An mTORC1 to HRI signaling axis promotes cytotoxicity of proteasome inhibitors in multiple myeloma.

Multiple myeloma (MM) causes approximately 20% of deaths from blood cancers. Notwithstanding significant therapeutic progress, such as with proteasome inhibitors (PIs), MM remains incurable due to the development of resistance. mTORC1 is a key metabolic regulator, which frequently becomes dysregulated in cancer. While mTORC1 inhibitors reduce MM viability and synergize with other therapies in vitro, clinically, mTORC1 inhibitors are not effective for MM. Here we show that the inactivation of mTORC1 is an intrinsic response of MM to PI treatment. Genetically enforced hyperactivation of mTORC1 in MM was sufficient to compromise tumorigenicity in mice. In vitro, mTORC1-hyperactivated MM cells gained sensitivity to PIs and hypoxia. This was accompanied by increased mitochondrial stress and activation of the eIF2α kinase HRI, which initiates the integrated stress response. Deletion of HRI elevated the toxicity of PIs in wt and mTORC1-activated MM. Finally, we identified the drug PMA as a robust inducer of mTORC1 activity, which synergized with PIs in inducing MM cell death. These results help explain the clinical inefficacy of mTORC1 inhibitors in MM. Our data implicate mTORC1 induction and/or HRI inhibition as pharmacological strategies to enhance MM therapy by PIs.

Endoplasmic Reticulum Homeostasis Regulates TLR4 Expression and Signaling in Mast Cells.

The endoplasmic reticulum (ER) is a dynamic organelle that responds to demand in secretory proteins by undergoing expansion. The mechanisms that control the homeostasis of ER size and function involve the activation of the unfolded protein response (UPR). The UPR plays a role in various effector functions of immune cells. Mast cells (MCs) are highly granular tissue-resident cells and key drivers of allergic inflammation. Their diverse secretory functions in response to activation through the high-affinity receptor for IgE (FcεRI) suggest a role for the UPR in their function. Using human cord blood-derived MCs, we found that FcεRI triggering elevated the expression level and induced activation of the UPR transducers IRE1α and PERK, accompanied by expansion of the ER. In mouse bone marrow-derived MCs and peritoneal MCs, the ER underwent a more moderate expansion, and the UPR was not induced following MC activation. The deletion of IRE1α in mouse MCs did not affect proliferation, survival, degranulation, or cytokine stimulation following FcεRI triggering, but it did diminish the surface expression of TLR4 and the consequent response to LPS. A similar phenotype was observed in human MCs using an IRE1α inhibitor. Our data indicate that the ER of MCs, primarily of humans, undergoes a rapid remodeling in response to activation that promotes responses to TLR4. We suggest that IRE1α inhibition can be a strategy for inhibiting the hyperactivation of MCs by LPS over the course of allergic responses.

Macrophage-derived secretome is sufficient to confer olanzapine-mediated insulin resistance in human adipocytes.

Objective: Olanzapine and Aripiprazole are widely used second-generation antipsychotic drugs. Olanzapine, more than Aripiprazole, leads to considerable metabolic side effects including obesity and diabetes. While the underlying mechanisms are not fully understood, these side effects are likely associated with mild inflammation in the metabolic organs. An in vitro model that accurately recapitulates the metabolic impact of olanzapine and aripiprazole should be useful to elucidate the underlying mechanisms. Methods: We established co-cultures of matured adipocytes derived from the human SGBS cell line and the THP-1 human monocytic cell-derived or primary macrophages to explore the effects of both drugs on the response to insulin. Results: Olanzapine, but not aripiprazole induced insulin resistance in SGBS adipocytes only when co-cultured with THP-1 or primary macrophages, polarized either into M0, M1 or M2. Noteworthy, M2 macrophages induced olanzapine-dependent insulin resistance in the absence of induction of pro-inflammatory cytokines. Insulin resistance by olanzapine was stronger than induced by high concentration of pro-inflammatory cytokines even in combinations, suggesting the contribution of factors other than the classical inflammatory cytokines to promote insulin resistance in adipocytes by olanzapine. Conclusion: Macrophage/adipocyte co-cultures recapitulate the features of olanzapine-induced insulin resistance and implicate the existence of yet unknown factors in mediating this effect.

The integrated stress response promotes B7H6 expression.

The B7 family member, B7H6, is a ligand for the natural killer cell receptor NKp30. B7H6 is hardly expressed on normal tissues, but undergoes upregulation on different types of tumors, implicating it as an attractive target for cancer immunotherapy. The molecular mechanisms that control B7H6 expression are poorly understood. We report that in contrast to other NK cell ligands, endoplasmic reticulum (ER) stress upregulates B7H6 mRNA levels and surface expression. B7H6 induction by ER stress requires protein kinase R-like ER kinase (PERK), one of the three canonical sensors of the unfolded protein response. PERK phosphorylates eIF2α, which regulates protein synthesis and gene expression. Because eIF2α is phosphorylated by several kinases following different stress conditions, the program downstream to eIF2α phosphorylation is called the integrated stress response (ISR). Several drugs were reported to promote the ISR. Nelfinavir and lopinavir, two clinically approved HIV protease inhibitors, promote eIF2α phosphorylation by different mechanisms. We show that nelfinavir and lopinavir sustainably instigate B7H6 expression at their pharmacologically relevant concentrations. As such, ER stress and ISR conditions sensitize melanoma targets to CAR-T cells directed against B7H6. Our study highlights a novel mechanism to induce B7H6 expression and suggests a pharmacological approach to improve B7H6-directed immunotherapy. KEY MESSAGES: B7H6 is induced by ER stress in a PERK-dependent mechanism. Induction of B7H6 is obtained pharmacologically by HIV protease inhibitors. Exposure of tumor cells to the HIV protease inhibitor nelfinavir improves the recognition by B7H6-directed CAR-T.

Monocytes as Carriers of Magnetic Nanoparticles for Tracking Inflammation in the Epileptic Rat Brain.

BACKGROUND: Inflammation is a hallmark of epileptogenic brain tissue. Previously, we have shown that inflammation in epilepsy can be delineated using systemically-injected fluorescent and magnetite- laden nanoparticles. Suggested mechanisms included distribution of free nanoparticles across a compromised blood-brain barrier or their transfer by monocytes that infiltrate the epileptic brain. OBJECTIVE: In the current study, we evaluated monocytes as vehicles that deliver nanoparticles into the epileptic brain. We also assessed the effect of epilepsy on the systemic distribution of nanoparticleloaded monocytes. METHODS: The in vitro uptake of 300-nm nanoparticles labeled with magnetite and BODIPY (for optical imaging) was evaluated using rat monocytes and fluorescence detection. For in vivo studies we used the rat lithium-pilocarpine model of temporal lobe epilepsy. In vivo nanoparticle distribution was evaluated using immunohistochemistry. RESULTS: 89% of nanoparticle loading into rat monocytes was accomplished within 8 hours, enabling overnight nanoparticle loading ex vivo. The dose-normalized distribution of nanoparticle-loaded monocytes into the hippocampal CA1 and dentate gyrus of rats with spontaneous seizures was 176-fold and 380-fold higher compared to the free nanoparticles (p<0.05). Seizures were associated with greater nanoparticle accumulation within the liver and the spleen (p<0.05). CONCLUSION: Nanoparticle-loaded monocytes are attracted to epileptogenic brain tissue and may be used for labeling or targeting it, while significantly reducing the systemic dose of potentially toxic compounds. The effect of seizures on monocyte biodistribution should be further explored to better understand the systemic effects of epilepsy.

The effects of valproic acid on early pregnancy human placentas: Pilot ex vivo analysis in cultured placental villi.

Valproic acid is an established structural and neurodevelopmental teratogen. Recently, we demonstrated that valproate alters the barrier function of perfused term human placentas. Here, we conducted a pilot study to evaluate the effects of subchronic valproate exposure on carrier expression in cultured placental villous explants from early human pregnancies. Placental tissue of gestational age 6-13 weeks was collected from elective pregnancy terminations in women without known epilepsy. The effects of valproate (42, 83, or 166 µg/mL) on the mRNA expression of 37 major placental carriers and related genes were evaluated by a customized gene expression array (n = 5, 5 days). Five-day exposure to valproate was associated with high variability in gene expression. However, two main gene clusters were identified, including a cluster of three major folate carriers. Exposure to low therapeutic levels of valproate (42 µg/mL) was associated with a tendency toward reduced mRNA expression of genes encoding folate and amino acid and fatty acid carriers (P = 0.065, paired analysis). Our initial findings suggest that valproate can affect the function of the human placenta during early pregnancy.

Adverse placental effects of valproic acid: Studies in perfused human placentas.

OBJECTIVE: In utero exposure to valproic acid (VPA) has been associated with worse pregnancy outcomes compared to all other antiepileptic drugs. We have previously shown that VPA alters the expression of placental transporters for hormones and nutrients in vitro and in pregnant mice. Here, our aim was to characterize the effects of short exposure to VPA on the expression of carriers for compounds essential for fetal development in human placentas ex vivo, under controlled conditions. METHODS: Placentas were obtained from cesarean deliveries of women with no known epilepsy. Cotyledons were cannulated and perfused in the absence or the presence of VPA (42, 83, or 166 µg/mL; n = 6/group) in the maternal perfusate over 180 minutes. A customized gene panel array was used to analyze the expression of carrier genes in the perfused cotyledons. We additionally measured in the perfused placentas folic acid concentrations and histone acetylation. RESULTS: VPA significantly altered the mRNA levels of major carriers for folic acid, glucose, choline, thyroid hormones, and serotonin (P < .05) and reduced placental folate concentrations by 25%-35% (P = .059). The effects were observed at therapeutic concentrations sufficient to enhance placental histone acetylation, and some were concentration-dependent. SIGNIFICANCE: Our results point to the placenta as a novel target of VPA, implying potential involvement of the placenta in VPA's adverse fetal outcomes.

Folate homeostasis in epileptic rats.

Folate is involved in metabolic processes and it has been implicated in both aggravation and amelioration of seizures. The aim of the current work was to study the effect of chronic temporal lobe epilepsy (TLE) on the plasma and brain concentrations of folate and on its uptake carriers in the brain - the reduced folate carrier (RFC), folate receptor α (FRα) and proton coupled folate transporter (PCFT). We utilized the rat lithium pilocarpine model for TLE. Approximately two months following status epilepticus, rats with spontaneous recurrent seizures (SRS) were sacrificed for brain and plasma folate concentration analyses and folate uptake carrier expression studies. RT-PCR and western blot analyses were utilized for quantification of folate carriers' mRNAs and proteins, respectively. The distribution of folate carriers in the brain was studied using immunohistochemistry. In the SRS rats we found lower plasma concentrations (10 ±â€¯0.9 in control vs. 6.6 ±â€¯1.6 ng/ml in SRS, P < 0.05), but preserved cortical and increased hippocampal levels of folate (0.5 ±â€¯0.1 in control vs. 0.9 ±â€¯0.2 ng/mg in SRS, P = 0.055). Hippocampus - to - plasma ratio of folate concentration was 3-fold higher in the SRS group, compared with the controls (0.13 ±â€¯0.03 vs. 0.04 ±â€¯0.02, respectively; P < 0.01). mRNA and protein levels of the folate uptake carriers did not differ between SRS rats and controls. However, immunofluorescent staining quantification revealed that the emission intensity of both RFC and FRα was elevated 8-fold and 4-fold, respectively, in hippocampal CA1 neurons of SRS rats, compared to controls (P < 0.01). PCFT was unquantifiable. If corroborated by complementary research in humans, the findings of this study may be utilized clinically for supplemental therapy planning, in imaging the epileptic focus, and for drug delivery into the epileptic brain. Further studies are required for better elucidating the clinical and mechanistic significance of altered folate balances in the epileptic brain.

Evaluation of Near Infrared Dyes as Markers of P-Glycoprotein Activity in Tumors.

Aim: The multidrug resistance protein 1 (MDR1; P-glycoprotein) has been associated with efflux of chemotherapeutic agents from tumor cells and with poor patient prognosis. This study evaluated the feasibility of non-invasive, non-radioactive near infrared (NIR) imaging methodology for detection of MDR1 functional activity in tumors. Methods: Initial accumulation assays were conducted in MDR1-overexpressing MDCK cells (MDCK-MDR1) and control MDCK cells (MDCK-CT) using the NIR dyes indocyanine green (ICG), IR-783, IR-775, rhodamine 800, XenoLight DiR, and Genhance 750, at 0.4 µM-100 µM. ICG and IR-783 were also evaluated in HT-29 cells in which MDR1 overexpression was induced by colchicine (HT-29-MDR1) and their controls (HT-29-CT). In vivo optical imaging studies were conducted using immunodeficient mice bearing HT-29-CT and HT-29-MDR1 xenografts. Results: ICG's emission intensity was 2.0- and 2.2-fold higher in control versus MDR1-overexpressing cells, in MDCK and HT-29 cell lines, respectively. The respective IR-783 control:MDR1 ratio was 1.4 in both MDCK and HT-29 cells. Optical imaging of mice bearing HT-29-CT and HT-29-MDR1 xenografts revealed a statistically non-significant, 1.7-fold difference (p > 0.05) in ICG emission intensity between control and MDR1 tumors. No such differences were observed with IR-783. Conclusion: ICG and IR-783 appear to be weak MDR1 substrates. In vivo, low sensitivity and high between-subject variability impair the ability to use the currently studied probes as markers of tumor MDR1 activity. The results suggest that, for future use of this technology, additional NIR probes should be screened as MDR1 substrates.

Maternal-fetal transfer of indocyanine green across the perfused human placenta.

Indocyanine green (ICG) is an FDA-approved near-infrared imaging probe, given also to pregnant women. We aimed to characterize ICG's transplacental transfer using the ex-vivo perfusion model. Placentas were obtained from caesarean deliveries. Cotyledons were cannulated and dually perfused. ICG, 9.6µg/mL and antipyrine (50µg/mL) were added to the maternal circulation in the absence (n=4) or the presence of the organic anion transporting polypeptide (OATPs) inhibitor rifampin (10µg/mL; n=5) or the P-glycoprotein inhibitor valspodar (2µg/mL; n=3). ICG's maternal-to-fetal transfer was evaluated over 180min. The cumulative percent of ICG in the fetal reservoir was minor. When ICG transfer was normalized to that of antipyrine, it was lower in the presence of rifampin (a 41% decrease; p<0.05). Valspodar did not appear to modify the kinetics of ICG. ICG's transplacental transfer is minimal and is probably OATP-mediated. The placenta is an effective protective barrier to ICG's distribution into the fetus.

Tracking inflammation in the epileptic rat brain by bi-functional fluorescent and magnetic nanoparticles.

Correct localization of epileptic foci can improve surgical outcome in patients with drug-resistant seizures. Our aim was to demonstrate that systemically injected nanoparticles identify activated immune cells, which have been reported to accumulate in epileptogenic brain tissue. Fluorescent and magnetite-labeled nanoparticles were injected intravenously to rats with lithium-pilocarpine-induced chronic epilepsy. Cerebral uptake was studied ex vivo by confocal microscopy and MRI. Cellular uptake and biological effects were characterized in vitro in murine monocytes and microglia cell lines. Microscopy confirmed that the nanoparticles selectively accumulate within myeloid cells in the hippocampus, in association with inflammation. The nanoparticle signal was also detectable by MRI. The in vitro studies demonstrate rapid nanoparticle uptake and good cellular tolerability. We show that nanoparticles can target myeloid cells in epileptogenic brain tissue. This system can contribute to pre-surgical and intra-surgical localization of epileptic foci, and assist in detecting immune system involvement in epilepsy.

Indocyanine Green Liposomes for Diagnosis and Therapeutic Monitoring of Cerebral Malaria.

Cerebral malaria (CM) is a major cause of death of Plasmodium falciparum infection. Misdiagnosis of CM often leads to treatment delay and mortality. Conventional brain imaging technologies are rarely applicable in endemic areas. Here we address the unmet need for a simple, non-invasive imaging methodology for early diagnosis of CM. This study presents the diagnostic and therapeutic monitoring using liposomes containing the FDA-approved fluorescent dye indocyanine green (ICG) in a CM murine model. Increased emission intensity of liposomal ICG was demonstrated in comparison with free ICG. The Liposomal ICG's emission was greater in the brains of the infected mice compared to naïve mice and drug treated mice (where CM was prevented). Histological analyses suggest that the accumulation of liposomal ICG in the cerebral vasculature is due to extensive uptake mediated by activated phagocytes. Overall, liposomal ICG offers a valuable diagnostic tool and a biomarker for effectiveness of CM treatment, as well as other diseases that involve inflammation and blood vessel occlusion.

Imaging the urinary pathways in mice by liposomal indocyanine green.

Intraoperative ureter identification can assist in the prevention of ureteral injury and consequently improve surgery outcomes. Our aim was to take advantage of the altered pharmacokinetics of liposomal indocyanine green (ICG), the only FDA-approved near-infrared (NIR) dye, for imaging of ureters during surgeries. ICG was passively adsorbed to liposomes. NIR whole mice body and isolated tissue imaging were used to study liposomal ICG properties vs. free ICG. In vivo, the urinary bladder could be clearly observed in most of the liposome-treated mice. Liposomal encapsulation of ICG enhanced ureteral emission up to 1.9 fold compared to free ICG (P<0.01). Increase in liposomal micropolarity and microviscosity and differential scanning calorimetry supported ICG localization within the liposomal bilayer. Our findings suggest that liposomal ICG could be utilized for ureteral imaging intra-operatively, thus potentially improving surgical outcomes. FROM THE CLINICAL EDITOR: Iatrogenic ureteral injury is a serious complication of abdominal surgery and intra-operative recognition of the ureters is usually the best method of injury prevention. In this article, the authors developed liposomal indocyanine green, which could be excreted via the urinary system and investigated its in-vivo use in mice.

Interactions of ABCG2 (BCRP) with epidermal growth factor receptor kinase inhibitors developed for molecular imaging.

The objective of this study was to investigate in vitro the interactions between novel epidermal growth factor receptor kinase inhibitors (EGFRIs) developed for positron emission tomography (PET) imaging and the major efflux transporter breast cancer resistance protein (BCRP/ABCG2). Seven compounds were evaluated, using the ATPase activity assays and Madin-Darbey canine kidney (MDCK) cells overexpressing BCRP. Five of the tested compounds activated BCRP ATPase to various extent. Overexpression of BCRP conferred resistance to ML04, ML06, methoxy-Br-ML03, and PEG6-ML05 (IC50 values for inhibition of control cell proliferation 2.1 ± 0.6, 2.2 ± 0.7, 1.8 ± 1.2, and 2.8 ± 3.1 µM, respectively, compared to >50 µM in MDCK-BCRP cells). At submicromolar concentrations, none of the EGFRIs significantly inhibited BCRP. Immunoblotting studies indicated that BCRP expression is evident in cell lines utilized for in vivo tumor grafting in small animal PET imaging studies. Thus, the intensity of EGFRIs radioactivity signals previously observed in tumor xenografts reflects an interplay between transporter-mediated distribution of the probe into tumor cells and target binding. Concomitant use of efflux transporter inhibitors may help distinguish between the contribution of efflux transport and EGFR binding to the tissue signal.

Toxicity mechanisms of amphotericin B and its neutralization by conjugation with arabinogalactan.

Amphotericin B (AMB) is an effective antifungal agent. However, its therapeutic use is hampered by its toxicity, mainly due to channel formation across kidney cell membranes and the disruption of postendocytic trafficking. We previously described a safe injectable AMB-arabinogalactan (AG) conjugate with neutralized toxicity. Here we studied the mechanism of the toxicity of free AMB and its neutralization by conjugation with AG. AMB treatment of a kidney cell line modulated the trafficking of three receptors (C-X-C chemokine receptor type 4 [CXCR4], M1 receptor, and human transferrin receptor [hTfnR]) due to an increase in endosomal pH. Similar data were also obtained in yeast but with an increase in vacuolar pH and the perturbation of Hxt2-green fluorescent protein (GFP) trafficking. The conjugation of AMB with AG neutralized all elements of the toxic activity of AMB in mammalian but not in fungal cells. Based on these results, we provide an explanation of how the conjugation of AMB with AG neutralizes its toxicity in mammalian cells and add to the knowledge of the mechanism of action of free AMB in both fungal and mammalian cells.

Multiple triphenylphosphonium cations as a platform for the delivery of a pro-apoptotic peptide.

PURPOSE: Triphenyl phosphonium cations (TPPs) are delocalized lipophilic cations that accumulate in the mitochondria of cells. We have explore the effect of increasing the number of TPPs on delivery of a cell-impermeable pro-apoptotic peptide to intact cells. METHODS: The pro-apoptotic peptide D-(KLAKLAK)(2) (KLA) was extended with 0-3 L-Lysines modified at their ε-amine with TPP. Peptides were studied in HeLa cells to determine their cytotoxic activity and cellular uptake. RESULTS: In HeLa cells, the increased cytotoxicity correlates with the number of TPPs; the peptide with 3 TPP molecules (3-KLA) exerts the highest cytotoxic activity. This FITC-labeled peptide is found to accumulate in intact HeLa cells, whereas peptides with 0-2 TPPs are not detected at the same peptide concentration. Mitochondria-dependent apoptosis of HeLa cells in the presence of 3-KLA was followed by propidium iodide, Annexin-V and DiOC fluorescence by FACS. CONCLUSION: A facile synthetic methodology has been presented for the delivery of a biologically active peptide into mitochondria of intact cells by attaching multiple TPP moieties to the peptide. This approach was shown to dramatically increase biological activity of the peptide as a pro-apoptotic agent.

Beta-tubulin cofactor D and ARL2 take part in apical junctional complex disassembly and abrogate epithelial structure.

In epithelial cells, the apical junctional complex (AJC), composed of tight junctions (TJs) and adherens junctions (AJs), maintains cell-surface polarity by forming a fence that prevents lateral movement and diffusion of proteins and lipids between the apical and basolateral PM and holds the epithelial monolayer intact through cell-cell contacts. Disassembly of this complex is a prime event in development and cell transformation. Maintenance of the AJC has been shown to involve mainly the actin cytoskeleton. Recent findings also point to the involvement of the microtubule (MT) system. Here we show the first evidence that in polarized epithelial MDCK cells, ARF-like protein 2 (ARL2) and beta-tubulin cofactor D, known to be involved in MT dynamics, have a role in disassembly of the AJC followed by cell dissociation from the epithelial monolayer, which is not dependent on MT depolymerization. In addition, we show that beta-tubulin cofactor D is partially localized to the lateral PM through its 15 C-terminal amino acids and intact MTs. ARL2 inhibited beta-tubulin cofactor D-dependent cell dissociation from the monolayer and AJC disassembly. To our knowledge, this is the first evidence that beta-tubulin cofactor D plays a role in cells independent of its presumed role in folding tubulin heterodimers. We conclude that ARL2 and beta-tubulin cofactor D participate in AJC disassembly and epithelial depolarization.

Caveolin 2 regulates endocytosis and trafficking of the M1 muscarinic receptor in MDCK epithelial cells.

Clathrin and caveolins are known for their involvement in the internalization of numerous receptors. Here we show that in polarized epithelial Madin-Darby canine kidney cells, both the clathrin machinery and caveolins are involved in the endocytosis and delivery to the plasma membrane (PM) of the M1 muscarinic acetylcholine receptor (mAChR). We initially localized this receptor to the lateral membrane, where it accumulates proximal to the tight junctions. From there it is internalized through the clathrin-mediated pathway. In addition, the receptor may associate on the PM with caveolin (cav) 2 or in intracellular compartments with either cav 2, or monomeric or oligomeric cav 1. Association of the PM M1 mAChR with cav 2 inhibits receptor endocytosis through the clathrin-mediated pathway or retains the receptor in an intracellular compartment. This intracellular association attenuates receptor trafficking. Expression of cav 1 with cav 2 rescues the latter's inhibitory effect. The caveolins stimulate M1 mAChR oligomerization thus maintaining a constant amount of monomeric receptor. These results provide evidence that caveolins play a role in the attenuation of the M1 muscarinic receptor's intracellular trafficking to and from the PM.

EFA6 regulates endosomal trafficking and affects early endosomes in polarized MDCK cells.

The small-GTPase family of ADP ribosylation factors (ARFs) recruit coat proteins to promote vesicle budding. ARFs are activated by an association with sec7-containing exchange factors which load them with GTP. In epithelial cells, the small GTPase ARF6 operates within the endocytic system and has been shown to associate with ARNO to promote apical endocytosis and early to late endosomal trafficking. EFA6 has been shown to stimulate tight-junction formation and maintenance. Here, we show that in polarized epithelial MDCK cells, EFA6 is localized to early endosomes, causes their dramatic enlargement, and promotes basolateral targeting of IgA, which is normally targeted to the apical PM. These results suggest that the physiological function of ARF6 within the endocytic system is regulated by the exchange factor it associates with.

ARNO through its coiled-coil domain regulates endocytosis at the apical surface of polarized epithelial cells.

ARNO is a guanine-nucleotide exchange protein for the ARF family of GTPases. Here we show that in polarized epithelial cells, ARNO is localized exclusively to the apical plasma membrane, where it regulates endocytosis. Expression of ARNO stimulates apical endocytosis of the polymeric immunoglobulin receptor, and coexpression of ARF6 with ARNO leads to a synergistic stimulation of apical endocytosis. Expression of a dominant negative ARF6 mutant, ARF6-T27N, antagonizes this stimulatory effect. Deletion of the N-terminal coiled-coil (CC) domain of ARNO causes the mutant ARNO to localize to both the apical and basolateral plasma membranes. Expression of the CC domain alone abolishes ARNO-induced apical endocytosis as well as co-localization of IgA-receptor complexes with ARNO and clathrin. These results suggest that the CC domain contributes to the specificity of apical localization of ARNO through association with components of the apical plasma membrane. We conclude that ARNO acts together with ARF6 to regulate apical endocytosis.