Scanning electron microscopy study of neutrophil membrane tubulovesicular extensions (cytonemes) and their role in anchoring, aggregation and phagocytosis. The effect of nitric oxide

We have shown that human neutrophils develop dynamic thin and very long tubulovesicular extensions (cytonemes) upon adhesion to fibronectin, if cell spreading was blocked by Na(+)-free medium or by 4-bromophenacyl bromide, N-ethylmaleimide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and cytochalasin D (S. I. Galkina, G. F. Sud'ina and V. Ullrich, (2001). Exp. Cell Res. 266, 222-228). In the present work we found that similar in size and behavior tubulovesicular extensions were formed on the neutrophil cell bodies upon adhesion to fibronectin-coated substrata in the presence of the nitric oxide donor diethylamine NONOate. In the presence of the nitric oxide synthase inhibitor N-omega-nitro-L-arginine methyl ester, neutrophils were well spread and had no microextensions. Using scanning electron microscopy, we demonstrated that tubulovesicular extensions of neutrophils executed long-range adhesion and binding objects for phagocytosis, such as serum-opsonized zymosan particles and erythrocytes. Tubulovesicular extensions anchored neutrophils to substrata in a beta1 and beta2 integrin-independent, but L-selectin-dependent manner. BODIPY-sphingomyelin impaired development of tubulovesicular extension, and heparitinase 1 played a role in their destruction. Membrane tubulovesicular extensions are supposed to represent protrusions of an intracellular exocytotic traffic and serve as cellular sensory and adhesive organelles. Nitric oxide seems to play a role in regulation of tubulovesicular extensions formation, thus affecting neutrophil adhesive interactions and phagocytosis.     

Inhibition of neutrophil spreading during adhesion to fibronectin reveals formation of long tubulovesicular cell extensions (cytonemes)

Human neutrophils developed long thin tubulovesicular extensions (cytonemes) upon adhesion to fibronectin-coated substrata, when spreading was blocked. We observed extension formation when neutrophils were plated to fibronectin-coated substrata in Na(+)-free extracellular medium or in the presence of drugs capable of inhibiting spreading: 4-bromophenacyl bromide, N-ethylmaleimide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and cytochalasin D. Addition of Na(+) ions or washing of inhibitors restored neutrophil spreading. Phase-contrast and scanning electron microscopy revealed two types of extensions: (1) highly dynamic, flexible tubulovesicular extensions with unattached tips 0.2-0.4 microm in diameter, which can achieve 70-80 microm in length during 20 min, and (2) thinner straight extensions with flattened tips, which were formed in the presence of phorbol 12-myristate 13-acetate and connected cells to substratum or to the neighboring cells several cell diameters away. The latter may have derived from the former through tension after attachment of the tips. Spreading and extension formation may represent two states of the cell adhesive and communicative mechanism.     

Intracellular pH regulation during spreading of human neutrophils

The regulation of the intracelluar pH (pHi) during spreading of human neutrophils was studied by a combination of fluorescence imaging and video microscopy. Spreading on adhesive substrates caused a rapid and sustained cytosolic alkalinization. This pHi increase was prevented by the omission of external Na+, suggesting that it results from the activation of Na+/H+ exchange. Spreading-induced alkalinization was also precluded by the compound HOE 694 at concentrations that selectively block the NHE-1 isoform of the Na+H+ antiporter. Inhibition of Na+/H+ exchange by either procedure unmasked a sizable cytosolic acidification upon spreading, indicative of intracellular acid production. The excess acid generation was caused, at least in part, by the activation of the respiratory burst, since the acidification closely correlated with superoxide production, measured in single spreading neutrophils with dihydrorhodamine-123, and little acid production was observed in the presence of diphenylene iodonium, a blocker of the NADPH oxidase. Moreover, neutrophils from chronic granulomatous disease patients, which do not produce superoxide, failed to acidify. Comparable pHi changes were observed when beta 2 integrins were selectively activated during spreading on surfaces coated with anti-CD18 antibodies. When integrin engagement was precluded by pretreatment with soluble anti-CD18 antibody, the pHi changes associated with spreading on fibrinogen were markedly reduced. Inhibition of microfilament assembly with cytochalasin D precluded spreading and concomitantly abolished superoxide production and the associated pHi changes, indicating that cytoskeletal reorganization and/or an increase in the number of adherence receptors engaged are required for the responses. Neutrophils spread normally when the oxidase was blocked or when pHi was clamped near physiological values with nigericin. Spreading, however, was strongly inhibited when pHi was clamped at acidic values. Our results indicate that neutrophils release superoxide upon spreading, generating a burst of intracellular acid production. The concomitant activation of the Na+/H+ antiport not only prevents the deleterious effects of the acid released by the NADPH oxidase, but induces a net cytosolic alkalinization. Since several functions of neutrophils are inhibited at an acidic pHi, the coordinated activation of pHi regulatory mechanisms along with the oxidase is essential for sustained microbicidal activity.     

Activation of vacuolar-type proton pumps by protein kinase C. Role in neutrophil pH regulation.

Activated neutrophils undergo a large burst of metabolic acid generation, yet maintain their cytosolic pH (pHi) within physiological limits. To analyze the underlying regulatory mechanisms, pHi was measured fluorimetrically in suspensions of human neutrophils. In acid loaded but otherwise unstimulated cells, pHi recovered rapidly via Na+/H+ exchange. Upon Na+ removal, recovery from an imposed acid load was negligible. Phorbol ester activation of acidified cells induced a rapid recovery of pHi partly due to a Zn(2+)-sensitive H(+)-conductive pathway. A third component of the regulatory response was apparent in Na(+)-free media containing Zn2+. Acid extrusion through this alternate pathway was voltage sensitive and capable of translocating H+ equivalents against their electrochemical gradient. This active H+ transport was inhibited by N-ethylmaleimide, by N,N'-dicyclohexylcarbodiimide and by nanomolar doses of bafilomycins A1 or B1, suggesting the involvement of vacuolar (V)-type H+ pumps. Cytosolic alkalinization was accompanied by extracellular acidification, indicative of translocation of H+ equivalents across the surface membrane and consistent with the sensitivity of the alkalinization to changes in plasma membrane potential. The activity of the V-type H+ pumps was virtually undetectable in resting cells, becoming apparent only after treatment with phorbol esters or other, chemically unrelated agonists of protein kinase C. These H+ pumps are likely to play a role in pHi homeostasis during the metabolic burst that accompanies neutrophil activation during infection and inflammation.     

Nitric oxide-induced membrane tubulovesicular extensions (cytonemes) of human neutrophils catch and hold Salmonella enterica serovar Typhimurium at a distance from the cell surface

Nitric oxide (NO) plays an important role in host defense against bacterial infections such as salmonellosis. NO and 4-bromophenacyl bromide (BPB) induce the formation of long tubulovesicular extensions (TVE, cytonemes, membrane tethers) from human neutrophils. These TVE serve as cellular sensory and adhesive organelles. In the present study, we demonstrated that in the presence of the NO donor, diethylamine NONOate or BPB human neutrophils bound and aggregated Salmonella enterica serovar Typhimurium bacteria extracellularly by TVE. In contrast, inhibition of NO-synthase activity by N ^ω-nitro- l -arginine methyl ester stimulated neutrophil phagocytosis (ingestion) of bacteria. Neutrophil TVE consisted of membrane-covered cytoplasm as was shown by the fluorescent cytoplasmic dye 2',7'-bis(2carboxyethyl)-5,(6)-carboxyfluorescein, and the fluorescent lipid, BODIPY-labeled sulfatide. Disruption and shedding of TVE were accompanied by the appearance of specific invaginations (porosomes) on neutrophil cell bodies. These invaginations corresponded to the variations in diameter of TVE (160–240 nm). We hypothesized that TVE represented protrusions of neutrophil exocytotic trafficking through special structures on the neutrophil surface. In conclusion, we propose a novel mechanism by which NO-induced TVE formation enables neutrophils to bind and aggregate bacteria at a distance.     

Impairment of Na+/H+ exchange underlies inhibitory effects of Na+-free media on leukocyte function

Inhibition of activation has been reported when neutrophils are suspended in Na+-free media. We considered the possibility that impairment of cellular pH (pHi) regulation due to elimination of Na+/H+ exchange underlies this effect. In the absence of Na+, the phorbol ester-induced respiratory burst was partially inhibited and a concomitant cytoplasmic acidification recorded. Using nigericin/K+ to clamp pHi we demonstrated that the acidification accounts for the inhibition of O2 uptake. Moreover, in Na+-free media, relieving the acidification by means of ionophores restored maximal O2 consumption. It was concluded that Na+ is not directly involved in signal transduction during stimulation. Instead, omission of Na+ affects neutrophils activation indirectly, by impairing pHi regulation.     

The vasodilator-stimulated phosphoprotein is regulated by cyclic GMP-dependent protein kinase during neutrophil spreading

The expression and phosphorylation state of the vasodilator-stimulated phosphoprotein (VASP), a membrane-associated focal adhesion protein, was investigated in human neutrophils. Adhesion and spreading of neutrophils induced the rapid phosphorylation of VASP. The phosphorylation of VASP was dependent on cell spreading, as VASP was expressed as a dephosphorylated protein in round adherent cells and was phosphorylated at the onset of changes in cell shape from round to spread cells. Immunofluorescence microscopy demonstrated that VASP was localized at the cell cortex in round cells and redistributed to focal adhesions at the ventral surface of the cell body during cell spreading. Dual labeling of spread cells indicated that VASP was colocalized with F-actin in filopodia and in focal adhesions, suggesting that the phosphorylation of VASP during cell spreading may be involved in focal adhesion complex organization and actin dynamics. VASP is a prominent substrate for both cGMP-dependent protein kinase (cGK) and cAMP-dependent protein kinase. Evidence suggested that cGK regulated neutrophil spreading, as both VASP phosphorylation and neutrophil spreading were inhibited by Rp-8-pCPT-cGMPS (cGK inhibitor), but not KT5720 (cAMP-dependent protein kinase inhibitor). In contrast, neutrophil spreading was accelerated when cGMP levels were elevated with 8-Br-cGMP, a direct activator of cGK. Furthermore, the same conditions that lead to VASP phosphorylation during neutrophil adherence and spreading induced significant elevations of cGMP in neutrophils. These results indicate that cGMP/cGK signal transduction is required for neutrophil spreading, and that VASP is a target for cGK regulation.     

The V-type H+-ATPase in vesicular trafficking: targeting, regulation and function

Vacuolar-type H+-ATPase (V-ATPase)-driven proton pumping and organellar acidification is essential for vesicular trafficking along both the exocytotic and endocytotic pathways of eukaryotic cells. Deficient function of V-ATPase and defects of vesicular acidification have been recently recognized as important mechanisms in a variety of human diseases and are emerging as potential therapeutic targets. In the past few years, significant progress has been made in our understanding of function, regulation, and the cell biological role of V-ATPase. Here, we will review these studies with emphasis on novel direct roles of V-ATPase in the regulation of vesicular trafficking events.     

Na(+), Cl(-), Ca(2+) and Zn(2+) transport by fish gills: retrospective review and prospective synthesis

The secondary active Cl(-) secretion in seawater (SW) teleost fish gills and elasmobranch rectal gland involves basolateral Na(+),K(+)-ATPase and NKCC, apical membrane CFTR anion channels, and a paracellular Na(+)-selective conductance. In freshwater (FW) teleost gill, the mechanism of NaCl uptake is more controversial and involves apical V-type H(+)-ATPase linked to an apical Na(+) channel, apical Cl(-)-HCO-3 exchange and basolateral Na(+),K(+)-ATPase. Ca(2+) uptake (in FW and SW) is via Ca(2+) channels in the apical membrane and Ca(2+)-ATPase in the basolateral membrane. Mainly this transport occurs in mitochondria rich (MR) chloride cells, but there is a role for the pavement cells also. Future research will likely expand in two major directions, molded by methodology: first in physiological genomics of all the transporters, including their expression, trafficking, operation, and regulation at the molecular level, and second in biotelemetry to examine multivariable components in behavioral physiological ecology, thus widening the integration of physiology from the molecular to the environmental levels while deepening understanding at all levels.     

Intracellular pH regulation and metabolic interactions in hepatic tissues.

Intracellular pH (pHi) regulation in the vertebrate liver relies heavily on ionic transport mechanisms. Liver, in common with many tissues, has plasma membrane Na(+)-H+ and Cl(-)-HCO3- electroneutral exchangers which work in opposition to tightly control pHi. Mammalian livers also possess electrogenic Na(+)-HCO3- exchangers, capable of base uptake, which, when coupled to pHi-mediated changes in membrane potential, probably confer an additional measure of pHi control, compared to fish livers, where the transporter appears to be functionally absent. It is suggested that this may be a fundamental difference between aquatic and aerial breathing. pHi regulation has barely been examined in invertebrate hepatic tissues, but already some interesting differences are apparent. Notably, an electrogenic 2Na(+)-1H+ acid-extrusion system is present in apical membranes of crustacean hepatopancreas. Despite these ionic control systems, complex acid-base disturbances (e.g., "metabolic" acidosis) have been known for some time to influence hepatic metabolism in vertebrates, but few studies have carefully examined the independent effects of the acid-base variables involved. Thus mechanistic explanations for the effects of acid-base disturbances are scarce. Ureogenesis in mammals has been well studied, and several pH-related mechanisms are evident. In contrast, the pH-insensitivity of ureogenesis in fish liver may represent a second difference between aquatic and terrestrial species. In summary, by virtue of its metabolic diversity, liver represents a potentially important organ in acid-base balance, and an interesting study tissue for interrelationships between metabolism and acid-base balance.     

Regulation of intracellular chloride concentration in rat lactotrope cells and its relation to the membrane resting potential

Rat lactotrope cells in primary culture exhibit physiological properties closely associated with chloride ions (Cl-) homeostasis. In this work, we studied the regulation of intracellular Cl- concentrations ([Cl-]i) and its relation to the membrane resting potential, using a combination of electrophysiology and spectrofluorimetry. Variations in [Cl-]i resulting from the patch clamp technique, pHi, antagonists of Cl(-)-Ca(2+)-dependent channels, an anion exchanger antagonist, and an antagonist of K(+)-Cl- cotransport were considered with respect to their involvement in membrane potential. We show that: (i) The patch-pipette does not always impose its Cl- concentration. (ii) In rat lactotrope cells, membrane resting potential is partially determined by [Cl-]i. (iii) Besides ion channel activity, electroneutral ion transports (cotransports such as K(+)-Cl- and Na(+)-K(+)-2Cl-) participate actively in maintaining a high [Cl-]i. (iv) Finally, Cl- homeostasis is probably linked to cell energetics.     

Immunolocalization of Vacuolar-Type H(+)-ATPase in the Yolk-Sac Membrane of Tilapia (Oreochromis mossambicus) Larvae

To investigate the involvement of the yolk-sac membrane in ion absorption, developmental changes in whole-body cation contents, cellular localization of vacuolar-type H(+)-ATPase (V-ATPase), and size and density of pavement and chloride cells in the yolk-sac membrane were examined in tilapia (Oreochromis mossambicus) larvae in fresh water (FW) and those transferred to seawater (SW) at 2 days before hatching (day-2). The whole-body content of Na(+) in embryos and larvae adapted to both FW and SW increased constantly from day-2 to day 10, although they were not fed through the experiment. The yolk-sac membrane of FW larvae at days 0 and 2 showed V-ATPase immunoreactivity in pavement cells, but not in chloride cells. No positive immunoreactivity was detected in SW larvae. Whole-mount immunocytochemistry showed that some pavement cells were intensively immunoreactive, whereas others were less or not immunoreactive. Electron-microscopic immunocytochemistry revealed that V-ATPase immunoreactivity was present in the apical regions of pavement cells in FW larvae, especially in their ridges. The pavement cells in FW larvae were significantly smaller in size but higher in density than those in SW. These results suggest that pavement cells are the site of active Na(+) uptake in exchange for H(+) secretion through V-ATPase in FW-adapted tilapia during early life stages.     

Intracellular pH regulation in isolated hepatopancreas cells from the Roman snail (Helix pomatia)

The mechanisms of intracellular pH (pHi) regulation were studied in isolated hepatopancreas cells from the Roman snail, Helix pomatia. The relationship between intracellular and extracellular pH indicated that pHi is actively regulated in these cells. At least three pHi-regulatory ion transporters were found to be present in these cells and to be responsible for the maintenance of pHi: an amiloride-sensitive Na+/H+ exchanger, a 4-acetamido-4'-isothiocyanostilbene-2,2'disulfonic acid (SITS)-sensitive, presumably Na(+)-dependent, Cl-/HCO3-exchanger, and a bafilomycin-sensitive H(+)-pump. Inhibition of one of these transporters alone did not affect steady state pHi, whereas incubation with amiloride and SITS in combination resulted in a significant intracellular acidification. Following the induction of intracellular acidosis by addition of the weak acid Na+propionate, the Na+/H+ exchanger was immediately activated leading to a rapid recovery of pHi towards the baseline level. Both the SITS-sensitive mechanism and the H(+)-pump responded more slowly, but were of similar importance for pHi recovery. Measurement of pHi recovery from acidification in the three discernible types of hepatopancreas cells with a video fluorescence image system revealed slightly differing response patterns, the physiological significance of which remains to be determined.     

Interrelationship between insulin-like growth factor I-induced activation of the Na+/H(+)-antiporter and intracellular Ca(2+)-mobilization in thyroid cells.

Insulin-like growth factor I (IGF-I) increased cytoplamic pH (pHi) and cytoplasmic Ca2+ [( Ca2+]i) in cultured porcine thyroid cells. Inhibition of the Na+/H(+)-antiporter by dimethylamiloride or a reduction of external Na(+)-concentrations attenuates the increases in pHi and [Ca2+]i. The [Ca2+]i response to IGF-I is a pHi-dependent process. IGF-I activates Na+/H(+)-antiporter and alkalinizes thyroid cells. The resulting increase in pHi facilitates the [Ca2+]i response by adjusting the pHi closer to the pHi-optimum of the intracellular Ca(2+)-mobilizing system. One of the biological functions of IGF-I-induced activation of the Na+/H(+)-antiporter is to shift the pHi to an optimal value for the [Ca2+]i response.     

Microbial alkaloid staurosporine induces formation of nanometer-wide membrane tubular extensions (cytonemes,membrane tethers) in human neutrophils

In the present work, we demonstrate that microbial alkaloid staurosporine (STS) and Ro 31-8220, structurally related to STS protein kinase C inhibitor, caused development of membrane tubular extensions in human neutrophils upon adhesion to fibronectin-coated substrata. STS-induced tubular extensions interconnected neutrophils in a network and bound serum-opsonized bacteria Salmonella enterica serovar Typhimurium. The diameter of STS-induced extensions varied in the range 160-200 nm. The extensions were filled with cytoplasm and covered with membrane, as they included fluorescent cytoplasmic and lipid dyes. Neither protein kinase C inhibitors H-7 and bisindolylmaleimide VII, nor tyrosine protein kinase inhibitors tyrphostin AG 82 and genistein caused such extensions formation. Supposedly, STS induces membrane tubular extension formation promoting actin cytoskeleton depolymerization or affecting NO synthesis.     

Spreading of HeLa cells on a collagen substratum requires a second messenger formed by the lipoxygenase metabolism of arachidonic acid released by collagen receptor clustering.

HeLa cells attach to a variety of substrata but spread only on collagen or gelatin. Spreading is dependent on collagen-receptor upregulation, clustering, and binding to the cytoskeleton. This study examines whether second messengers are involved in initiating the spreading process on gelatin. The levels of cytosolic free calcium ([Ca++]i), cAMP, and cytoplasmic pH (pHi) do not change during cell attachment and spreading. However, a basal level of [Ca++]i and an alkaline pH(i) are required for spreading. There is an activation of protein kinase C (PKC) and a release of arachidonic acid (AA) on attachment and before cell spreading. Inhibition of PKC does not block cell spreading, indicating that PKC activation is not essential for spreading. Inhibition of phospholipase A2 blocks cell spreading, whereas addition of exogeneous AA overcomes this inhibitory effect. Among AA metabolic pathways, inhibitors of lipoxygenase (LOX) block cell spreading, suggesting that a LOX product(s) formed from AA initiates spreading. Clustering receptors for collagen with polyclonal antibodies, or with anti-collagen-receptor antigen-binding fragments (Fab) in combination with a secondary antibody, induce AA release. Also, AA is released when cells attach to either immobilized gelatin or immobilized Arg-Gly-Asp (RGD) peptide. Thus, AA is released whenever receptor clustering is observed. Receptor occupancy is not sufficient to release AA; when cells are treated with gelatin or RGD peptide in solution or anti-collagen-receptor Fab fragments without secondary antibody, conditions where receptor clustering is not observed, AA is not released. Thus, a LOX metabolite(s) of AA formed by collagen-receptor clustering is a second messenger(s) that initiates HeLa cell spreading. LOX inhibitors also block the spreading of bovine aortic endothelial cells, chicken embryo fibroblasts, and CV-1 fibroblasts on gelatin or fibronectin, indicating that other cells might use the same second messenger system in initiating cell-substratum adhesion.     

Intracellular ph and the substrate dependence of Chinese hamster fibroblast proliferation]

Data have been presented on the effect of serum and of cell adhesion to a solid substrate on the intracellular pH (pHi) value in anchorage-dependent Chinese hamster fibroblasts. Proliferation of cells was observed in a pHi range from 7.0 to 7.5, which exceeded by 0.3-0.4 the pHi of quiescent cells. It was shown that attachment and spreading of cells in a bicarbonate-containing media without serum produced elevation of pHi from 6.7 to 7.1 but did not provide such an alkalization value for times greater than the lag period of cell growth. By the end of the first day after plating the pHi value of the spread cells in a serum-free medium reduced to 6.75, and the cells ceased to grow. The serum without cell attachment produced only a short-time (less than 1 h) pHi increase from 6.7 to 6.88, which was inadequate for cell growth. However, the serum produced a prolongation in cytoplasm alkalization characteristic of proliferation and caused by attachment of cells to a solid substrates. Evidence also was obtained for the absence of Na-independent HCO3-/Cl- exchange, the presence of a slow HCO3- transport into the cell and the key role of Na+/H+ exchange in pHi regulation. Addition to the bicarbonate-containing medium of amiloride, a blocker of Na+/H+ exchange, resulted in acidification of the cytoplasm to 6.5, and inhibited cell attachment and proliferation.     

Intracellular pH and Catecholamine Secretion from Bovine Adrenal Chromaffin Cells

To study the role of intracellular pH (pHi) in catecholamine secretion and the regulation of pHi in bovine chromaffin cells, the pH-sensitive fluorescent indicator [2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein] was used to monitor the on-line changes in pHi. The pHi of chromaffin cells at resting state is approximately 7.2. The pHi was manipulated first by incubation of the cells with NH4+, and then the solution was replaced with a NH4(+)-free solution to induce acidification of the cytoplasm. The pHi returned toward the basal pH value after acidification within 5-10 min in the presence of Na+ or Li+, but the pHi stayed acidic when Na(+)-free buffers were used or in the presence of amiloride and its analogues. These results suggest that the pH recovery process after an acid load is due to the Na+/H+ exchange activity in the plasma membrane of the chromaffin cells. The catecholamine secretion evoked by carbachol and Na+ removal was enhanced after the cytoplasm had been made more acidic. It appears that acidic pH favors the occurrence of exocytosis.     

Tamoxifen does not inhibit the swell activated chloride channel in human neutrophils during the respiratory burst

Effective functioning of neutrophils relies upon electron translocation through the NADPH oxidase (NOX). The electron current generated (I_e) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential in activated human neutrophils. Swelling activated chloride channels have been demonstrated in part to counteract the depolarisation generated by the NADPH oxidase I_e. In the present study, the effects of inhibitors of swell activated chloride channels on ROS production and on the swelling activated chloride conductance was investigated in activated human neutrophils. Tamoxifen (10 μM), a specific inhibitor for swell activated chloride channels in neutrophils, completely inhibited both the PMA and FMLP stimulated respiratory burst. This inhibition of the neutrophil respiratory burst was not due to the blocking effect of tamoxifen on the swelling activated chloride conductance in these cells. These results demonstrate that a tamoxifen insensitive swell activated chloride channel has important significance during the neutrophil respiratory burst.     

V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway

The recruitment of the small GTPase Arf6 and ARNO from cytosol to endosomal membranes is driven by V-ATPase-dependent intra-endosomal acidification. The molecular mechanism that mediates this pH-sensitive recruitment and its role are unknown. Here, we demonstrate that Arf6 interacts with the c-subunit, and ARNO with the a2-isoform of V-ATPase. The a2-isoform is targeted to early endosomes, interacts with ARNO in an intra-endosomal acidification-dependent manner, and disruption of this interaction results in reversible inhibition of endocytosis. Inhibition of endosomal acidification abrogates protein trafficking between early and late endosomal compartments. These data demonstrate the crucial role of early endosomal acidification and V-ATPase/ARNO/Arf6 interactions in the regulation of the endocytic degradative pathway. They also indicate that V-ATPase could modulate membrane trafficking by recruiting and interacting with ARNO and Arf6; characteristics that are consistent with the role of V-ATPase as an essential component of the endosomal pH-sensing machinery.