. Additionally, unlike wild-type Arf6, neither Arf6 mutant could generate protrusions or recruit the Arf6 GTPase activating protein (GAP) ACAP1 onto the endosome in the presence of aluminum fluoride. Remarkably, all of these phenotypes, including accumulated tubular endosomes, blocked recycling, and failure to make protrusions and recruit ACAP effectively, could be recreated in either untransfected cells or cells expressing wild-type Arf6 by treatment with 1-butanol to inhibit the formation of phosphatidic acid (PA), the product of PLD. Moreover, most of the defects present in cells expressing Arf6N48R or N48I could be reversed by treatment with agents expected to elevate PA levels in cells. Together, these observations provide compelling evidence that Arf6 stimulation of PLD is required for endosomal membrane recycling and GAP recruitment.

SNAP receptor (SNARE)-mediated fusion is regarded as a core event in exocytosis. Exocytosis is supported by other proteins that set up SNARE interactions between secretory vesicle and plasma membranes or facilitate fusion pore formation. Secretory carrier membrane proteins (SCAMPs) are candidate proteins for functioning in these events. In neuroendocrine PC12 cells, SCAMP2 colocalizes on the cell surface with three other proteins required for dense-core vesicle exocytosis: phospholipase D1 (PLD1), the small GTPase Arf6, and Arf6 guanine nucleotide exchange protein ARNO. Arf6 and PLD1 coimmunoprecipitate (coIP) with SCAMP2. These associations have been implicated in exocytosis by observing enhanced coIP of Arf6 with SCAMP2 after cell depolarization and in the presence of guanosine 5'-O-(3-thio)triphosphate and by inhibition of coIP by a SCAMP-derived peptide that inhibits exocytosis. The peptide also suppresses PLD activity associated with exocytosis. Using amperometry to analyze exocytosis, we show that expression of a point mutant of SCAMP2 that exhibits decreased association with Arf6 and of mutant Arf6 deficient in activating PLD1 have the same inhibitory effects on early events in membrane fusion. However, mutant SCAMP2 also uniquely inhibits fusion pore dilation. Thus, SCAMP2 couples Arf6-stimulated PLD activity to exocytosis and links this process to formation of fusion pores.

The vacuolating cytotoxin VacA is a major virulence factor of Helicobacter pylori, a bacterium responsible for gastroduodenal ulcers and cancer. VacA associates with lipid rafts, is endocytosed, and reaches the late endocytic compartment where it induces vacuolation. We have investigated the endocytic and intracellular trafficking pathways used by VacA, in HeLa and gastric AGS cells. We report here that VacA was first bound to plasma-membrane domains localized above F-actin structures that were controlled by the Rac1 GTPase. VacA was subsequently pinocytosed by a clathrin-independent mechanism into cell peripheral early endocytic compartments lacking caveolin 1, the Rab5 effector early endosomes antigen-1 (EEA1) and transferrin. These compartments took up fluid-phase (as evidenced by the accumulation of fluorescent dextran) and glycosylphosphatidylinositol-anchored proteins (GPI-APs). VacA pinocytosis was controlled by Cdc42 and did not require cellular tyrosine kinases, dynamin 2, ADP-ribosylating factor 6, or RhoA GTPase activities. VacA was subsequently routed to EEA1-sorting endosomes and then sorted to late endosomes. During all these different endocytic steps, VacA was continuously associated with detergent resistant membrane domains. From these results we propose that VacA might be a valuable probe to study raft-associated molecules, pinocytosed by a clathrin-independent mechanism, and routed to the degradative compartment.

ADP-ribosylation factor 6 (Arf6) is a small-GTPase that regulates the membrane trafficking between the plasma membrane and endosome. It is also involved in the reorganization of the actin cytoskeleton. GTPase-activating protein (GAP) is a critical regulator of Arf function as it inactivates Arf. Here, we identified a novel species of GAP denoted as SMAP1 that preferentially acts on Arf6. Although overexpression of SMAP1 did not alter the subcellular distribution of the actin cytoskeleton, it did block the endocytosis of transferrin receptors. Knock down of endogenous SMAP1 also abolished transferrin internalization, which confirms that SMAP1 is needed for this endocytic process. SMAP1 overexpression had no effect on clathrin-independent endocytosis, however. Intriguingly, SMAP1 binds directly to the clathrin heavy chain via its clathrin-box and mutation studies revealed that its GAP domain and clathrin-box both contribute to the role SMAP1 plays in clathrin-dependent endocytosis. These observations suggest that SMAP1 may be an Arf6GAP that specifically regulates one of the multiple functions of Arf6, namely, clathrin-dependent endocytosis, and that it does so by binding directly to clathrin.

Clathrin-independent endocytosis internalizes plasma membrane proteins that lack cytoplasmic sequences recognized by clathrin adaptor proteins. There is evidence for different clathrin-independent pathways but whether they share common features has not been systematically tested. Here, we examined whether CD59, an endogenous glycosylphosphatidyl inositol-anchored protein (GPI-AP), and major histocompatibility protein class I (MHCI), an endogenous, integral membrane protein, entered cells through a common mechanism and followed a similar itinerary. At early times of internalization, CD59 and MHCI were found in the same Arf6-associated endosomes before joining clathrin cargo proteins such as transferrin in common sorting endosomes. CD59 and MHCI, but not transferrin, also were observed in the Arf6-associated tubular recycling membranes. Endocytosis of CD59 and MHCI required free membrane cholesterol because it was inhibited by filipin binding to the cell surface. Expression of active Arf6 stimulated endocytosis of GPI-APs and MHCI to the same extent and led to their accumulation in Arf6 endosomes that labeled intensely with filipin. This blocked delivery of GPI-APs and MHCI to early sorting endosomes and to lysosomes for degradation. Endocytosis of transferrin was not affected by any of these treatments. These observations suggest common mechanisms for endocytosis without clathrin.

The adaptor protein Numb is necessary for the cell fate specification of progenitor cells in the Drosophila nervous system. Numb is evolutionarily conserved and previous studies have provided evidence for a similar functional role during mammalian development. The Numb protein has multiple protein-protein interaction regions including a phosphotyrosine binding (PTB) domain and a carboxy-terminal domain that contains conserved interaction motifs including an EH (Eps15 Homology) domain binding motif and alpha-adaptin binding site. In this study we identify the EHD/Rme-1/Pincher family of endocytic proteins as Numb interacting partners in mammals and Drosophila. The EHD/Rme-1 proteins function in recycling of plasma membrane receptors internalized by both clathrin-mediated endocytosis and a clathrin-independent pathway regulated by ADP ribosylation factor 6 (Arf6). Here we report that Numb colocalizes with endogenous EHD4/Pincher and Arf6 and that Arf6 mutants alter Numb subcellular localization. In addition, we present evidence that Numb has a novel function in endosomal recycling and intracellular trafficking of receptors.

Cholera toxin (CT) and related AB(5) toxins bind to glycolipids at the plasma membrane and are then transported in a retrograde manner, first to the Golgi and then to the endoplasmic reticulum (ER). In the ER, the catalytic subunit of CT is translocated into the cytosol, resulting in toxicity. Using fluorescence microscopy, we found that CT is internalized by multiple endocytic pathways. Inhibition of the clathrin-, caveolin-, or Arf6-dependent pathways by overexpression of appropriate dominant mutants had no effect on retrograde traffic of CT to the Golgi and ER, and it did not affect CT toxicity. Unexpectedly, when we blocked all three endocytic pathways at once, although fluorescent CT in the Golgi and ER became undetectable, CT-induced toxicity was largely unaffected. These results are consistent with the existence of an additional retrograde pathway used by CT to reach the ER.

Although neuronal axons and dendrites with their associated filopodia and spines exhibit a profound cell polarity, the mechanism by which they develop is largely unknown. Here, we demonstrate that specific palmitoylated protein motifs, characterized by two adjacent cysteines and nearby basic residues, are sufficient to induce filopodial extensions in heterologous cells and to increase the number of filopodia and the branching of dendrites and axons in neurons. Such motifs are present at the N-terminus of GAP-43 and the C-terminus of paralemmin, two neuronal proteins implicated in cytoskeletal organization and filopodial outgrowth. Filopodia induction is blocked by mutations of the palmitoylated sites or by treatment with 2-bromopalmitate, an agent that inhibits protein palmitoylation. Moreover, overexpression of a constitutively active form of ARF6, a GTPase that regulates membrane cycling and dendritic branching reversed the effects of the acylated protein motifs. Filopodia induction by the specific palmitoylated motifs was also reduced upon overexpression of a dominant negative form of the GTPase cdc42. These results demonstrate that select dually lipidated protein motifs trigger changes in the development and growth of neuronal processes.

We addressed the role of EFA6, exchange factor for ARF6, during the development of epithelial cell polarity in Madin-Darby canine kidney cells. EFA6 is located primarily at the apical pole of polarized cells, including the plasma membrane. After calcium-triggered E-cadherin-mediated cell adhesion, EFA6 is recruited to a Triton X-100-insoluble fraction and its protein level is increased concomitantly to the accelerated formation of a functional tight junction (TJ). The expression of EFA6 results in the selective retention at the cell surface of the TJ protein occludin. This effect is due to EFA6 capacities to promote selectively the stability of the apical actin ring onto which the TJ is anchored, resulting in the exclusion of TJ proteins from endocytosis. Finally, our data suggest that EFA6 effects are achieved by the coordinate action of both its exchange activity and its actin remodeling C-terminal domain. We conclude that EFA6 is a signaling molecule that responds to E-cadherin engagement and is involved in TJ formation and stability.

MTM1, MTMR2, and SBF2 belong to a family of proteins called the myotubularins. X-linked myotubular myopathy, a severe congenital disorder characterized by hypotonia and generalized muscle weakness in newborn males, is caused by mutations in MTM1 (Laporte et al., 1996). Charcot-Marie-Tooth types 4B1 and 4B2 are severe demyelinating neuropathies caused by mutations in MTMR2 (Bolino et al., 2000) and SBF2/MTMR13 (Senderek et al., 2003), respectively. Although several myotubularins are known to regulate phosphoinositide-phosphate levels in cells, little is known about the actual cellular process that is defective in patients with these diseases. Mutations in worm MTM-6 and MTM-9, myotubularins belonging to two subgroups, disorganize phosphoinositide 3-phosphate localization and block endocytosis in the coelomocytes of Caenorhabditis elegans. We demonstrate that MTM-6 and MTM-9 function as part of a complex to regulate an endocytic pathway that involves the Arf6 GTPase, and we define protein domains required for MTM-6 activity.

In the developing nervous system, controlled neurite extension and branching are critical for the establishment of connections between neurons and their targets. Although much is known about the regulation of axonal development, many of the molecular events that regulate axonal extension remain unknown. ADP-ribosylation factor nucleotide-binding site opener (ARNO) and ADP-ribosylation factor (ARF)6 have important roles in the regulation of the cytoskeleton as well as membrane trafficking. To investigate the role of these molecules in axonogenesis, we expressed ARNO and ARF6 in cultured rat hippocampal neurons. Expression of catalytically inactive ARNO or dominant negative ARF6 resulted in enhanced axonal extension and branching and this effect was abrogated by coexpression of constitutively active ARF6. We sought to identify the downstream effectors of ARF6 during neurite extension by coexpressing phosphatidyl-inositol-4-phosphate 5-Kinase alpha [PI(4)P 5-Kinase alpha] with catalytically inactive ARNO and dominant negative ARF6. We found that PI(4)P 5-Kinase alpha plays a role in neurite extension and branching downstream of ARF6. Also, expression of inactive ARNO/ARF6 depleted the actin binding protein mammalian ena (Mena) from the growth cone leading edge, indicating that these effects on axonogenesis may be mediated by changes in cytoskeletal dynamics. These results suggest that ARNO and ARF6, through PI(4)P 5-Kinase alpha, regulate axonal elongation and branching during neuronal development.

HIV Nef has a number of important biological effects, including the down-modulation of several immunological important molecules (CD4, major histocompatibility complex [MHC] class I). Down-modulation of CD4 seems to be via clathrin-dependent endocytosis, whereas down-modulation of MHC class I remains unexplained. Several mutant proteins, including mutations in the small GTPase Arf6, have been used to probe membrane traffic pathways. One such mutant has recently been used to propose that Nef acts through Arf6 to activate the endocytosis of MHC class I. Here, we show that MHC class I down-modulation is unaffected by other Arf6 mutants that provide more specific perturbations in the GDP-GTP cycling of Arf6. Inhibition of phosphatidylinositol-3-phosphate kinase, an upstream activator of Arf6, also had no effect on the internalization step, but its activity is required to direct MHC class I to the trans-Golgi network. We conclude that the apparent Arf6 dependency of Nef-mediated MHC class I down-modulation is due to nonspecific perturbations in membrane traffic.

Little is known about the molecular mechanism of recycling of intracellular receptors and lipid raft-associated proteins. Here, we have investigated the recycling pathway and internalization mechanism of a transmembrane, lipid raft-associated intracellular prohormone sorting receptor, carboxypeptidase E (CPE). CPE is found in the trans-Golgi network (TGN) and secretory granules of (neuro)endocrine cells. An extracellular domain of the IL2 receptor alpha-subunit (Tac) fused to the transmembrane domain and cytoplasmic tail of CPE (Tac-CPE25) was used as a marker to track recycling of CPE. We show in (neuro)endocrine cells, that upon stimulated secretory granule exocytosis, raft-associated Tac-CPE25 was rapidly internalized from the plasma membrane in a clathrin-independent manner into early endosomes and then transported through the endocytic recycling compartment to the TGN. A yeast two-hybrid screen and in vitro binding assay identified the CPE cytoplasmic tail sequence S472ETLNF477 as an interactor with active small GTPase ADP-ribosylation factor (ARF) 6, but not ARF1. Expression of a dominant negative, inactive ARF6 mutant blocked this recycling. Mutation of residues S472 or E473 to A in the cytoplasmic tail of CPE obliterated its binding to ARF6, and internalization from the plasma membrane of Tac-CPE25 mutated at S472 or E473 was significantly reduced. Thus, CPE recycles back to the TGN by a novel mechanism requiring ARF6 interaction and activity.

To understand the role that ARF6 plays in regulating isoactin dynamics and cell motility, we transfected endothelial cells (EC) with HA-tagged ARF6: the wild-type form (WT), a constitutively-active form unable to hydrolyze GTP (Q67L), and two dominant-negative forms, which are either unable to release GDP (T27N) or fail to bind nucleotide (N122I). Motility was assessed by digital imaging microscopy before Western blot analysis, coimmunoprecipitation, or colocalization studies using ARF6, beta-actin, or beta-actin-binding protein-specific antibodies. EC expressing ARF6-Q67L spread and close in vitro wounds at twice the control rates. EC expressing dominant-negative ARF6 fail to develop a leading edge, are unable to ruffle their membranes (N122I), and possess arborized processes. Colocalization studies reveal that the Q67L and WT ARF6-HA are enriched at the leading edge with beta-actin; but T27N and N122I ARF6-HA are localized on endosomes together with the beta-actin capping protein, betacap73. Coimmunoprecipitation and Western blot analyses reveal the direct association of ARF6-HA with betacap73, defining a role for ARF6 in signaling cytoskeletal remodeling during motility. Knowledge of the role that ARF6 plays in orchestrating membrane and beta-actin dynamics will help to reveal molecular mechanisms regulating actin-based motility during development and disease.

ADP-ribosylation factors (ARFs) are ubiquitous regulators of virtually every step of vesicular membrane traffic. Yeast Arf3p, which is most similar to mammalian ARF6, is not essential for cell viability and not required for endoplasmic reticulum-to-Golgi protein transport. Although mammalian ARF6 has been implicated in the regulation of early endocytic transport, we found that Arf3p was not required for fluid-phase, membrane internalization, or mating-type receptor-mediated endocytosis. Arf3p was partially localized to the cell periphery, but was not detected on endocytic structures. The nucleotide-binding, N-terminal region, and N-terminal myristate of Arf3p are important for its proper localization. C-Terminally green fluorescent protein-tagged Arf3, expressed from the endogenous promoter, exhibited a polarized localization to the cell periphery and buds, in a cell cycle-dependent manner. Arf3-GFP achieved its proper localization during polarity growth through an actin-independent pathway. Both haploid and homologous diploid arf3 mutants exhibit a random budding defect, and the overexpression of the GTP-bound form Arf3p(Q71L) or GDP-binding defective Arf3p(T31N) mutant interfered with budding-site selection. We conclude that the GTPase cycle of Arf3p is likely to be important for the function of Arf3p in polarizing growth of the emerging bud and/or an unidentified vesicular trafficking pathway.

The mechanisms coordinating adhesion, actin organization, and membrane traffic during growth cone migration are poorly understood. Neuritogenesis and branching from retinal neurons are regulated by the Rac1B/Rac3 GTPase. We have identified a functional connection between ADP-ribosylation factor (Arf) 6 and p95-APP1 during the regulation of Rac1B-mediated neuritogenesis. P95-APP1 is an ADP-ribosylation factor GTPase-activating protein (ArfGAP) of the GIT family expressed in the developing nervous system. We show that Arf6 has a predominant role in neurite extension compared with Arf1 and Arf5. Cotransfection experiments indicate a specific and cooperative potentiation of neurite extension by Arf6 and the carboxy-terminal portion of p95-APP1. Localization studies in neurons expressing different p95-derived constructs show a codistribution of p95-APP1 with Arf6, but not Arf1. Moreover, p95-APP1-derived proteins with a mutated or deleted ArfGAP domain prevent Rac1B-induced neuritogenesis, leading to PIX-mediated accumulation at large Rab11-positive endocytic vesicles. Our data support a role of p95-APP1 as a specific regulator of Arf6 in the control of membrane trafficking during neuritogenesis.

The trafficking of two plasma membrane (PM) proteins that lack clathrin internalization sequences, major histocompatibility complex class I (MHCI), and interleukin 2 receptor alpha subunit (Tac) was compared with that of PM proteins internalized via clathrin. MHCI and Tac were internalized into endosomes that were distinct from those containing clathrin cargo. At later times, a fraction of these internalized membranes were observed in Arf6-associated, tubular recycling endosomes whereas another fraction acquired early endosomal autoantigen 1 (EEA1) before fusion with the "classical" early endosomes containing the clathrin-dependent cargo, LDL. After convergence, cargo molecules from both pathways eventually arrived, in a Rab7-dependent manner, at late endosomes and were degraded. Expression of a constitutively active mutant of Arf6, Q67L, caused MHCI and Tac to accumulate in enlarged PIP(2)-enriched vacuoles, devoid of EEA1 and inhibited their fusion with clathrin cargo-containing endosomes and hence blocked degradation. By contrast, trafficking and degradation of clathrin-cargo was not affected. A similar block in transport of MHCI and Tac was reversibly induced by a PI3-kinase inhibitor, implying that inactivation of Arf6 and acquisition of PI3P are required for convergence of endosomes arising from these two pathways.

Phospholipase Ds (PLDs) are regulated enzymes that generate phosphatidic acid (PA), a putative second messenger implicated in the regulation of vesicular trafficking and cytoskeletal reorganization. Mast cells, when stimulated with antigen, show a dramatic alteration in their cytoskeleton and also release their secretory granules by exocytosis. Butan-1-ol, which diverts the production of PA generated by PLD to the corresponding phosphatidylalcohol, was found to inhibit membrane ruffling when added together with antigen or when added after antigen. Inhibition by butan-1-ol was completely reversible because removal of butan-1-ol restored membrane ruffling. Measurements of PLD activation by antigen indicate a requirement for continual PA production during membrane ruffling, which was maintained for at least 30 min. PLD1 and PLD2 are both expressed in mast cells and green fluorescent protein-tagged proteins were used to identify PLD2 localizing to membrane ruffles of antigen-stimulated mast cells together with endogenous ADP ribosylation factor 6 (ARF6). In contrast, green fluorescent protein-PLD1 localized to intracellular vesicles and remained in this location after stimulation with antigen. Membrane ruffling was independent of exocytosis of secretory granules because phorbol 12-myristate 13-acetate increased membrane ruffling in the absence of exocytosis. Antigen or phorbol 12-myristate 13-acetate stimulation increased both PLD1 and PLD2 activity when expressed individually in RBL-2H3 cells. Although basal activity of PLD2-overexpressing cells is very high, membrane ruffling was still dependent on antigen stimulation. In permeabilized cells, antigen-stimulated phosphatidylinositol(4,5)bisphosphate synthesis was dependent on both ARF6 and PA generated from PLD. We conclude that both activation of ARF6 by antigen and a continual PLD2 activity are essential for local phosphatidylinositol(4,5)bisphosphate generation that regulates dynamic actin cytoskeletal rearrangements.

Phospholipase D (PLD) activity can be detected in response to many agonists in most cell types; however, the pathway from receptor occupation to enzyme activation remains unclear. In vitro PLD1b activity is phosphatidylinositol 4,5-bisphosphate dependent via an N-terminal PH domain and is stimulated by Rho, ARF, and PKC family proteins, combinations of which cooperatively increase this activity. Here we provide the first evidence for the in vivo regulation of PLD1b at the molecular level. Antigen stimulation of RBL-2H3 cells induces the colocalization of PLD1b with Rac1, ARF6, and PKCalpha at the plasma membrane in actin-rich structures, simultaneously with cooperatively increasing PLD activity. Activation is both specific and direct because dominant negative mutants of Rac1 and ARF6 inhibit stimulated PLD activity, and surface plasmon resonance reveals that the regulatory proteins bind directly and independently to PLD1b. This also indicates that PLD1b can concurrently interact with a member from each regulator family. Our results show that in contrast to PLD1b's translocation to the plasma membrane, PLD activation is phosphatidylinositol 3-kinase dependent. Therefore, because inactive, dominant negative GTPases do not activate PLD1b, we propose that activation results from phosphatidylinositol 3-kinase-dependent stimulation of Rac1, ARF6, and PKCalpha.