Exocytosis

Fluorescent fusion proteins are an important tool for the study of vesicle trafficking and exocytosis, especially when combined with newer types of microscopy. We previously reported that the design of a vesicle-targeted fluorescent fusion construct strongly influences the kinetics of fluorescence change at exocytosis. In the present study we demonstrate that the cell in which a construct is expressed also affects the kinetics of fluorescence change at exocytosis. We fused enhanced green fluorescent protein to the carboxy terminus of the vesicular cargo protein rodent islet amyloid polypeptide. The two proteins were separated by a “linker” sequence of 18 amino acids. We then compared kinetics of fluorescence change at exocytosis for this fluorescent cargo protein expressed in three different types of peptidergic endocrine cell: pancreatic alpha cell, pancreatic beta cell, and adrenal chromaffin cell. In resting cells of all three types, fluorescent spots of similar size and membrane-proximal density appeared near the plasma membrane as expected if the probe is stored in large dense-core secretory vesicles. Upon stimulation, the fluorescent spots displayed sudden changes in fluorescence intensity that were consistent with exocytosis. In beta and alpha cells the fluorescent spots consistently brightened and persisted, whereas in chromaffin cells the fluorescent spots always dispersed rapidly. Thus, for fluorescent cargo proteins in peptidergic endocrine cells, cell type influences the kinetics of fluorescence change at exocytosis. Together with our previous findings, this observation strongly highlights the fact that the behavior of vesicle-targeted fluorescent cargo may be unrelated to that of native cargo, and it emphasizes the need for caution in interpreting fluorescence kinetics in terms of an exocytosis mechanism.

Acrosomal loss was induced in marsupial spermatozoa by an intermediate of the phosphoinositide pathway. The diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DiC8; 100 \g=m\moll \ m=-\ 1) induced acrosomal loss in 70% of brushtail possum (Trichosurus vulpecula) spermatozoa and in 80% of tammar wallaby (Macropus eugenii) spermatozoa. The DiC8-induced acrosomal loss was not enhanced by co-incubation with calcium ionophore A23187 and occurred in Ca2+free medium and in the presence of the calcium chelator EGTA (3 mmol l \m=-\1). There was no evidence of uptake of 45Ca2+ during the DiC8-induced acrosomal loss. Inhibitors of protein kinase C [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] and phospholipase A2 dexamethasone] did not effect DiC8-induced acrosomal loss in wallaby spermatozoa. The phorbol ester, phorbol 12-myristate 13-acetate, at a concentration of 10 \g=m\mol l \m=-\1had no effect on possum spermatozoa and induced acrosomal loss in only 6% of wallaby spermatozoa. It appears that the DiC8-induced acrosome reaction is not mediated by activation of the phosphoinositide pathway and that extracellular calcium is not required for the membrane fusion event. As acrosomal loss was seen only at relatively high concentrations of diacylglycerol (> 50 \g=m\moll \ m=-\ 1) and there is no evidence of involvement of other phosphoinositide intermediates or analogues, it is likely that its role is as a direct membrane fusogen.

Abstraet---Catecholamine secretion induced by various secretagogues in cultured bovine chromaffin cells has been correlated with Ca 2+ influx and intracellular Ca z+ concentrations. Nicotine and high K + caused prompt secretion of catecholamines from cells. Coincidently, both seeretagogues evoked 45[Ca2+] influx with a parallel increase in free intracellular Ca 2+ concentration, as determined by Quin 2 fluorescence. However, the rate of return of Ca 2+ level to baseline after nicotine stimulation was more rapid than after K + stimulation. In comparison, stimulation with veratridine produced a slow and prolonged Ca 2+ influx accompanied by lower levels of intracellular Ca 2+ than those observed after nicotine or K + stimulation. Yet, during 15 min of stimulation, veratridine induced a substantial catecholamine release, which was larger than that obtained after nicotine or K + stimulations. The Ca 2+ ionophore A23187 (1/~M) induced a pronounced increase in intracellular Ca 2+ levels, but did not evoke any significant catecholamine release. Finally, addition of the Ca 2 ÷ channel blocker verapamil following stimulation, at a time when intracellular Ca 2+ concentration was at its peak level, did not affect the rate of decline in intracellular free Ca 2+ concentration but promptly blocked Ca 2+ uptake and catecholamine secretion. These findings suggest that the rate of Ca 2+ influx, rather than the absolute level of intracellular Ca 2+ concentration, determines the rate and extent of catecholamine release.

In the central nervous system, astrocytes form an intimately connected network with neurons, and their processes closely enwrap synapses. The critical role of these cells in metabolic and trophic support to neurons, ion buffering and clearance of neurotransmitters is well established. However, recent accumulating evidence suggests that astrocytes are active partners of neurons in additional and more complex functions. In particular, astrocytes express a repertoire of neurotransmitter receptors mirroring that of neighbouring synapses. Such receptors are stimulated during synaptic activity and start calcium signalling into the astrocyte network. Intracellular oscillations and intercellular calcium waves represent the astrocyte’s own form of excitability, as they trigger release of transmitter (i.e. glutamate) via a novel process sensitive to blockers of exocytosis and involving cyclooxygenase eicosanoids. Astrocyte-released glutamate activates receptors on the surrounding neurons and modifies their electrical and intracellular calcium ([Ca2+]i) state. These exciting new findings reveal an active participation of astrocytes in synaptic transmission and the involvement of neuron-astrocyte circuits in the processing of information in the brain.

Immunocytochemistry of dystrophin, spectrin, vinculin (sarcolemmaspecific proteins), and desmin (an intermediate filament protein) were investigated in 5 patients with acid maltase deficiency (AMD). One patient had infant onset, 2 had childhood onset, and 2 had adult onset. All had a vacuolar myopathy with autophagic vacuoles containing glycogen and cytoplasmic degradation products. Dystrophin, spectrin, and vinculin were localized at the sarcolemma as in normal muscle fibers. Within the cytoplasm of many fibers, immunoreactivity for the three proteins was seen as single or multiple spots or as circular structures, which most likely corresponded to the limiting membrane of vacuoles. Desmin was overexpressed at the periphery of some vacuoles. It is plausible that, before exocytosis occurs, sarcolemma-specific proteins appear within the vacuole membrane. Vacuole immunolabeling frequently occurred in the patients with childhood and adult onset AMD, but very rarely occurred in the case with infant onset. We hypothesize that a reduced exocytosis rate might explain the infrequent vacuole immunolabeling and the early onset of the infant form of the disease. © 1994 John Wiley & Sons, Inc.

Cell-cell communication is essential for the development and homeostasis of multicellular organisms. Recently, a new type of cell-cell communication was discovered that is based on the formation of thin membranous nanotubes between remote cells 1,2 . These long membrane tethers, termed tunneling nanotubes (TNTs), form an intercellular conduit and have been shown to enable the transport of various cellular components and signals. However, the molecular basis for TNT formation remains to be elucidated. Here we report that a mammalian protein, M-Sec, induces de novo formation of numerous membrane protrusions extending from the plasma membrane, some of which tether onto adjacent cells and subsequently form TNT-like structures. Depletion of M-Sec by RNA interference (RNAi) greatly reduced endogenous TNT formation as well as intercellular propagation of a calcium flux in a macrophage cell line. Furthermore, blockage of the interaction of M-Sec with Ral and the exocyst complex, which serves as a downstream effector of Ral, attenuated the formation of membrane nanotubes. Our results reveal that M-Sec functions as a key regulator of membrane nanotube formation through interaction with the Ral-exocyst pathway.

The small vesicles shed from the surface of many cells upon stimulation, considered for a long time to be artefacts, are now recognized as specific structures that are distinct from the exosomes released upon exocytosis of multivesicular bodies. Recent reports indicate that shedding vesicles participate in important biological processes, such as the surface-membrane traffic and the horizontal transfer of protein and RNAs among neighboring cells, which are necessary for the rapid phenotype adjustments in a variety of conditions. In addition, shedding vesicles have important physiological and pathological roles: in coagulation, by mediating the coordinate contribution of platelets, macrophages and neutrophils; in inflammatory diseases, via the release of cytokines; and in tumor progression, facilitating the spreading and release of cancer cells to generate metastases.

The small chemical vacuolin-1 induces rapid formation of large vacuoles in various cell types. In epithelial cells, vacuolin-1 has been shown to inhibit Ca 2+ ionophore-induced exocytosis depending on experimental conditions used but had no effect on repair of damaged membranes. However, it is not known whether vacuolin-1 could inhibit exocytosis induced by immunoreceptor triggering in professional secretory cells and whether there is any correlation between effect of vacuolin-1 on exocytosis and membrane repair in such cells. Here we show that in rat basophilic leukemia (RBL-2H3) cells activated by the high-affinity IgE receptor (FcεRI) triggering vacuolin-1 enhanced exocytosis. Under identical conditions of activation, vacuolin-1 inhibited exocytosis in mouse bone marrow-derived mast cells (BMMCs). This inhibition was not reflected by decreased phosphorylation of the FcεRI α and β subunits, linker for activation of T cells, non-T cell activation linker, Akt and MAP kinase Erk, and uptake of extracellular Ca 2+ , indicating that early activation events are not affected. In both cell types vacuolin-1 led to formation of numerous vacuoles, a process which was inhibited by bafilomycin A1, an inhibitor of vacuolar H + -ATPase. Thapsigargin-or Ca 2+ ionophore A23187induced exocytosis also showed different sensitivity to the inhibitory effect of vacuolin-1. Pretreatment of the cells with vacuolin-1 followed by permeabilization with bacterial toxin streptolysin O enhanced Ca 2+dependent repair of plasma membrane lesions in RBL-2H3 cells but inhibited it in BMMCs. Our data indicate that lysosomal exocytosis exhibits different sensitivity to vacuolin-1 depending on the cell type analyzed and mode of activation. Furthermore, our results support the concept that lysosomal exocytosis is involved in the repair of injured plasma membranes.

Phaeocystis globosa, a leading agent in marine carbon cycling, releases its photosynthesized biopolymers via regulated exocytosis. Release is elicited by blue light and relayed by a characteristic cytosolic Ca 2+ signal. However, the source of Ca 2+ in these cells has not been established. The present studies indicate that Phaeocystis' secretory granules work as an intracellular Ca 2+ oscillator. Optical tomography reveals that photo-stimulation induces InsP 3 -triggered periodic lumenal [Ca 2+ ] oscillations in the granule and corresponding out-of-phase cytosolic oscillations of [Ca 2+ ] that trigger exocytosis. This Ca 2+ dynamics results from an interplay between the intragranular polyanionic matrix, and two Ca 2+ -sensitive ion channels located on the granule membrane: an InsP 3 -receptor-Ca 2+ channel, and an apaminsensitive K + channel.

The harderian gland in the Wistar rat consists of tubules with wide lumina lined by a single layer of columnar epithelial cells possessing myoepithelial cells within their basal laminae. The gland contains porphyrin pigment which is stored as solid intraluminal deposits. The glandular epithelium possesses 2 cell types, termed A and B. These are characterised by an extraordinarily well-developed tubular smooth endoplasmic reticulum and numerous lipid vacuoles. Type A cells can be distinguished from type B by the number, size and content of the lipid vacuoles. Type A cells are more numerous. They contain large lipid vacuoles with dense ribbon-like material identical in form to the material in the luminal masses of porphyrin pigment, whereas those of type B cells are small with crescentic dense lamellar material. The content of the vacuoles is essentially released by exocytosis, but holocrine secretion also occurs. The lipids and the ribbonlike material represent the bulk of the intraluminal secretory product. The secretion of porphyrins seems to be associated with type A cells. The single excretory duct is lined by a stratified epithelium. The duct epithelium comprises serous cell types, designated C1 and C2 and scarce mucus-secreting cells. Type C1 cells are characterised by numerous dense granules, whereas type C2 cells are distinguished by lysosomal structures. Fibroblasts, macrophages, mast cells, plasma cells, fenestrated capillaries and unmyelinated axons are frequently observed in the connective tissue. The gland is surrounded by a collagenous capsule and an outer layer of endothelial cells of the orbital venous sinus.

The main function of background two-pore potassium (K(2P)) channels KCNK3/9/15 is to stabilize the cell membrane potential. We previously observed that membrane potential depolarization enhances the release of HIV-1 viruses. Because membrane polarization affects the biomembrane directly, here we examined the effects of KCNK3/9/15 on the budding of nonviral vesicles. We found that depolarization by knocking down endogenous KCNK3/9/15 promoted secretion of cell-derived vesicles. We further used Vpu (an antagonist of KCNK3) as a model for the in vivo study of depolarization-stimulated secretion. Vpu is a HIV-1-encoded, ion channel-like protein (viroporin) capable of enhancing virus release and depolarizing the cell membrane potential. We found that Vpu could also promote nonviral vesicle release, perhaps through a similar mechanism that Vpu utilizes to promote viral particle release. Notably, T cells expressing Vpu alone became pathologically low in intracellular K(+) and insensitive to extracellular K(+) or membrane potential stimulation. In contrast, heterologous expression of KCNK3 in T cells stabilized the cell potentials by maintaining intracellular K(+). We thus concluded that KCNK3/9/15 expression limits membrane depolarization and depolarization-induced secretion at least in part by maintaining intracellular K(+).

L’exocytose de neurotransmetteurs est un mécanisme essentiel à la communication cellulaire. Au cours de ce processus, des vésicules de sécrétions remplies de messagers chimiques ou biochimiques libèrent leur contenu dans le milieu extracellulaire. Ce mécanisme (impliqué par exemple lors de la libération d’adrénaline dans le sang par les cellules chromaffines) se déroule en plusieurs étapes : les vésicules contenant des molécules messagers se rapprochent puis s’arriment à la membrane cellulaire. Après fusion de leur membrane avec celle de la cellule, elles libèrent leur contenu vers l’extérieur par l’intermédiaire d’un pore de fusion.

Depuis les vingt dernières années, plusieurs méthodes ont été développées en vue d’étudier ce mécanisme biologique sur cellule vivante en temps réel. Parmi ces techniques, principalement basées sur des mesures électriques ou optiques, l’ampérométrie sur fibre de carbone 1 et la microscopie de fluorescence à excitation par onde évanescente (TIRFM) se sont révélées être les plus puissantes 2.

Les propriétés physico-chimiques des microélectrodes leur confèrent une sensibilité de détection élevée et les rend particulièrement adaptées au suivi en temps réel de la libération de molécules électroactives par les cellules. Cependant, l’ampérométrie ne permet de détecter ces molécules qu’à partir de leur libération par le pore de fusion. Le couplage de cette technique avec la microscopie de fluorescence permettrait de visualiser le statut des vésicules se rapprochant de la membrane plasmique et ainsi de suivre également leur comportement avant et pendant la fusion. A cet égard, le couplage des deux techniques doit impliquer un modèle cellulaire dont les vésicules peuvent être à la fois marquées par une sonde fluorescente et dont le contenu est électroactif.

De premières études ampérométriques sur microélectrodes à fibre de carbone ont permis de mettre en évidence la sécrétion de sérotonine par les cellules de type BON et de choisir le modèle cellulaire le mieux adapté au couplage. En l’occurrence, la fréquence de sécrétion des cellules BON N13 (de l’ordre de 8 évènements par minutes) est suffisante sans être trop élevée pour corréler les informations électrochimiques et optiques. Actuellement, le couplage ampérométrie-microscopie est en cours de développement. La mise au point des premiers dispositifs nécessaires à cette étude a été réalisée. L’ITO, semi-conducteur et transparent sous forme de film, joue le rôle d’électrode sans entraver la détection optique des vésicules marquées par une sonde fluorescente. Les premiers résultats obtenus dans ces conditions sur cellule unique seront décrits et discutés.


(1) Wightman, R. M.; Jankowski, J. A.; Kennedy, R. T.; Kawagoe, K. T.; Schroeder, T. J.; Leszczyszyn, D. J.; Near, J. A.; Diliberto, E. J.; Viveros, O. H. PNAS 1991, 88, 10754-10758.
(2) Amatore, C.; Arbault, S.; Chen, Y.; Crozatier, C.; Lemaître, F.; Verchier, Y. Angewandte Chemie 2006, 45, 4000-4003.

Glucose entry into muscle cells is precisely regulated by insulin, through recruitment of GLUT4 (glucose transporter-4) to the membrane of muscle and fat cells. Work done over more than two decades has contributed to mapping the insulin signalling and GLUT4 vesicle trafficking events underpinning this response. In spite of this intensive scientific research, there are outstanding questions that continue to challenge us today. The present review summarizes the knowledge in the field, with emphasis on the latest breakthroughs in insulin signalling at the level of AS160 (Akt substrate of 160 kDa), TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) and their target Rab proteins; in vesicle trafficking at the level of vesicle mobilization, tethering, docking and fusion with the membrane; and in the participation of the cytoskeleton to achieve optimal temporal and spatial location of insulin-derived signals and GLUT4 vesicles.

In neuroendocrine cells, actin reorganization is a prerequisite for regulated exocytosis. Small GTPases, Rho proteins, represent potential candidates coupling actin dynamics to membrane trafficking events. We previously reported that Cdc42 plays an active role in regulated exocytosis in chromaffin cells. The aim of the present work was to dissect the molecular effector pathway integrating Cdc42 to the actin architecture required for the secretory reaction in neuroendocrine cells. Using PC12 cells as a secretory model, we show that Cdc42 is activated at the plasma membrane during exocytosis. Expression of the constitutively active Cdc42 L61 mutant increases the secretory response, recruits neural Wiskott-Aldrich syndrome protein (N-WASP), and enhances actin polymerization in the subplasmalemmal region. Moreover, expression of N-WASP stimulates secretion by a mechanism dependent on its ability to induce actin polymerization at the cell periphery. Finally, we observed that actin-related protein-2/3 (Arp2/3) is associated with secretory granules and that it accompanies granules to the docking sites at the plasma membrane upon cell activation. Our results demonstrate for the first time that secretagogue-evoked stimulation induces the sequential ordering of Cdc42, N-WASP, and Arp2/3 at the interface between granules and the plasma membrane, thereby providing an actin structure that makes the exocytotic machinery more efficient.

Excellent spatial and temporal resolution and physico-chemical properties of ultramicroélectrodes (UME) induce high detection sensitivity, thus being particularly well adapted to the study of biological mechanisms of secretion from a single cell. In the « semi-artificial synapse » configuration, the short distance between the transmitting cell and the UME causes appropriate local concentration to be electrochemically detected. UME allows the detection of minute amount of electroactive molecules in real time. This analytical technique was used, completed or adapted to study two major biological phenomena: vesicular exocytosis and cellular oxidative stress.
Amperometric investigations of exocytosis, a key mechanism involved in cell communication, allow quantitative monitoring of the kinetics of intravesicular species release into extracellular medium. An UME, placed in the external environment, does not provide any information about vesicles status before fusion. In order to complement this information, we have developed, through microfabrication techniques, a device constituted by conductive and transparent ITO electrodes for combinated detection by amperometry and optical microscopy (TIRFM) for the study of BON BC21 cells secretion.
Amperometry at four different constant potentials, used in the laboratory for the detection of ROS/RNS released by macrophages, immune cells, requires a large number of experiments to overcome the cell variability and differences in UME sensitivity. To significantly reduce the experimental duration, we have developed a device constituted by four measuring chambers, each containing a set of three electrodes. These four chambers will allow real time and simultaneous monitoring of changes in production of H2O2, ONOO-, NO• and NO2- by a single cell.

This review attempts to touch on the history and application of amperometry at PC12 cells for fundamental investigation into the exocytosis process. PC12 cells have been widely used as a model for neural differentiation and as such they have been used to examine the effects of differentiation on exocytotic release and specifically release at varicosities. In addition, dexamethasonedifferentiated cells have been shown to have an increased number of releasable vesicles with increased quantal size, thereby allowing for an even broader range of applications including neuropharmacological and neurotoxicological studies. PC12 cells exhibiting large numbers of events have two distinct pools of vesicles, one about twice the quantal size of the other and each about half the total releasable vesicles. As will be outlined in this review, these cells have served as an extremely useful model of exocytosis in the study of the latency of stimulation-release coupling, the role of exocytotic proteins in regulation of release, effect of drugs on quantal size, autoreceptors, fusion pore biophysics, environmental factors, health and disease. As PC12 cells have some advantages over other models for neurosecretion, including chromaffin cells, it is more than likely that in the following decade PC12 cells will continue to serve as a model to study exocytosis.

This paper suggests that mental units called psychons by Eccles could be tachyons defined theoretically by physicists sometime ago. Although experiments to detect faster-than-light particles have not been successful so far, recently, there has been renewed interest in tachyon theories in various branches of physics. We suggest that tachyon theories may be applicable to brain physics. Eccles proposed an association between psychons and what he called dendrons which are dendrite bundles and basic anatomical units of the neocortex for reception. We show that a zero-energy tachyon could act as a trigger for exocytosis (modeled by Friedrich Beck as a quantum tunneling process), not merely at a single presynaptic terminal but at all selected terminals in the interacting dendron by momentarily transferring momentum to vesicles, thereby decreasing the effective barrier potential and increasing the probability of exocytosis at all boutons at the same time. This is consistent with the view of tachyons, which treats them as strictly non-local phenomenon produced and absorbed instantaneously and non-locally by detectors acting in a coherent and cooperative way.

We review the evidence of exocytosis from extrasynaptic sites in the soma, dendrites, and axonal varicosities of central and peripheral neurons of vertebrates and invertebrates, with emphasis on somatic exocytosis, and how it contributes to signaling in the nervous system. The finding of secretory vesicles in extrasynaptic sites of neurons, the presence of signaling molecules (namely transmitters or peptides) in the extracellular space outside synaptic clefts, and the mismatch between exocytosis sites and the location of receptors for these molecules in neurons and glial cells, have long suggested that in addition to synaptic communication, transmitters are released, and act extrasynaptically. The catalog of these molecules includes low molecular weight transmitters such as monoamines, acetylcholine, glutamate, gama-aminobutiric acid (GABA), adenosine-5-triphosphate (ATP), and a list of peptides including substance P, brain-derived neurotrophic factor (BDNF), and oxytocin. By comparing the mechanisms of extrasynaptic exocytosis of different signaling molecules by various neuron types we show that it is a widespread mechanism for communication in the nervous system that uses certain common mechanisms, which are different from those of synaptic exocytosis but similar to those of exocytosis from excitable endocrine cells. Somatic exocytosis has been measured directly in different neuron types. It starts after high-frequency electrical activity or long experimental depolarizations and may continue for several minutes after the end of stimulation. Activation of L-type calcium channels, calcium release from intracellular stores and vesicle transport towards the plasma membrane couple excitation and exocytosis from small clear or large dense core vesicles in release sites lacking postsynaptic counterparts. The presence of synaptic and extrasynaptic exocytosis endows individual neurons with a wide variety of time-and space-dependent communication possibilities. Extrasynaptic exocytosis may be the major source of signaling molecules producing volume transmission and by doing so may be part of a long duration signaling mode in the nervous system.

Weibel-Palade bodies (WPB) are the regulated secretory organelles of endothelial cells. These cigar-shaped membrane-bound structures function in both hemostasis and inflammation but their biogenesis is poorly understood. Here, we review what is currently known about their formation. The content of WPBs is dominated by the hemostatic factor von Willebrand factor (VWF), whose complex biogenesis ends in the formation of high molecular weight multimers. VWF is also organized into proteinaceous tubules which underlie the striated interior of WPBs as seen in the EM. VWF expression is necessary for formation of WPBs, and its heterologous expression can even lead to the specific recruitment of WPB membrane proteins, including the leukocyte receptor P-selectin, the tetraspanin CD63, and Rab27a. Unusually, the VWF propeptide is implicated in the biogenesis of WPBs, being essential for formation of the storage compartment. The elongation of the cigars and the formation of the tubules are determined by non-covalent interactions between pro-and mature VWF proteins. Surprisingly, high molecular weight multimers seem neither necessary nor sufficient to trigger formation of a storage compartment, and do not seem to have any role in WPB biogenesis. Von Willebrand's disease, usually caused by mutations within VWF, has provided many of the insights into the way in which VWF drives the formation of these organelles.

In neuroscience, myosin V motor proteins have attracted attention since they are highly expressed in brain, and absence of myosin Va in man leads to a severe neurological disease called Griscelli syndrome. While in some cells myosin V is described to act as a vesicle transport motor, an additional role in exocytosis has emerged recently. In neurons, myosin V has been linked to exocytosis of secretory vesicles and recycling endosomes. Through these functions, it is implied in regulating important brain functions including the release of neuropeptides by exocytosis of large dense-core vesicles and the insertion of neurotransmitter receptors into post-synaptic membranes. This review focuses on the role of myosin V in (i) axonal transport and stimulated exocytosis of large dense-core vesicles to regulate the secretion of neuroactive substances, (ii) tethering of the endoplasmic reticulum at cerebellar synapses to permit long-term depression, (iii) recycling of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors at hippocampal synapses during long-term potentiation, and (iv) recycling of nicotinic acetylcholine receptors at the neuromuscular junction. Myosin V is thus discussed as an important modulator of synaptic plasticity.

Cone-driven responses of second-order retinal neurons are considerably faster than rod-driven responses. We examined whether differences in the kinetics of synaptic transmitter release from rods and cones may contribute to differences in postsynaptic response kinetics. Exocytosis from rods and cones was triggered by membrane depolarization and monitored in two ways: (1) by measuring EPSCs evoked in second-order neurons by depolarizing steps applied to presynaptic rods or cones during simultaneous paired whole-cell recordings or (2) by direct measurements of exocytotic increases in membrane capacitance. The kinetics of release was assessed by varying the length of the depolarizing test step. Both measures of release revealed two kinetic components to the increase in exocytosis as a function of the duration of a step depolarization. In addition to slow sustained components in both cell types, the initial fast component of exocytosis had a time constant of <5 ms in cones, >10-fold faster than that of rods. Rod/cone differences in the kinetics of release were substantiated by a linear correlation between depolarization-evoked capacitance increases and EPSC charge transfer. Experiments on isolated rods indicate that the slower kinetics of exocytosis from rods was not a result of rod-rod coupling. The initial rapid release of vesicles from cones can shape the postsynaptic response and may contribute to the faster responses of cone-driven cells observed at light offset.

consists of corticostriatal glutamatergic projections Stanislav S. Zakharenko, 4 Leonard Zablow, 4 (Wilson, 1987) that form asymmetric synaptic contacts and David Sulzer 2,3,6, * with the dendrites of medium spiny neurons (MSNs) 1 Department of Neurology and nearby nigrostriatal dopaminergic projections from Department of Pediatrics ventral midbrain neurons that form symmetrical syn-University of Washington apses on MSN dendrites (Graybiel et al., 1981). This Children's Hospital and Regional Medical Center configuration suggests that dopamine release, which is Seattle, Washington 98105 elicited by salient behavioral stimuli that evoke burst 2 Department of Neurology firing in ventral midbrain neurons or by dopamine-3 Department of Psychiatry releasing drugs such as amphetamine, may not only 4 Center for Neurobiology and Behavior modulate the excitability of MSNs (Nicola et al., 2000) Howard Hughes Medical Institute but may also diffuse from the synaptic cleft to activate Columbia University College of Physicians and nearby dopamine receptors on cortical presynaptic ter-Surgeons minals (Garris et al.Maura et al., Dopamine input to the striatum is required for volun-1988; O'Donnell and Grace, 1994; Tang et al., 2001; West tary motor movement, behavioral reinforcement, and and Grace, 2002) receptors. Some studies found no efresponses to drugs of abuse. It is speculated that fects unless animals were subjected to interventions these functions are dependent on either excitatory or designed to drive dopamine receptor signaling into a inhibitory modulation of corticostriatal synapses onto supersensitive state (Nicola et al., 1996). Indirect evimedium spiny neurons (MSNs). While dopamine moddence suggests that presynaptic D1-like receptors modulates MSN excitability, a direct presynaptic effect on ulate activity of glutamatergic terminals in the ventral the corticostriatal input has not been clearly demon-(Harvey and Lacey, 1996; Nicola et al., 1996; Nicola and strated. We combined optical monitoring of synaptic Malenka, 1998) but not dorsal (Nicola and Malenka, vesicle exocytosis from motor area corticostriatal af-1998) striatum. Although some of the studies above sugferents and electrochemical recordings of striatal dogest an inhibition of corticostriatal transmission by D2pamine release to directly measure effects of dopalike receptors, it remains unclear whether these effects mine at the level of individual presynaptic terminals. are due to pre-or postsynaptic effects on the corticostri-Dopamine released by either electrical stimulation or atal synapse. amphetamine acted via D2 receptors to inhibit the Immunohistochemical analyses of rat motor striatum activity of subsets of corticostriatal terminals. Optical by electron microscopy have also been controversial, and electrophysiological data suggest that heterosyrevealing exceedingly rare (Hersch et al., 1995) or low naptic inhibition was enhanced by higher frequency (Fisher et al., 1994; Sesack et al., 1994; Wang and Pickel, stimulation and was selective for the least active ter-2002) levels of dopamine receptors on corticostriatal minals. Thus, dopamine, by filtering less active inputs, terminals. D1 receptors may be exclusively localized on appears to reinforce specific sets of corticostriatal postsynaptic elements (Caille et al., 1996; Hersch et al., synaptic connections. 1995; Levey et al., 1993), while D2 receptors may be localized on pre-and postsynaptic elements, including Introduction a subpopulation of corticostriatal terminals (Fisher et al., 1994; Hersch et al., 1995; Sesack et al., 1994; Wang Axon-to-axon communication by modulatory neuroand Pickel . Thus, the action and even presence of transmitters acting on presynaptic G protein-coupled dopamine receptors on corticostriatal terminals remain receptors has long been suggested to provide a major unestablished, although these issues are paramount to form of central neurotransmission and modulation. A understanding striatal function. Here, we introduce an approach to address these questions at the level of individual synaptic terminals and find direct evidence *Correspondence: [email protected] 43 loading procedure before unloading. The half-time decay of intensity during destaining (t 1/2 ) was determined graphically, in some We thank Drs. Sara Glickstein, Francois Gonon, Richard Palmiter, instances using a 5 th degree polynomial smoothing function on Sig-Dietmar Plenz, Steve Siegelbaum, and Lisa Zimberg for helpful ad-maPlot (SPSS, Chicago, IL) software. The fractional release paramevice. The authors thank the Colleen Giblin Charitable Foundation ter ƒ was calculated from ln (F 1 /F 2 )/⌬AP (Isaacson and for Pediatric Neurology and the Vision Research Center, University where ln is the natural logarithm, F 1 and F 2 are the fluorescent intensiof Washington. N.S.B. was a Neurological Sciences Academic Deties at t 1 and t 2 , respectively, and ⌬AP is the number of action velopment Awardee at Columbia University. Supported by Chilpotentials delivered during that period. Unless otherwise stated, dren'

Needle-type voltammetric ultramicroelectrodes show exceptional sensitivity for the detection of redox-active substances, rapid response times, and total tip diameters in the lower micrometer range. These characteristics make them ideal for analyzing the chemical environment and the activity of isolated living cells, which in their various forms are the microscopic building blocks of human, animal, and other life forms. Prerequisites for successful local electrochemical measurements in the vicinity of the tiny biological objects are gentle, stress-free, and accurate placement of the tip at the cell, exact knowledge of the tip-to-cell distance, and appropriate selectivity of the ultramicroelectrode tip for species that may change in concentration as a result of cellular actions such as growth, respiration, or transmitter and metabolite uptake or release. The concepts of single-cell microelectrochemistry are considered and an overview is given of recent results on the fundamental mechanisms of cell functions.

Pancreatic b-cell function is of critical importance in the regulation of fuel homoeostasis, and metabolic dysregulation is a hallmark of diabetes mellitus (DM). The b-cell is an intricately designed cell type that couples metabolism of dietary sources of carbohydrates, amino acids and lipids to insulin secretory mechanisms, such that insulin release occurs at appropriate times to ensure efficient nutrient uptake and storage by target tissues. However, chronic exposure to high nutrient concentrations results in altered metabolism that impacts negatively on insulin exocytosis, insulin action and may ultimately lead to development of DM. Reduced action of insulin in target tissues is associated with impairment of insulin signalling and contributes to insulin resistance (IR), a condition often associated with obesity and a major risk factor for DM. The altered metabolism of nutrients by insulin-sensitive target tissues (muscle, adipose tissue and liver) can result in high circulating levels of glucose and various lipids, which further impact on pancreatic b-cell function, IR and progression of the metabolic syndrome. Here, we have considered the role played by the major nutrient groups, carbohydrates, amino acids and lipids, in mediating b-cell insulin secretion, while also exploring the interplay between amino acids and insulin action in muscle. We also focus on the effects of altered lipid metabolism in adipose tissue and liver resulting from activation of inflammatory processes commonly observed in DM pathophysiology. The aim of this review is to describe commonalities and differences in metabolism related to insulin secretion and action, pertinent to the development of DM.

Purpose: The study aimed to evaluate the (i) regulatory role of G i-like protein during the acrosome reaction (AR) of normal sperm donors and (ii) the role of intact acrosomes during sperm–zona binding. Methods: The acrosomal exocytosis of spermatozoa incubated with solubilized zona pellucida (ZP) at a final concentration of 1 ZP/μl was compared with 10 μM calcium ionophore A23187 and 30% (v/v) pooled human follicular fluid (HFF). Spermatozoa were incubated with 1, 10, and 100 ng/ml pertussis toxin (PT) during capacitation to functionally inactivate the G i-like protein. The sperm–zona binding potential of 100 ng/ml PT-treated spermatozoa followed, by exposure to 1 ZP/μl, revealed significantly higher zona-bound spermatozoa compared to controls treated with 1 ZP/μl only. Results: PT treatment of spermatozoa did not affect sperm motility, however, inhibited the percentage AR induced by solubilized ZP. In contrast, the A23187- and HFF-induced ARs were not sensitive to PT treatment. PT inhibition of the ZP-induced AR occurred in a concentration-dependent manner, with maximal effects observed at 100 ng/ml PT. Conclusions: In conclusion, it seems that PT-sensitive G i-like protein in human spermatozoa plays an important regulatory role in the AR induced by the human ZP, and this underlines the importance of intact acrosomes during sperm–zona binding.

The ethyl acetate-soluble portion of the MeOH extract of Ehretia microphylla showed inhibitory activity on exocytosis in antigen-stimulated rat basophils. The bioassay-gnided separation of this fraction afforded five biologically active compounds. By means of chemical and spectroscopic methods, the structures of these compounds, which include microphyllone, a unique dimeric prenylbenzoquinone, and its congeners, were elucidated.

In order to understand exocytosis and endocytosis, it is necessary to study these processes directly. an elegant way to do this is by measuring plasma membrane capacitance (C m ), a parameter proportional to cell surface area, the fluctuations of which are due to fusion and fission of secretory and other vesicles. Here we describe protocols that enable high-resolution C m measurements in macroscopic and microscopic modes. Macroscopic mode, performed in whole-cell configuration, is used for measuring bulk C m changes in the entire membrane area, and it enables the introduction of exocytosis stimulators or inhibitors into the cytosol through the patch pipette. Microscopic mode, performed in cell-attached configuration, enables measurements of C m with attofarad resolution and allows characterization of fusion pore properties. although we usually apply these protocols to primary pituitary cells and astrocytes, they can be adapted and used for other cell types. after initial hardware setup and culture preparation, several C m measurements can be performed daily.

Synaptotagmins (Syts), comprise a gene family of proteins, implicated in the control of protein traffic. Rat basophilic leukemia cells (RBL-2H3), a tumor analogue of mucosal mast cells (MMC), express at least four distinct Syt homologues, including Syt II, Syt III, Syt V and Syt IX. Synaptotagmin II is located at the late/endosomal/lysosomal compartment, where it negatively regulates lysosomal exocytosis. Mast cells may contribute to immune defense mechanisms by presenting MHC class II/antigen complexes and triggering T cell-dependent immune responses. We now demonstrate that RBL-2H3 mast cells, which express reduced levels of Syt II (<5%) by transfection with Syt II antisense cDNA, are able to release MHC class II molecules. We further show that release of both MHC class II molecules and of the lysosomal enzyme cathepsin D is stimulated by lipopolysaccharide (LPS, 1 g/ml, 48 h). We show further that LPS reduces by >40% the level of Syt II expression in both RBL-2H3 and bone marrow-derived mast cells (BMMC). This effect is both dose and time-dependent. These results indicate that Syt II can be down-regulated by external inflammatory signals, resulting in the amplification of mast cell function. Finally, our results implicate Syt II as an important and novel regulator of MHC class II presentation.

The GTPases Rab3a and Rab27a and their effectors Granuphilin/Slp4 and Noc2 are essential regulators of neuroendocrine secretion. Chronic exposure of pancreatic b-cells to supraphysiological glucose levels decreased selectively the expression of these proteins. This glucotoxic effect was mimicked by cAMP-raising agents and blocked by PKA inhibitors. We demonstrate that the transcriptional repressor ICER, which is induced in a PKA-dependent manner by chronic hyperglycemia and cAMP-raising agents, is responsible for the decline of the four genes. ICER overexpression diminished the level of Granuphilin, Noc2, Rab3a and Rab27a by binding to cAMP responsive elements located in the promoters of these genes and inhibited exocytosis of b-cells in response to secretagogues. Moreover, the loss in the expression of the genes of the secretory machinery caused by glucose and cAMPraising agents was prevented by an antisense construct that reduces ICER levels. We propose that induction of inappropriate ICER levels lead to defects in the secretory process of pancreatic b-cells possibly contributing, in conjunction with other known deleterious effects of hyperglycemia, to defective insulin release in type 2 diabetes.

The secretory cycle of dense-core vesicles (DCVs) in physiologically stimulated patch-clamped PC12 cells was analyzed using both amperometry and capacitance measurements. Untreated cells had low or undetectable Ca currents and sparse secretory responses to short depolarizations. Dexamethasone (5 M) treatment for 5-7 d tripled Ca current magnitude and dramatically increased quantal secretion in response to depolarization with action potentials. Such cells expressed L-, N-, and P-type Ca channels, and depolarization evoked rapid catecholamine secretion recorded as amperometric spikes; the average latency was ϳ50 msec. These spikes were much smaller and shorter than those of primary adrenal chromaffin cells, reflecting the smaller size of DCVs in PC12 cells. Depolarizing pulse trains also elicited a rapid increase in membrane capacitance corresponding to exocytosis in differentiated but not in naïve cells. On termination of stimulation, membrane capacitance declined within 20 sec to baseline indicative of rapid endocytosis (RE). RE did not take place when secretion was stimulated in the presence of Ba or Sr, indicating that RE is Ca-specific. RE was blocked when either anti-dynamin antibodies or the pleckstrin homology domain of dynamin-1 was loaded into the cell via the patch pipette. These studies indicate that neuroendocrine differentiation of PC12 cells with glucocorticoids enhances the development of the excitable membrane and increases the coupling between Ca channels and vesicle release sites, leading to rapid exocytosis and endocytosis. Slow catecholamine secretion in undifferentiated cells may be caused in part by a lack of localized secretory machinery rather than being an intrinsic property of dense-core vesicles.

MicroRNAs (miRNAs) are short nucleotides sequences that regulate the expression of genes in different eukaryotic cell types. A tremendous amount of knowledge on miRNAs has rapidly accumulated over the last few years, revealing the growing interest in this field of research. On the other hand, clarifying the physiological regulation of gene expression in the central nervous system is important for establishing a reference for comparison to the diseased state. It is well known that the fine tuning of neuronal networks relies on intricate molecular mechanisms, such as the adjustment of the synaptic transmission. As determined by recent studies, regulation of neuronal interactions by miRNAs has critical consequences in the development, adaptation to ambient demands, and degeneration of the nervous system. In contrast, activation of synaptic receptors triggers downstream signaling cascades that generate a vast array of effects, which includes the regulation of novel genes involved in the control of the miRNA life cycle. In this review, we have examined the hot topics on miRNA gene-regulatory activities in the broad field of neuronal communication-related processes. Furthermore, in addition to indicating the newly described effect of miRNAs on the regulation of specific neurotransmitter systems, we have pointed out how these systems affect the expression, transport, and stability of miRNAs. Moreover, we discuss newly described and under-investigation mechanisms involving the intercellular transfer of miRNAs, aided by exosomes and gap junctions. Thus, in the current review, we were able to highlight recent findings related to miRNAs that indisputably contributed towards the understanding of the nervous system in health and disease.

The cellular processes that govern neuronal function are highly complex, with many basic cell biological pathways uniquely adapted to perform the elaborate information processing achieved by the brain. This is particularly evident in the trafficking and regulation of membrane proteins to and from synapses, which can be a long distance away from the cell body and number in the thousands. The regulation of neurotransmitter receptors, such as the AMPA-type glutamate receptors (AMPARs), the major excitatory neurotransmitter receptors in the brain, is a crucial mechanism for the modulation of synaptic transmission. The levels of AMPARs at synapses are very dynamic, and it is these plastic changes in synaptic function that are thought to underlie information storage in the brain. Thus, understanding the cellular machinery that controls AMPAR trafficking will be critical for understanding the cellular basis of behavior as well as many neurological diseases. Here we describe the life cycle of AMPARs, from their biogenesis, through their journey to the synapse, and ultimately through their demise, and discuss how the modulation of this process is essential for brain function. 613 Annu. Rev. Cell Dev. Biol. 2007.23:613-643. Downloaded from arjournals.annualreviews.org by University of California -San Diego on 04/10/08. For personal use only.