Dynamic multimerization of Dab2-Myosin VI complexes regulates cargo processivity while minimizing cortical actin reorganization.

Myosin VI ensembles on endocytic cargo facilitate directed transport through a dense cortical actin network. Myosin VI is recruited to clathrin-coated endosomes via the cargo adaptor Dab2. Canonically, it has been assumed that the interactions between a motor and its cargo adaptor are stable. However, it has been demonstrated that the force generated by multiple stably attached motors disrupts local cytoskeletal architecture, potentially compromising transport. In this study, we demonstrate that dynamic multimerization of myosin VI-Dab2 complexes facilitates cargo processivity without significant reorganization of cortical actin networks. Specifically, we find that Dab2 myosin interacting region (MIR) binds myosin VI with a moderate affinity (184 nM) and single-molecule kinetic measurements demonstrate a high rate of turnover (1 s-1) of the Dab2 MIR-myosin VI interaction. Single-molecule motility shows that saturating Dab2-MIR concentration (2 µM) promotes myosin VI homodimerization and processivity with run lengths comparable with constitutive myosin VI dimers. Cargo-mimetic DNA origami scaffolds patterned with Dab2 MIR-myosin VI complexes are weakly processive, displaying sparse motility on single actin filaments and "stop-and-go" motion on a cellular actin network. On a minimal actin cortex assembled on lipid bilayers, unregulated processive movement by either constitutive myosin V or VI dimers results in actin remodeling and foci formation. In contrast, Dab2 MIR-myosin VI interactions preserve the integrity of a minimal cortical actin network. Taken together, our study demonstrates the importance of dynamic motor-cargo association in enabling cargo transportation without disrupting cytoskeletal organization.

Mechanical Regulation of Endocytosis: New Insights and Recent Advances.

Endocytosis is a mechanosensitive process. It involves remodeling of the plasma membrane from a flat shape to a budded morphology, often at the sub-micrometer scale. This remodeling process is energy-intensive and is influenced by mechanical factors such as membrane tension, membrane rigidity, and physical properties of cargo and extracellular surroundings. The cellular responses to a variety of mechanical factors by distinct endocytic pathways are important for cells to counteract rapid and extreme disruptions in the mechanohomeostasis of cells. Recent advances in microscopy and mechanical manipulation at the cellular scale have led to new discoveries of mechanoregulation of endocytosis by the aforementioned factors. While factors such as membrane tension and membrane rigidity are generally shown to inhibit endocytosis, other mechanical stimuli have complex relationships with endocytic pathways. At this juncture, it is now possible to utilize experimental techniques to interrogate theoretical predictions on mechanoregulation of endocytosis in cells and even living organisms.

ß-Adrenergic receptor structure and function: molecular insights guiding the development of novel therapeutic strategies to treat malignancy.

ß adrenergic receptors mediate effects via activation of G proteins, transactivation of membrane growth factor receptors, or ß adrenergic receptor-ß arrestin-facilitated scaffold-mediated signaling. Agonist occupancy of the ß adrenergic receptor induces desensitization by promoting ß adrenergic receptor kinase phosphorylation of the carboxyl terminal domain, facilitating binding of the amino terminal of the ß arrestin, which sterically inhibits interactions between ß adrenergic receptors and G proteins and induces clathrin-coated pit-mediated receptor endocytosis. Scaffold formation promoted by ß arrestin binding to the ß adrenergic receptor activates extracellular regulated kinase 1/2 in a manner which elicits cytosolic retention of, and prevents promotion of nuclear transcriptional activity by, mitogen-activated protein kinase. The ß adrenergic receptor kinase also interacts with a yet to be determined microsomal membrane protein via high-affinity electrostatic interactions. We evaluate ß adrenergic receptor structure, function, and downstream signaling and ß arrestin-mediated desensitization, receptor endocytosis, and scaffold-facilitated signal transduction in order to illumine therapeutic strategies designed to modulate these pathways. We trust these approaches may arm us with the capacity to selectively modulate signal transduction pathways regulating cellular proliferation, immunogenicity, angiogenesis, and invasive and metastatic potential implicated in cancer initiation, promotion, and progression.

Dynamics of Auxilin 1 and GAK in clathrin-mediated traffic.

Clathrin-coated vesicles lose their clathrin lattice within seconds of pinching off, through the action of the Hsc70 "uncoating ATPase." The J- and PTEN-like domain-containing proteins, auxilin 1 (Aux1) and auxilin 2 (GAK), recruit Hsc70. The PTEN-like domain has no phosphatase activity, but it can recognize phosphatidylinositol phosphate head groups. Aux1 and GAK appear on coated vesicles in successive transient bursts, immediately after dynamin-mediated membrane scission has released the vesicle from the plasma membrane. These bursts contain a very small number of auxilins, and even four to six molecules are sufficient to mediate uncoating. In contrast, we could not detect auxilins in abortive pits or at any time during coated pit assembly. We previously showed that clathrin-coated vesicles have a dynamic phosphoinositide landscape, and we have proposed that lipid head group recognition might determine the timing of Aux1 and GAK appearance. The differential recruitment of Aux1 and GAK correlates with temporal variations in phosphoinositide composition, consistent with a lipid-switch timing mechanism.

Temporal Ordering in Endocytic Clathrin-Coated Vesicle Formation via AP2 Phosphorylation.

Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the µ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. µ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with µ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.

Chlamydia trachomatis CT229 Subverts Rab GTPase-Dependent CCV Trafficking Pathways to Promote Chlamydial Infection.

Chlamydial infection requires the formation of a membrane-bound vacuole, termed the inclusion, that undergoes extensive interactions with select host organelles. The importance of the Inc protein CT229 in the formation and maintenance of the chlamydial inclusion was recently highlighted by studies demonstrating that its absence during infection results in reduced bacterial replication, premature inclusion lysis, and host cell death. Previous reports have indicated that CT229 binds Rab GTPases; however, the physiological implications of this interaction are unknown. Here, we show that CT229 regulates host multivesicular trafficking by recruiting multiple Rab GTPases and their cognate effectors to the inclusion. We demonstrate that CT229 specifically modulates clathrin-coated vesicle trafficking and regulates the trafficking of transferrin and the mannose-6-phosphate receptor, both of which are crucial for proper chlamydial development. This study highlights CT229 as a master regulator of multiple host vesicular trafficking pathways essential for chlamydial infection.

Role of clathrin-mediated endocytosis in the use of heme and hemoglobin by the fungal pathogen Cryptococcus neoformans.

Heme is a major source of iron for pathogens of humans, and its use is critical in determining the outcome of infection and disease. Cryptococcus neoformans is an encapsulated fungal pathogen that causes life-threatening infections in immunocompromised individuals. C. neoformans effectively uses heme as an iron source, but the underlying mechanisms are poorly defined. Non-iron metalloporphyrins (MPPs) are toxic analogues of heme and are thought to enter microbial cells via endogenous heme acquisition systems. We therefore carried out a mutant screen for susceptibility against manganese MPP (MnMPP) to identify new components for heme uptake in C. neoformans. We identified several genes involved in signalling, DNA repair, sugar metabolism, and trafficking that play important roles in susceptibility to MnMPP and in the use of heme as an iron source. We focused on investigating the role of clathrin-mediated endocytosis (CME) and found that several components of CME including Chc1, Las17, Rvs161, and Rvs167 are required for growth on heme and hemoglobin and for endocytosis and intracellular trafficking of these molecules. We show that the hemoglobin uptake process in C. neoformans involves clathrin heavy chain, Chc1, which appears to colocalise with hemoglobin-containing vesicles and to potentially assist in proper delivery of hemoglobin to the vacuole. Additionally, C. neoformans strains lacking Chc1, Las17, Rvs161, or Rvs167 were defective in the elaboration of several key virulence factors, and a las17 mutant was avirulent in a mouse model of cryptococcosis. Overall, this study unveils crucial functions of CME in the use of heme iron by C. neoformans and reveals a role for CME in fungal pathogenesis.

Varicella-Zoster Virus ORF9p Binding to Cellular Adaptor Protein Complex 1 Is Important for Viral Infectivity.

ORF9p (homologous to herpes simplex virus 1 [HSV-1] VP22) is a varicella-zoster virus (VZV) tegument protein essential for viral replication. Even though its precise functions are far from being fully described, a role in the secondary envelopment of the virus has long been suggested. We performed a yeast two-hybrid screen to identify cellular proteins interacting with ORF9p that might be important for this function. We found 31 ORF9p interaction partners, among which was AP1M1, the µ subunit of the adaptor protein complex 1 (AP-1). AP-1 is a heterotetramer involved in intracellular vesicle-mediated transport and regulates the shuttling of cargo proteins between endosomes and the trans-Golgi network via clathrin-coated vesicles. We confirmed that AP-1 interacts with ORF9p in infected cells and mapped potential interaction motifs within ORF9p. We generated VZV mutants in which each of these motifs was individually impaired and identified leucine 231 in ORF9p to be critical for the interaction with AP-1. Disrupting ORF9p binding to AP-1 by mutating leucine 231 to alanine in ORF9p strongly impaired viral growth, most likely by preventing efficient secondary envelopment of the virus. Leucine 231 is part of a dileucine motif conserved among alphaherpesviruses, and we showed that VP22 of Marek's disease virus and HSV-2 also interacts with AP-1. This indicates that the function of this interaction in secondary envelopment might be conserved as well.IMPORTANCE Herpesviruses are responsible for infections that, especially in immunocompromised patients, can lead to severe complications, including neurological symptoms and strokes. The constant emergence of viral strains resistant to classical antivirals (mainly acyclovir and its derivatives) pleads for the identification of new targets for future antiviral treatments. Cellular adaptor protein (AP) complexes have been implicated in the correct addressing of herpesvirus glycoproteins in infected cells, and the discovery that a major constituent of the varicella-zoster virus tegument interacts with AP-1 reveals a previously unsuspected role of this tegument protein. Unraveling the complex mechanisms leading to virion production will certainly be an important step in the discovery of future therapeutic targets.

Mechanisms of Carrier Formation during Clathrin-Independent Endocytosis.

Clathrin-independent endocytosis (CIE) mediates the cellular uptake of many extracellular ligands, receptors, and pathogens, including several life-threatening bacterial toxins and viruses. So far, our understanding of CIE carrier formation has lagged behind that of clathrin-coated vesicles. Impediments have been the imprecise definition of some CIE pathways, the lack of specific cargoes being transported and of exclusive cytosolic markers and regulators. Notwithstanding these limitations, three distinct molecular mechanisms by which CIE carriers form can be defined. Cargo capture by cytosolic proteins is the main mechanism used by fast endophilin-mediated endocytosis (FEME) and interleukin 2 receptor (IL-2R) endocytosis. Acute signaling-induced membrane remodeling drives macropinocytosis. Finally, extracellular lipid or cargo clustering by the glycolipid-lectin (GL-Lect) hypothesis mediates the uptake of Shiga and cholera toxins and receptors by the CLIC/GEEC pathway. Here, we review these mechanisms and highlight current gaps in knowledge that will need to be addressed to complete our understanding of CIE.

Precise tracking of the dynamics of multiple proteins in endocytic events.

Endocytosis is a complex and dynamic process that involves dozens of different proteins to define the site of endocytosis, form a membrane invagination, and pinch off a membrane vesicle into the cytoplasm. Fluorescent light microscopy is a powerful tool to visualize the dynamic behaviors of the proteins taking part in the endocytic process. The resolution of light microscopy is, however, a serious limitation. Here, we detail a fluorescence microscope method that we have developed to visualize the dynamics of the clathrin-mediated endocytic protein machinery in yeast cells. This method is based on subpixel centroid tracking of endocytic proteins. For each endocytic protein, the centroid trajectories obtained from multiple endocytic events are used to compute an average trajectory that describes, at nanometer scale, the assembly and movement of the protein during endocytosis. The average trajectories of the different endocytic proteins are then aligned together in space and time to reconstruct how the different proteins behave relative to each other during the endocytic process.

NECAP2 controls clathrin coat recruitment to early endosomes for fast endocytic recycling.

Endocytic recycling returns receptors to the plasma membrane following internalization and is essential to maintain receptor levels on the cell surface, re-sensitize cells to extracellular ligands and for continued nutrient uptake. Yet, the protein machineries and mechanisms that drive endocytic recycling remain ill-defined. Here, we establish that NECAP2 regulates the endocytic recycling of EGFR and transferrin receptor. Our analysis of the recycling dynamics revealed that NECAP2 functions in the fast recycling pathway that directly returns cargo from early endosomes to the cell surface. In contrast, NECAP2 does not regulate the clathrin-mediated endocytosis of these cargos, the degradation of EGFR or the recycling of transferrin along the slow, Rab11-dependent recycling pathway. We show that protein knockdown of NECAP2 leads to enlarged early endosomes and causes the loss of the clathrin adapter AP-1 from the organelle. Through structure-function analysis, we define the protein-binding interfaces in NECAP2 that are crucial for AP-1 recruitment to early endosomes. Together, our data identify NECAP2 as a pathway-specific regulator of clathrin coat formation on early endosomes for fast endocytic recycling.

Auxilin facilitates membrane traffic in the early secretory pathway.

Coat protein complexes contain an inner shell that sorts cargo and an outer shell that helps deform the membrane to give the vesicle its shape. There are three major types of coated vesicles in the cell: COPII, COPI, and clathrin. The COPII coat complex facilitates vesicle budding from the endoplasmic reticulum (ER), while the COPI coat complex performs an analogous function in the Golgi. Clathrin-coated vesicles mediate traffic from the cell surface and between the trans-Golgi and endosome. While the assembly and structure of these coat complexes has been extensively studied, the disassembly of COPII and COPI coats from membranes is less well understood. We describe a proteomic and genetic approach that connects the J-domain chaperone auxilin, which uncoats clathrin-coated vesicles, to COPII and COPI coat complexes. Consistent with a functional role for auxilin in the early secretory pathway, auxilin binds to COPII and COPI coat subunits. Furthermore, ER-Golgi and intra-Golgi traffic is delayed at 15°C in swa2Δ mutant cells, which lack auxilin. In the case of COPII vesicles, we link this delay to a defect in vesicle fusion. We propose that auxilin acts as a chaperone and/or uncoating factor for transport vesicles that act in the early secretory pathway.

A phosphotyrosine switch for cargo sequestration at clathrin-coated buds.

The AP-2 clathrin adaptor complex oversees endocytic cargo selection in two parallel but independent manners. First, by physically engaging peptide-based endocytic sorting signals, a subset of clathrin-dependent transmembrane cargo is directly collected into assembling buds. Synchronously, by interacting with an assortment of clathrin-associated sorting proteins (CLASPs) that independently select different integral membrane cargo for inclusion within the incipient bud, AP-2 handles additional cargo capture indirectly. The distal platform subdomain of the AP-2 ß2 subunit appendage is a privileged CLASP-binding surface that recognizes a cognate, short α-helical interaction motif. This signal, found in the CLASPs ß-arrestin and the autosomal recessive hypercholesterolemia (ARH) protein, docks into an elongated groove on the ß2 appendage platform. Tyr-888 is a critical constituent of this spatially confined ß2 appendage contact interface and is phosphorylated in numerous high-throughput proteomic studies. We find that a phosphomimetic Y888E substitution does not interfere with incorporation of expressed ß2-YFP subunit into AP-2 or alter AP-2 deposition at surface clathrin-coated structures. The Y888E mutation does not affect interactions involving the sandwich subdomain of the ß2 appendage, indicating that the mutated appendage is folded and operational. However, the Y888E, but not Y888F, switch selectively uncouples interactions with ARH and ß-arrestin. Phyogenetic conservation of Tyr-888 suggests that this residue can reversibly control occupancy of the ß2 platform-binding site and, hence, cargo sorting.

Caveolae regulate Smad signaling as verified by novel imaging and system biology approaches.

The contribution of caveolae in Bone Morphogenetic Protein 2 (BMP2) activated Smad signaling was quantified using a system biology approach. BMP2 plays crucial roles during processes such as hematopoiesis, embryogenesis, and skeletal development. BMP2 signaling is tightly regulated on the plasma membrane by its receptors. The localization of BMP receptors in caveolae and endocytosis through clathrin-coated pits are thought to regulate the signaling; however the conclusions in the current literature are inconsistent. Therefore published literature was used to establish a mathematical model that was validated using confocal AFM (atomic force microscopy), confocal microscopy, and sucrose density centrifugation followed by Western blots, and reporter gene assays. The model and experiments confirmed that both caveolae and CCPs regulate the Smad-dependent signaling pathway, however caveolae are centers at the plasma membrane where receptor-ligand interaction is crucial, Smad phosphorylation occurs, and a high degree of Smad signaling is regulated. This demonstrates a role for caveolae that needs to be considered and further studied.

BRAG2/GEP100/IQSec1 interacts with clathrin and regulates α5ß1 integrin endocytosis through activation of ADP ribosylation factor 5 (Arf5).

ADP ribosylation factors (Arfs) are small GTP-binding proteins known for their role in vesicular transport, where they nucleate the assembly of coat protein complexes at sites of carrier vesicle formation. Similar to other GTPases, Arfs require guanine nucleotide exchange factors to catalyze GTP loading and activation. One subfamily of ArfGEFs, the BRAGs, has been shown to activate Arf6, which acts in the endocytic pathway to control the trafficking of a subset of cargo proteins including integrins. We have previously shown that BRAG2 modulates cell adhesion by regulating integrin surface expression. Here, we show that, in addition to Arf6, endogenous BRAG2 also activates the class II Arfs, Arf4 and Arf5, and that surprisingly, it is Arf5 that mediates integrin internalization. We observed that cell spreading on fibronectin is enhanced upon inhibition of BRAG2 or Arf5 but not Arf6. Similarly, spreading in BRAG2-depleted cells is reverted by expression of a rapid cycling Arf5 mutant (T161A) but not by a corresponding Arf6 construct (T157A). We also show that BRAG2 binds clathrin and the AP-2 adaptor complex and that both BRAG2 and Arf5 localize to clathrin-coated pits at the plasma membrane. Consistent with these observations, depletion of Arf5, but not Arf6 or Arf4, slows internalization of ß1 integrins without affecting transferrin receptor uptake. Together, these findings indicate that BRAG2 acts at clathrin-coated pits to promote integrin internalization by activating Arf5 and suggest a previously unrecognized role for Arf5 in clathrin-mediated endocytosis of specific cargoes.

TBC-8, a putative RAB-2 GAP, regulates dense core vesicle maturation in Caenorhabditis elegans.

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2-specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation.

Fast modulation of µ-opioid receptor (MOR) recycling is mediated by receptor agonists.

The µ-opioid receptor (MOR) is a member of the G protein-coupled receptor family and the main target of endogenous opioid neuropeptides and morphine. Upon activation by ligands, MORs are rapidly internalized via clathrin-coated pits in heterologous cells and dissociated striatal neurons. After initial endocytosis, resensitized receptors recycle back to the cell surface by vesicular delivery for subsequent cycles of activation. MOR trafficking has been linked to opioid tolerance after acute exposure to agonist, but it is also involved in the resensitization process. Several studies describe the regulation and mechanism of MOR endocytosis, but little is known about the recycling of resensitized receptors to the cell surface. To study this process, we induced internalization of MOR with [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) and morphine and imaged in real time single vesicles recycling receptors to the cell surface. We determined single vesicle recycling kinetics and the number of receptors contained in them. Then we demonstrated that rapid vesicular delivery of recycling MORs to the cell surface was mediated by the actin-microtubule cytoskeleton. Recycling was also dependent on Rab4, Rab11, and the Ca(2+)-sensitive motor protein myosin Vb. Finally, we showed that recycling is acutely modulated by the presence of agonists and the levels of cAMP. Our work identifies a novel trafficking mechanism that increases the number of cell surface MORs during acute agonist exposure, effectively reducing the development of opioid tolerance.

MiR-3120 is a mirror microRNA that targets heat shock cognate protein 70 and auxilin messenger RNAs and regulates clathrin vesicle uncoating.

We show that a single gene locus gives rise to two fully processed and functional miRNAs, i.e. that due to imperfect base pairing, two distinct microRNAs (miRNAs) can be produced from the fully complementary DNA strands. The antisense strand encodes miR-214, which is transcribed by its own promoter, whereas a novel miRNA, miR-3120, is co-expressed with its host gene mRNA. We also found that miR-3120 regulates important aspects of cellular function that are similar to that of its host gene, dynamin-3. miR-3120 was found to be located in neuronal cell bodies and to target Hsc70 and auxilin, and its lentivirus-mediated expression inhibited the uncoating of clathrin-coated vesicles. Finally, mirror miRNAs are likely to represent a new group of miRNAs with complex roles in coordinating gene expression.

NADPH oxidase is internalized by clathrin-coated pits and localizes to a Rab27A/B GTPase-regulated secretory compartment in activated macrophages.

Here, we report that activation of different types of tissue macrophages, including microglia, by lipopolysaccharide (LPS) or GM-CSF stimulation correlates with the quantitative redistribution of NADPH oxidase (cyt b(558)) from the plasma membrane to an intracellular stimulus-responsive storage compartment. Cryo-immunogold labeling of gp91(phox) and CeCl(3) cytochemistry showed the presence of gp91(phox) and oxidant production in numerous small (<100 nm) vesicles. Cell homogenization and sucrose gradient centrifugation in combination with transferrin-HRP/DAB ablation showed that more than half of cyt b(558) is present in fractions devoid of endosomal markers, which is supported by morphological evidence to show that the cyt b(558)-containing compartment is distinct from endosomes or biosynthetic organelles. Streptolysin-O-mediated guanosine 5'-3-O-(thio)triphosphate loading of Ra2 microglia caused exocytosis of a major complement of cyt b(558) under conditions where lysosomes or endosomes were not mobilized. We establish phagocytic particles and soluble mediators ATP, TNFα, and CD40L as physiological inducers of cyt b(558) exocytosis to the cell surface, and by shRNA knockdown, we identify Rab27A/B as positive or negative regulators of vesicular mobilization to the phagosome or the cell surface, respectively. Exocytosis was followed by clathrin-dependent internalization of cyt b(558), which could be blocked by a dominant negative mutant of the clathrin-coated pit-associated protein Eps15. Re-internalized cyt b(558) did not reach lysosomes but associated with recycling endosomes and undefined vesicular elements. In conclusion, cyt b(558) depends on clathrin for internalization, and in activated macrophages NADPH oxidase occupies a Rab27A/B-regulated secretory compartment, which allows rapid agonist-induced redistribution of superoxide production in the cell.

Disruption of kv1.3 channel forward vesicular trafficking by hypoxia in human T lymphocytes.

Hypoxia in solid tumors contributes to decreased immunosurveillance via down-regulation of Kv1.3 channels in T lymphocytes and associated T cell function inhibition. However, the mechanisms responsible for Kv1.3 down-regulation are not understood. We hypothesized that chronic hypoxia reduces Kv1.3 surface expression via alterations in membrane trafficking. Chronic hypoxia decreased Kv1.3 surface expression and current density in Jurkat T cells. Inhibition of either protein synthesis or degradation and endocytosis did not prevent this effect. Instead, blockade of clathrin-coated vesicle formation and forward trafficking prevented the Kv1.3 surface expression decrease in hypoxia. Confocal microscopy revealed an increased retention of Kv1.3 in the trans-Golgi during hypoxia. Expression of adaptor protein-1 (AP1), responsible for clathrin-coated vesicle formation at the trans-Golgi, was selectively down-regulated by hypoxia. Furthermore, AP1 down-regulation increased Kv1.3 retention in the trans-Golgi and reduced Kv1.3 currents. Our results indicate that hypoxia disrupts AP1/clathrin-mediated forward trafficking of Kv1.3 from the trans-Golgi to the plasma membrane thus contributing to decreased Kv1.3 surface expression in T lymphocytes.