Exocytosis of CTLA-4 is dependent on phospholipase D and ADP ribosylation factor-1 and stimulated during activation of regulatory T cells

CTLA-4 is an essential protein in the regulation of T cell responses that interacts with two ligands found on the surface of APCs (CD80 and CD86). CTLA-4 is itself poorly expressed on the T cell surface and is predominantly localized to intracellular compartments. We have studied the mechanisms involved in the delivery of CTLA-4 to the cell surface using a model Chinese hamster ovary cell system and compared this with activated and regulatory human T cells. We have shown that expression of CTLA-4 at the plasma membrane (PM) is controlled by exocytosis of CTLA-4-containing vesicles and followed by rapid endocytosis. Using selective inhibitors and dominant negative mutants, we have shown that exocytosis of CTLA-4 is dependent on the activity of the GTPase ADP ribosylation factor-1 and on phospholipase D activity. CTLA-4 was identified in a perinuclear compartment overlapping with the cis-Golgi marker GM-130 but did not colocalize strongly with lysosomal markers such as CD63 and lysosome-associated membrane protein. In regulatory T cells, activation of phospholipase D was sufficient to trigger release of CTLA-4 to the PM but did not inhibit endocytosis. Taken together, these data suggest that CTLA-4 may be stored in a specialized compartment in regulatory T cells that can be triggered rapidly for deployment to the PM in a phospholipase D- and ADP ribosylation factor-1-dependent manner.     

Lipid remodelling in neuroendocrine secretion

Neuroendocrine cells secrete hormones and polypeptides through a complex membrane trafficking process that involves the transport of specific organelles, called large dense core secretory granules, from the Golgi apparatus to specialised sites at the plasma membrane where these vesicles are successively exocytosed and recaptured by endocytosis through tightly coupled reactions. The minimal machinery required for exocytosis has been defined as SNARE proteins associated with few accessory proteins. On the other side, clathrin and dynamin constitute major components of some of the most important endocytotic pathways. Although many protein contributors of both exocytosis and endocytosis are now identified, their actual interplay is not well resolved. Furthermore, the necessary tight coupling of exocytosis and compensatory endocytosis to maintain membrane homeostasis in neuroendocrine cells is far from being understood. In this review, we focus on the more recently identified role of lipids in these important processes that are above all membrane remodelling events.     

Control of Ste6 recycling by ubiquitination in the early endocytic pathway in yeast

We present evidence that ubiquitination controls sorting of the ABC-transporter Ste6 in the early endocytic pathway. The intracellular distribution of Ste6 variants with reduced ubiquitination was examined. In contrast to wild-type Ste6, which was mainly localized to internal structures, these variants accumulated at the cell surface in a polar manner. When endocytic recycling was blocked by Ypt6 inactivation, the ubiquitination deficient variants were trapped inside the cell. This indicates that the polar distribution is maintained dynamically through endocytic recycling and localized exocytosis ("kinetic polarization"). Ste6 does not appear to recycle through late endosomes, because recycling was not blocked in class E vps (vacuolar protein sorting) mutants (Deltavps4, Deltavps27), which are affected in late endosome function and in the retromer mutant Deltavps35. Instead, recycling was partially affected in the sorting nexin mutant Deltasnx4, which serves as an indication that Ste6 recycles through early endosomes. Enhanced recycling of wild-type Ste6 was observed in class D vps mutants (Deltapep12, Deltavps8, and Deltavps21). The identification of putative recycling signals in Ste6 suggests that recycling is a signal-mediated process. Endocytic recycling and localized exocytosis could be important for Ste6 polarization during the mating process.     

Endocytosis and exocytosis in hyphal growth

Two ancient processes, endocytosis and exocytosis, are employed by eukaryotic cells to shape their plasma membrane and interact with their environment. Filamentous fungi have adapted them to roles compatible with their unique ecological niche and morphology. These organisms are optimal systems in which to address questions such as how endocytosis is localized, how endocytosis and exocytosis interact, and how large molecules traverse eukaryotic cell walls. In the tips of filamentous (hyphal) cells, a ring of endocytosis encircles an apical crescent of exocytosis, suggesting that this area is able to support an endocytic recycling route, although both processes can occur in subapical regions as well. Endocytosis and exocytosis underlie growth, but also facilitate disease progression and secretion of industrially relevant compounds in these organisms. Here we highlight recent work on endocytosis and exocytosis in filamentous fungi.     

Clathrin-mediated endocytosis at synapses

Neurons are communication specialists that convert electrical into chemical signals at specialized cell-cell junctions termed synapses. Arrival of an action potential triggers calcium-regulated exocytosis of neurotransmitter (NT) from small synaptic vesicles (SVs), which then diffuses across the synaptic cleft and binds to postsynaptic receptors to elicit specific changes within the postsynaptic cell. Endocytosis of pre- and postsynaptic membrane proteins including SV components and postsynaptic NT receptors is essential for the proper functioning of the synapse. During the past several years, we have witnessed enormous progress in our understanding of the mechanics of clathrin-mediated endocytosis (CME) and its role in regulating exo-endocytic vesicle cycling at synapses. Here we summarize the molecular machinery used for recognition of synaptic membrane protein cargo and its clathrin-dependent internalization, and describe the inventory of tools that can be used to monitor vesicle cycling at synapses or to inhibit CME in a stage-specific manner.     

Endocytosis at ribbon synapses

Unlike conventional synaptic terminals that release neurotransmitter episodically in response to action potentials, neurons of the visual, auditory and vestibular systems encode sensory information in graded signals that are transmitted at their synapses by modulating the rate of continuous release. The synaptic ribbon, a specialized structure found at the active zones of these neurons, is necessary to sustain the high rates of exocytosis required for continuous release. To maintain the fidelity of synaptic transmission, exocytosis must be balanced by high-capacity endocytosis, to retrieve excess membrane inserted during vesicle fusion. Capacitance measurements following vesicle release in ribbon-type neurons indicate two kinetically distinct phases of compensatory endocytosis, whose relative contributions vary with stimulus intensity. The two phases can be independently regulated and may reflect different underlying mechanisms operating on separate pools of recycling vesicles. Electron microscopy shows diversity among ribbon-type synapses in the relative importance of clathrin-mediated endocytosis versus bulk membrane retrieval as mechanisms of compensatory endocytosis. Ribbon synapses, like conventional synapses, make use of multiple endocytosis pathways to replenish synaptic vesicle pools, depending on the physiological needs of the particular cell type.     

Insulin and contraction stimulate exocytosis, but increased AMP-activated protein kinase activity resulting from oxidative metabolism stress slows endocytosis of GLUT4 in cardiomyocytes

Stimulations of glucose transport produced by insulin action, contraction, or through a change in cell energy status are mediated by separate signaling pathways. These are the wortmannin-sensitive phosphatidylinositol 3-kinase pathway leading to the intermediate Akt and the wortmannin-insensitive AMP-activated protein kinase (AMPK) pathway. Electrical stimulation of cardiomyocytes produced a rapid, insulin-like, wortmannin-sensitive stimulation of glucose transport activity, but this occurred without extensive activation of Akt. Although AMPK phosphorylation was increased by contraction, this response was not wortmannin-inhibitable and consequently did not correlate with the wortmannin sensitivity of the transport stimulation. Oxidative metabolism stress due to hypoxia or treatment with oligomycin led to increased AMPK activity with a corresponding increase in glucose transport activity. We show here that these separate signaling pathways converge on GLUT4 trafficking at separate steps. The rate of exocytosis of GLUT4 was rapidly stimulated by insulin, but insulin treatment did not alter the endocytosis rate. Like insulin stimulation, electrical stimulation of contraction led to a stimulation of GLUT4 exocytosis without any marked change in endocytosis. By contrast, after oxidative metabolism stress, no stimulation of GLUT4 exocytosis occurred; instead, this treatment led to a reduction in GLUT4 endocytosis.     

A quantitative imaging-based screen reveals the exocyst as a network hub connecting endocytosis and exocytosis

The coupling of endocytosis and exocytosis underlies fundamental biological processes ranging from fertilization to neuronal activity and cellular polarity. However, the mechanisms governing the spatial organization of endocytosis and exocytosis require clarification. Using a quantitative imaging-based screen in budding yeast, we identified 89 mutants displaying defects in the localization of either one or both pathways. High-resolution single-vesicle tracking revealed that the endocytic and exocytic mutants she4∆ and bud6∆ alter post-Golgi vesicle dynamics in opposite ways. The endocytic and exocytic pathways display strong interdependence during polarity establishment while being more independent during polarity maintenance. Systems analysis identified the exocyst complex as a key network hub, rich in genetic interactions with endocytic and exocytic components. Exocyst mutants displayed altered endocytic and post-Golgi vesicle dynamics and interspersed endocytic and exocytic domains compared with control cells. These data are consistent with an important role for the exocyst in coordinating endocytosis and exocytosis.     

Cyclical regulation of the exocyst and cell polarity determinants for polarized cell growth

Polarized exocytosis is important for morphogenesis and cell growth. The exocyst is a multiprotein complex implicated in tethering secretory vesicles at specific sites of the plasma membrane for exocytosis. In the budding yeast, the exocyst is localized to sites of bud emergence or the tips of small daughter cells, where it mediates secretion and cell surface expansion. To understand how exocytosis is spatially controlled, we systematically analyzed the localization of Sec15p, a member of the exocyst complex and downstream effector of the rab protein Sec4p, in various mutants. We found that the polarized localization of Sec15p relies on functional upstream membrane traffic, activated rab protein Sec4p, and its guanine exchange factor Sec2p. The initial targeting of both Sec4p and Sec15p to the bud tip depends on polarized actin cable. However, different recycling mechanisms for rab and Sec15p may account for the different kinetics of polarization for these two proteins. We also found that Sec3p and Sec15p, though both members of the exocyst complex, rely on distinctive targeting mechanisms for their localization. The assembly of the exocyst may integrate various cellular signals to ensure that exocytosis is tightly controlled. Key regulators of cell polarity such as Cdc42p are important for the recruitment of the exocyst to the budding site. Conversely, we found that the proper localization of these cell polarity regulators themselves also requires a functional exocytosis pathway. We further report that Bem1p, a protein essential for the recruitment of signaling molecules for the establishment of cell polarity, interacts with the exocyst complex. We propose that a cyclical regulatory network contributes to the establishment and maintenance of polarized cell growth in yeast.     

SNAP25, but not syntaxin 1A, recycles via an ARF6-regulated pathway in neuroendocrine cells

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.     

Basic fibroblast growth factor, a protein devoid of secretory signal sequence, is released by cells via a pathway independent of the endoplasmic reticulum-Golgi complex.

Basic fibroblast growth factor (bFGF) modulates functions of a variety of cell types. Whereas bFGF is known to act extracellularly, the protein lacks a transient signal peptide. No defined mechanism for bFGF secretion has been characterized besides release from dead or injured cells. To study this problem we devised an experimental system to examine bFGF-mediated migration of isolated single cells. Under these conditions individual cells are not affected by bFGF derived from other cells. By this method we have previously shown that bFGF released by NIH 3T3 cells transfected with bFGF cDNA modulates migration in an autocrine manner. We have now examined the effects on cell motility of drugs or treatments known to affect various pathways of protein secretion. Drugs that block secretion via the endoplasmic reticulum (ER)-Golgi complex or via multidrug resistance proteins did not inhibit cell motility. Migration was enhanced by the calcium ionophore A23187, which stimulates exocytosis, and was inhibited by methylamine, serum-free, and low temperature (18 degrees C) conditions, which block endo- and exocytosis. The reversal of these effects by the concomitant addition of affinity-purified anti-bFGF IgG or recombinant bFGF showed that the alterations in cell migration were mediated by changes in bFGF externalization. Thus bFGF can be released via a mechanism of exocytosis independent of the ER-Golgi pathway.     

Hrs and hbp: possible regulators of endocytosis and exocytosis

The molecular mechanisms of endocytosis and exocytosis are not yet fully understood. Hrs and Hbp, two tightly associated proteins in eukaryotic cells, have been implicated in these cellular processes. Hrs is homologous to Vps27p, an endosomal protein required for vacuolar and endocytic trafficking in yeast. Hrs is localized to early endosomes and is required for the normal morphology of early endosomes in mammalian cells. Hrs also associates with proteins implicated in endocytosis and exocytosis such as SNAP-25 and Eps15. Hrs treatment inhibits neurotransmitter release in permeabilized neuronal cells and its overexpression inhibits internalization of transferrin. Overexpression of dominant-negative Hbp mutants inhibits ligand-induced downregulation of growth factor/receptor complexes and immunoglobulin E receptor-triggered degranulation of secretory granules in mast cells. These observations suggest an important role for the Hrs/Hbp protein complex in vesicular trafficking during endocytosis and exocytosis.     

Voltage-dependent calcium channels at the plasma membrane, but not vesicular channels, couple exocytosis to endocytosis

Although calcium influx triggers endocytosis at many synapses and non-neuronal secretory cells, the identity of the calcium channel is unclear. The plasma membrane voltage-dependent calcium channel (VDCC) is a candidate, and it was recently proposed that exocytosis transiently inserts vesicular calcium channels at the plasma membrane, thus triggering endocytosis and coupling it to exocytosis, a mechanism suggested to be conserved from sea urchin to human. Here, we report that the vesicular membrane, when inserted into the plasma membrane upon exocytosis, does not generate a calcium current or calcium increase at a mammalian nerve terminal. Instead, VDCCs at the plasma membrane, including the P/Q-type, provide the calcium influx to trigger rapid and slow endocytosis and, thus, couple endocytosis to exocytosis. These findings call for reconsideration of the vesicular calcium channel hypothesis. They are likely to apply to many synapses and non-neuronal cells in which VDCCs control exocytosis, and exocytosis is coupled to endocytosis.     

HCO(3)(-) ions modify the role of PKC isoforms in the modulation of rat mast cell functions

PKC and the intracellular calcium signal are two well-known intracellular signaling pathways implicated in the induction of mast cell exocytosis. Both signals are modified by the presence or absence of HCO(3)(-) ions in the external medium. In this work, we studied the regulation of the exocytotic process by PKC isozymes and its relationship with HCO(3)(-) ions and PKC modulation of the calcium entry. The calcium entry, induced by thapsigargin and further addition of calcium, was inhibited by PMA, a PKC activator, and enhanced by 500 nM GF109203X, which inhibits Ca(2+)-independent PKC isoforms. PMA inhibition of the Ca(2+) entry was reverted by 500 and 50 nM GF109203X, which inhibit Ca(2+)-independent and Ca(2+)-dependent isoforms, respectively, and G?6976, a specific inhibitor of Ca(2+)-dependent PKCs. Thus, activation of Ca(2+)-dependent and Ca(2+)-independent PKC isoforms inhibit Ca(2+) entry in rat mast cells, either in a HCO(3)(-)-buffered or a HCO(3)(-)-free medium. PMA, GF109203X, G?6976 and rottlerin, a specific inhibitor of PKC delta, were also used to study the role of PKC isoforms in the regulation of exocytosis induced by thapsigargin, ionophore A23187 and PMA. The results demonstrate that Ca(2+)-dependent PKC isoforms inhibit exocytosis in a HCO(3)(-)-dependent way. Moreover, Ca(2+)-independent PKC delta was the main isoform implicated in promotion of Ca(2+)-dependent mast cell exocytosis in the presence or absence of HCO(3)(-). The role of PKC isoforms in the regulation of mast cell exocytosis depends on the stimulus and on the presence or absence of HCO(3)(-) ions in the medium, but it is independent of PKC modulation of the Ca(2+) entry.     

Analysis of sea urchin egg cortical transformation in the absence of cortical granule exocytosis

A burst of endocytosis accompanying microvillar elongation follows cortical granule exocytosis in normal sea urchin development. By 5 min postfertilization the burst is over and a lower level of endocytosis ensues (constitutive phase). To determine whether microvillar elongation and initiation of endocytosis are necessary concommitants of cortical granule exocytosis we utilized Chase's (1967, Ph.D. thesis, University of Washington, Seattle) high-hydrostatic pressure technique to block the latter and then examined developing eggs for endocytosis and microvillar elongation. To accomplish this, eggs were fertilized, after which hydrostatic pressure was quickly raised to 6000-7000 psi at the start of cortical granule exocytosis and maintained for 5 min. Only the cortical granules immediately surrounding the sperm penetration site were secreted (about 3% or less of the egg's total number of cortical granules). Blockage of major cortical granule exocytosis had the following consequences on surface events during first division: (1) The endocytosis burst normally associated with cortical granule exocytosis was effectively eliminated as was early microvillar elongation and elevation. Both occurred to a limited extent around the sperm penetration site which resulted in a highly localized surface transformation. (2) By 20 min after fertilization endocytosis began over the rest of the egg surface in the absence of any further cortical granule exocytosis. (3) Subsequently, during a 30-min period starting midway between fertilization and first cleavage microvilli more than doubled in length and endocytosis levels increased severalfold. These events brought about a complete surface transformation similar to that which normally occurs in early development but in the absence of cortical granule exocytosis. By first cleavage surfaces and cortices of high-pressure-treated and control eggs were nearly indistinguishable except for the presence of cortical granules in cortices of the former. Pressure-treated eggs cleaved normally and developed to larval forms overnight. The period of late surface transformation in high-pressure-treated Strongylocentrotus purpuratus eggs corresponds in timing and some of its characteristics to second phase microvillar elongation observed in normal development in this species and also in S. droebachiensis development. These observations suggest, therefore, that microvillar elongation and endocytosis are necessary membrane remodelling events which must occur for normal development even in the absence of membrane addition from the cortical granules.     

Low pH inhibits compensatory endocytosis at a step between depolarization and calcium influx

Cell function can be modulated by the insertion and removal of ion channels from the cell surface. The mechanism used to keep channels quiescent prior to delivery to the cell surface is not known. In eggs, cortical vesicle exocytosis inserts voltage-gated calcium channels into the cell surface. Calcium influx through these channels triggers compensatory endocytosis. Secretory vesicles contain high concentrations of calcium and hydrogen ions. We propose that lumenal hydrogen ions inhibit vesicular calcium channel gating prior to exocytosis, discharge of lumenal protons upon vesicle-plasma membrane fusion enables calcium channel gating. Consistent with this hypothesis we find that cortical vesicle lumens are acidic, and exocytosis releases lumenal hydrogen ions. Acidic extracellular pH reversibly blocks endocytosis, and the windows of opportunity for inhibition with a calcium-channel blocker or hydrogen ions are indistinguishable. Calcium ionophore treatment circumvents the low pH block, suggesting that calcium influx, or an upstream step, is obstructed. Inhibition of calcium influx by preventing membrane depolarization is unlikely, as elevation of the extracellular potassium concentration failed to overcome the pH block, and low extracellular pH was found to depolarize the membrane potential. We conclude that low pH inhibits endocytosis at a step between membrane depolarization and calcium influx .     

Interactions of cholera toxin with isolated hepatocytes. Effects of low pH, chloroquine and monensin on toxin internalization, processing and action.

The major steps in cholera-toxin action, i.e. binding, internalization, generation of A1 peptide and activation of adenylate cyclase, were examined in isolated hepatocytes. The binding of toxin involves a single class of high-affinity sites (KD congruent to 0.1 nM; Bmax. congruent to 10(7) sites/cell). At 37 degrees C, cell-associated toxin is progressively internalized, as judged by the loss of its accessibility to antibodies against whole toxin, A and B subunits (about 50, 75 and 30% of initially bound toxin after 40 min respectively). Two distinct pathways are involved in this process: endocytosis of the whole toxin, and selective penetration of the A subunit into the plasma membrane. Exposure of hepatocytes to an acidic medium (pH 5) results in a rapid and marked disappearance of the A subunit from the cell surface. Generation of A1 peptide and activation of adenylate cyclase by the toxin occur after a lag phase (10 min at 37 degrees C), and increase with time in a parallel manner up to 2-3% A1 peptide generated; they are unaffected by exposure of cells to an acidic medium. Chloroquine and monensin, which elevate the pH in acidic organelles, inhibit by 2-4-fold both the generation of A1 peptide and the activation of adenylate cyclase. Unexpectedly, these drugs also inhibit the internalization of the toxin. These results suggest that an acidic pH facilitates the penetration of A subunit into the plasma membrane and presumably the endosomal membrane as well, and that endocytosis of cholera toxin is required for generation of A1 peptide and activation of adenylate cyclase.     

Endocytosis and exocytosis protect cells against severe membrane tension variations

The ability of cells to regulate their shape and volume is critical for many cell functions. How endocytosis and exocytosis, as important ways of membrane trafficking, affect cellular volume regulation is still unclear. Here, we develop a theoretical framework to study the dynamics of cell volume, endocytosis, and exocytosis in response to osmotic shocks and mechanical loadings. This model can not only explain observed dynamics of endocytosis and exocytosis during osmotic shocks but also predict the dynamics of endocytosis and exocytosis during cell compressions. We find that a hypotonic shock stimulates exocytosis, while a hypertonic shock stimulates endocytosis; and exocytosis in turn allows cells to have a dramatic change in cell volume but a small change in membrane tension during hyposmotic swelling, protecting cells from rupture under high tension. In addition, we find that cell compressions with various loading speeds induce three distinct dynamic modes of endocytosis and exocytosis. Finally, we show that increasing endocytosis and exocytosis rates reduce the changes in cell volume and membrane tension under fast cell compression, whereas they enhance the changes in cell volume and membrane tension under slow cell compression. Together, our findings reveal critical roles of endocytosis and exocytosis in regulating cell volume and membrane tension.