To understand the intracellular mechanisms that control exocytosis it is necessary to have access to the cell interior. This is achieved by plasma membrane permeabilisation or by application of patch‐pipettes. These conditions permit control over the cytosol composition and also allow leakage of soluble factors that may have roles in the exocytotic mechanism. Different permeabilisation methods allow different extents of leakage and therefore provide complementary data. The exocytotic machinery itself remains intact and can be activated by providing Ca2+ and/or (...) a guanine nucleotide. In some cells there is evidence for the participation of two guanine nucleotide‐binding proteins (Gp and GE), as well as a Ca2+ ‐binding protein. In others Ca2+ is the only requirement. In a number of cell types, ATP is not required for the late steps in the secretory pathway. (shrink)

Neurons are now known to produce a variety of types of chemical transmitters. Classical transmitters are stored within synaptic vesicles which undergo synaptic exocytosis in association with presynaptic thickenings. The larger, dense‐cored secretory granules present in most neurons contain neuropeptides and mainly discharge their contents at morphologically undifferentiated (i.e.nonsynaptic) sites. The synaptic character of vesicle discharge enables transmitters to exercise a highly focal action, whereas nonsynaptic release probably relates to the slow rate of degradation of many neuropeptides and their (...) consequent widespread diffusion and sphere of action. However, one variant of the basic pattern, involving the restriction of granule discharge to areas of the terminal plasmalemma situated adjacent to the postsynaptic cells (i.e. a parasynaptic configuration), enables a degree of targeted peptide discharge to be achieved. The diversity of patterns of neural exocytosis adds a further dimension to the complexity of nervous function. (shrink)

Synaptophysin (Syp) was the first synaptic vesicle (SV) protein to be cloned. Since its discovery in 1985, it has been used by us and by many laboratories around the world as an invaluable marker to study the distribution of synapses in the brain and to uncover the basic features of the life cycle of SVs. Although single gene ablation of Syp does not lead to an overt phenotype, a large body of experimental data both in vitro and in vivo indicate (...) that Syp (alone or in association with homologous proteins) is involved in multiple, important aspects of SV exo‐endocytosis, including regulation of SNARE assembly into the fusion core complex, formation of the fusion pore initiating neurotransmitter release, activation of SV endocytosis and SV biogenesis. In this article, we summarise the main results of the studies on Syp carried out by our and other laboratories, and explain why we believe that Syp plays a major role in SV trafficking. BioEssays 26:445–453, 2004. © 2004 Wiley Periodicals, Inc. (shrink)

Twenty five years ago, Sir John Carew Eccles together with Friedrich Beck proposed a quantum mechanical model of neurotransmitter release at synapses in the human cerebral cortex. The model endorsed causal influence of human consciousness upon the functioning of synapses in the brain through quantum tunneling of unidentified quasiparticles that trigger the exocytosis of synaptic vesicles, thereby initiating the transmission of information from the presynaptic towards the postsynaptic neuron. Here, we provide a molecular upgrade of the Beck and Eccles (...) model by identifying the quantum quasiparticles as Davydov solitons that twist the protein α-helices and trigger exocytosis of synaptic vesicles through helical zipping of the SNARE protein complex. We also calculate the observable probabilities for exocytosis based on the mass of this quasiparticle, along with the characteristics of the potential energy barrier through which tunneling is necessary. We further review the current experimental evidence in support of this novel bio-molecular model as presented. (shrink)

Phosphatidylinositol 3‐phosphate (PtdIns3P) is generated on the cytosolic leaflet of cellular membranes, primarily by phosphorylation of phosphatidylinositol by class II and class III phosphatidylinositol 3‐kinases. The bulk of this lipid is found on the limiting and intraluminal membranes of endosomes, but it can also be detected in domains of phagosomes, autophagosome precursors, cytokinetic bridges, the plasma membrane and the nucleus. PtdIns3P controls cellular functions through recruitment of specific protein effectors, many of which contain FYVE or PX domains. Cellular processes known (...) to be controlled by PtdIns3P and its effectors include endosomal fusion, sorting and motility, autophagy, cytokinesis, regulated exocytosis and signal transduction. Here we discuss how Ptdins3P is generated on specific cellular membranes, how its localizations and functions can be studied, and how its effectors serve to control cellular functions.Editor's suggested further reading in BioEssays:Phosphatidylinositol 4,5‐bisphosphate: Targeted production and signaling AbstractHow does SHIP1/2 balance PtdIns(3,4)P2 and does it signal independently of its phosphatase activity? AbstractPhosphatidylinositol‐3,4,5‐trisphosphate: Tool of choice for class I PI 3‐kinases AbstractPhosphatidylinositol‐4‐phosphate: The Golgi and beyond Abstract. (shrink)

The remodeling of biological membranes is crucial for a vast number of cellular activities and is an inherently multiscale process in both time and space. Seminal work has provided important insights into nanometer‐scale membrane deformations, and highlighted the remarkable variation and complexity in the underlying molecular machineries and mechanisms. However, how membranes are remodeled at the micron‐scale, particularly in vivo, remains poorly understood. Here, we discuss how using regulated exocytosis of large (1.5–2.0 μm) membrane‐bound secretory granules in the salivary (...) gland of live mice as a model system, has provided evidence for the importance of the actomyosin cytoskeleton in micron‐scale membrane remodeling in physiological conditions. We highlight some of these advances, and present mechanistic hypotheses for how the various biochemical and biophysical properties of distinct actomyosin networks may drive this process. (shrink)

Mainstream cognitive neuroscience typically ignores the role of quantum physical effects in the neural processes underlying cogni¬tion and consciousness. However, many unsolved problems remain, suggesting the need to consider new approaches. We propose that quantum theory, especially through an ontological interpretation due to Bohm and Hiley, provides a fruitful framework for addressing the neural correlates of cognition and consciousness. In particular, the ontological interpretation suggests that a novel type of 'active information', connected with a novel type of 'quantum potential energy', (...) plays a key role in quantum physical processes. After introducing the ontological interpretation we illustrate its value for cognitive neuroscience by discussing it in the light of a proposal by Beck and Eccles about how quantum tunneling could play a role in controlling the frequency of synaptic exocytosis. In this proposal, quantum tunneling would enable the 'self' to control its brain without violating the energy conservation law. We argue that the ontological interpretation provides a sharper picture of what actually could be taking place in quantum tunneling in general and in synaptic exocytosis in particular. Based on the notions of active information and quantum potential energy, we propose a coherent way of understanding how mental processes (understood as involving non-classical physical processes) can act on traditional, classically describable neural processes without violating the energy conservation law. (shrink)

Synaptic ribbons, the organelles identified in electron micrographs of the sensory synapses involved in vision, hearing, and balance, have long been hypothesized to play an important role in regulating presynaptic function because they associate with synaptic vesicles at the active zone. Their physiology and molecular composition have, however, remained largely unknown. Recently, a series of elegant studies spurred by technical innovation have finally begun to shed light on the ultrastructure and function of ribbon synapses. Electrical capacitance measurements have provided sub‐millisecond (...) resolution of exocytosis, evanescent‐wave microscopy has filmed the fusion of single 30 nm synaptic vesicles, electron tomography has revealed the 3D architecture of the synapse, and molecular cloning has begun to identify the proteins that make up ribbons. These results are consistent with the ribbon serving as a vesicle “conveyor belt” to resupply the active zone, and with the suggestion that ribbon and conventional chemical synapses have much in common. BioEssays 23:831–840, 2001. © 2001 John Wiley & Sons, Inc. (shrink)

A Paramecium cell has a stereotypically patterned surface, with regularly arranged cilia, dense‐core secretory vesicles and subplasmalemmal calcium stores. Less strikingly, there is also a patterning of molecules; for instance, some ion channels are restricted to certain regions of the cell surface. This design may explain very effective and selective responses, such as that to Ca2+ upon stimulation. It enables the cell to respond to a Ca2+ signal precisely secretion (exocytosis) or by changing its ciliary activity. These responses depend (...) on the location and/or type of signal, even though these two target structures co‐exist side‐by‐side, and normally only limited overlap occurs between the different functions. Furthermore, the patterning of exocytotic sites and the possibility of synchronous exocytosis induction in the sub‐second time range have considerably facilitated analyses, and thus led to new concepts of exocytotic membrane fusion. It has been possible to dissect complicated events like overlapping Ca2+ fluxes produced from external sources and from internal stores. Since molecular genetic approaches have become available for Paramecium, many different gene products have been identified only some of which are known from “higher” eukaryotes. Although a variety of basic cellular functions are briefly addressed to demonstrate the uniqueness of this unicellular organism, this article focuses on exocytosis regulation. BioEssays 24:649–658, 2002. © 2002 Wiley Periodicals, Inc. (shrink)

Airway mucin secretion is important pathophysiologically and as a model of polarized epithelial regulated exocytosis. We find the trafficking protein, SNAP23, selectively expressed in secretory cells compared with ciliated and basal cells of airway epithelium by immunohistochemistry and FACS, suggesting that SNAP23 functions in regulated but not constitutive epithelial secretion. Heterozygous SNAP23 deletant mutant mice show spontaneous accumulation of intracellular mucin, indicating a defect in baseline secretion. However mucins are released from perfused tracheas of mutant and wild-type mice at (...) the same rate, suggesting that increased intracellular stores balance reduced release efficiency to yield a fully compensated baseline steady state. In contrast, acute stimulated release of intracellular mucin from mutant mice is impaired whether measured by a static imaging assay 5 min after exposure to the secretagogue ATP or by kinetic analysis of mucins released from perfused tracheas during the first 10 min of ATP exposure. Together, these data indicate that increased intracellular stores cannot fully compensate for the defect in release efficiency during intense stimulation. The lungs of mutant mice develop normally and clear bacteria and instilled polystyrene beads comparable to WT mice, consistent with these functions depending on baseline secretion that is fully compensated. (shrink)

Neurotransmitter release takes place by the exocytosis of loaded synaptic vesicles. The vesicles then fuse to the presynaptic membrane and are recycled by an endocytotic mechanism. A quantitative optical assay that detects uptake and release of a fluorescent dye during presynaptic activity was recently developed and used on the frog neauromuscular junction. I discuss a report(1) that demonstrates the effective application of this method to a Drosophila preparation. The authors use the shibire mutation and a spider venom to identify (...) two intermediates in vesicle recycling. Their report, along with other recent studies, demonstrates the power and promise of the genetic approach for the understanding of mechanisms of synapse function and development. (shrink)

Fluid-phase endocytosis (pinocytosis) kinetics were studied inDictyostelium discoideum amoebae from the axenic strain Ax-2 that exhibits high rates of fluid-phase endocytosis when cultured in liquid nutrient media. Fluorescein-labelled dextran (FITC-dextran) was used as a marker in continuous uptake- and in pulse-chase exocytosis experiments. In the latter case, efflux of the marker was monitored on cells loaded for short periods of time and resuspended in marker-free medium. A multicompartmental model was developed which describes satisfactorily fluid-phase endocytosis kinetics. In particular, it (...) accounts correctly for the extended latency period before exocytosis in pulse-chase experiments and it suggests the existence of some sorts of maturation stages in the pathway. (shrink)

The rate‐limiting step in the uptake and metabolism of Dglucose by insulin target cells is thought to be glucose transport mediated by glucose transporters (primarily the GLUT4 isoform) localized to the plasma membrane. However, subcellular fractionation, photolabelling and immunocytochemical studies have shown that the pool of GLUT4 present in the plasma membrane is only one of many subcellular pools of this protein. GLUT4 has been found in occluded vesicles at the plasma membrane, clathrin‐coated pits and vesicles, early endosomes, and tubulo‐vesicular (...) structures; the latter are analogous to known specialized secretory compartments. Tracking the movement of GLUT4 through these compartments, and defining the mechanism and site of action of insulin in stimulating this subcellular trafficking, are major topics of current investigation. Recent evidence focuses attention on the exocytosis of GLUT4 as the major site of insulin action. Increased exocytosis may be due to decreased retention of glucose transporters in an intracellular pool, or possibly to increased assembly of a vesicle docking and fusion complex. Although details are unknown, the presence in GLUT4 vesicles of a synaptobrevin homologue leads us to propose that a process analogous to that occurring in synaptic vesicle trafficking is involved in the assembly of GLUT4 vesicles into a form suitable for fusion with the plasma membrane. Evidence that the pathways of signalling from the insulin receptor and of GLUT4 vesicle exocytosis may converge at the level of the key signalling enzyme, phosphatidylinositol 3‐kinase, is discussed. (shrink)

The two theories of pancreatic enzyme secretion, those of exocytosis and transmembrane flow, are described. Data thought to support the theory of transmembrane flow of single molecules from pancreatic acinar cells are first reviewed, and the conditions which could allow these data to be explained by the theory of exocytosis of enzyme quanta, i.e. secretory granules, are then discussed.The evidence suggesting short‐term modulation of the composition of pancreatic juice is also considered, and its possible explanations at the organ (...) and cellular level are discussed, in the light of suggested theories on enzyme secretion pathways. (shrink)

Cyclin‐dependent kinase 5 (Cdk5) is recognized as an essential molecule in the brain, where it regulates several neuronal activities, including cytoskeletal remodeling and synaptic transmission. While activity of Cdk5 has primarily been associated with neurons, there are now substantial data indicating that the kinase's activity and function are more general. An increasing body of evidence has established Cdk5 kinase activity, the presence of the Cdk5 activators, p35 and p39, and Cdk5 functions in non‐neuronal cells, including myocytes, pancreatic β‐cells, monocytic and (...) neutrophilic leucocytes, glial cells and germ cells. In this review, we present the diverse roles of Cdk5 in several extraneuronal paradigms. The unique properties of each of the different cell types appear to involve distinct means of Cdk5 regulation and function. The potential mechanisms through which Cdk5 regulates extraneuronal cell activities such as exocytosis, gene transcription, wound healing and senescence are discussed. BioEssays 28: 1023–1034, 2006. © 2006 Wiley Periodicals, Inc. (shrink)

There are many blank areas in understanding the brain dynamics and especially how it gives rise to consciousness. Quantum mechanics is believed to be capable of explaining the enigma of conscious experience, however till now there is not good enough model considering both the data from clinical neurology and having some explanatory power! In this paper is presented a novel model in defence of macroscopic quantum events within and between neural cells. The beta-neurexin-neuroligin-1 link is claimed to be not just (...) the core of the central neural synapse, instead it is a device mediating entanglement between the cytoskeletons of the cortical neurons. Thus a macroscopic quantum state can extend throughout large brain cortical areas and the subsequent collapse of the wavefunction could affect simultaneously the subneuronal events in millions of neurons. The beta-neurexin-neuroligin-1 complex also controls the process of exocytosis and provides an interesting and simple mechanism for retrograde signalling during learning-dependent changes in synaptic connectivity. (shrink)

The inositol phosphate metabolism network has been found to be much more complex than previously thought, as more and more inositol phosphates and their metabolizing enzymes have been discovered. Some of the inositol phosphates have been shown to have biological activities, but little is known about their signal transduction mechanisms except for that of inositol 1,4,5‐trisphosphate. The recent discovery, however, of a number of binding proteins for inositol high polyphosphate [inositol 1,3,4,5‐tetrakisphosphate (IP4), inositol 1,3,4,5,6‐pentakisphosphate, or inositol hexakisphosphate] enables us to (...) speculate on the physiological function of these compounds. In this article we focus on two major issues: (1) the roles of inositol high polyphosphates in vesicular trafficking, especially exocytosis, and (2) pleckstrin homology domaincontaining IP4 binding proteins involved in the Ras signaling pathway. (shrink)

Different anesthetics are known to modulate different types of membrane-bound receptors. Their common mechanism of action is expected to alter the mechanism for consciousness. Consciousness is hypothesized as the integral of all the units of internal sensations induced by reactivation of inter-postsynaptic membrane functional LINKs during mechanisms that lead to oscillating potentials. The thermodynamics of the spontaneous lateral curvature of lipid membranes induced by lipophilic anesthetics can lead to the formation of non-specific inter-postsynaptic membrane functional LINKs by different mechanisms. These (...) include direct membrane contact by excluding the inter-membrane hydrophilic region and readily reversible partial membrane hemifusion. The constant reorganization of the lipid membranes at the lateral edges of the postsynaptic terminals (dendritic spines) resulting from AMPA receptor-subunit vesicle exocytosis and endocytosis can favor the effect of anesthetic molecules on lipid membranes at this location. Induction of a large number of non-specific LINKs can alter the conformation of the integral of the units of internal sensations that maintain consciousness. Anesthetic requirement is reduced in the presence of dopamine that causes enlargement of dendritic spines. Externally applied pressure can transduce from the middle ear through the perilymph, cerebrospinal fluid, and the recently discovered glymphatic pathway to the extracellular matrix space, and finally to the paravenular space. The pressure gradient reduce solubility and displace anesthetic molecules from the membranes into the paravenular space, explaining the pressure reversal of anesthesia. Changes in membrane composition and the conversion of membrane hemifusion to fusion due to defects in the checkpoint mechanisms can lead to cytoplasmic content mixing between neurons and cause neurodegenerative changes. The common mechanism of anesthetics presented here can operate along with the known specific actions of different anesthetics. (shrink)

An important feature of male fertility is the physiological priming of spermatozoa by a multifaceted process collectively referred to as capacitation. The end point of this evasive process is the hyperactivated spermatozoa capable of binding to terminal sugar residues on the egg's extracellular coat, the zona pellucida (ZP), and undergoing acrosomal exocytosis (i.e., induction of the acrosome reaction). The hydrolytic action of acrosomal enzymes released at the site of zona binding, along with the enhanced thrust generated by the hyperactivated (...) beat pattern of the bound spermatozoa, are important factors that regulate the penetration of ZP and fertilization of the egg. Despite many advances in identifying sperm components that promote capacitation, the mechanism underlying the calcium‐triggered process remains elusive. The purpose of this review article is to focus on new advances that have enhanced our understanding of in vivo/in vitro capacitation, a prerequisite event resulting from a dramatic modification and reorganization of the sperm membrane molecules. Special emphasis has been laid on accumulating evidence suggesting potential similarities between the sperm capacitation and early phases of calcium‐triggered membrane fusion (i.e., tethering and docking) during secretory and endocytotic pathways among eukaryotes. BioEssays 26:281–290, 2004. © 2004 Wiley Periodicals, Inc. (shrink)

The role of intuition in scientific endeavor is examined through the lens of three philosophers/historians of science—Karl Popper, Thomas Kuhn, and Gerald Holton. All three attribute an important role to imagination/intuition in scientific endeavor. As a case study, the article examines the controversy between the generally accepted Vesicular Sequestration/Exocytosis Model of pancreatic digestive enzyme secretion and an alternative view called the Equilibrium Model. It highlights the intertwining of intuition and reason in the genesis of the Equilibrium Model developed in (...) response to findings that could not readily be explained by the consensus view. It suggests that tacit knowledge/understanding works in conjunction with philosophical and aesthetic presuppositions that function to a greater or lesser extent below awareness. Together they guide judgments about whether falsification has occurred and help frame alternative theories. (shrink)