Focal exocytosis by eosinophils--compound exocytosis and cumulative fusion.

We have investigated the granule fusion events during exocytosis in horse eosinophils by time-resolved patch-clamp capacitance measurements. Stimulation with intracellular GTP gamma S leads to a stepwise capacitance increase by 4.0 +/- 0.9 pF. At GTP gamma S concentrations < 20 microM the step size distribution is in agreement with the granule size distribution in resting cells. Above 80 microM the number of steps is reduced and very large steps occur. The total capacitance increase, however, is unaffected. These results show that at high GTP gamma S concentrations granule--granule fusion occurs inside the cell forming large compound granules, which then fuse with the plasma membrane (compound exocytosis). The electrical equivalent circuit of the cell during degranulation indicates the formation of a degranulation sac by cumulative fusion events. Fusion of the first granule with the plasma membrane induces fusion of further granules with this granule directing the release of all the granular material to the first fusion pore. The physiological function of eosinophils is the killing of parasites. Compound exocytosis and cumulative fusion enable the cells to focus the release of cytotoxic proteins to well defined target regions and prevent uncontrolled diffusion of this material, which would damage intact host cells.     

v-SNAREs control exocytosis of vesicles from priming to fusion

SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca(2+)-dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.     

Presynaptic calcium and control of vesicle fusion

Vesicle fusion and transmitter release at synapses is driven by a highly localized Ca2+ signal that rapidly builds up around open Ca2+-channels at and near presynaptic active zones. It has been difficult to estimate the amplitude and the kinetics of this 'microdomain' signal by direct Ca2+-imaging approaches. Recently, Ca2+ uncaging at large CNS synapses, among them the calyx of Held, has shown that the intrinsic cooperativity of Ca2+ in inducing vesicle fusion is high, with 4-5 Ca2+ ions needed to trigger vesicle fusion. Given the Ca2+-sensitivity of vesicle fusion as determined by Ca2+-uncaging, it was found that a surprisingly small (10-25 microM) and brief (<1 ms) local Ca2+ signal is sufficient to achieve the amount, and the kinetics of the physiological transmitter release. The high cooperativity of Ca2+ in inducing vesicle fusion and the non-saturation of the Ca2+-sensor for vesicle fusion renders small changes of the local Ca2+-signal highly effective in changing the release probability; an insight that is important for our understanding of short-term modulation of synaptic strength.     

In vitro fusion of Acanthamoeba phagolysosomes. III. Evidence that cyclic nucleotides and vacuole subpopulations respectively control the rate and the extent of vacuole fusion in Acanthamoeba homogenates

Fusion of phagolysosomes has been previously demonstrated to occur during the incubation of phagolysosome-containing homogenates of Acanthamoeba (Oates and Touster, 1978, J. Cell Biol. 79:217-234). Further studies on this system have shown that methylxanthines (0.2 mM) and/or cAMP (0.5-1 mM) markedly accelerate the average rate, but not the extent, of the in vitro phagolysosome fusion process. Adenosine, 5'- AMP, and ADP (0.5-1 mM) were without effect. ATP (0.5-1 mM) caused variable stimulation, whereas beta, gamma-methylene-ATP (1 mM) caused pronounced inhibition, as did GTP (1 mM) and cGMP (1 mM). Stimulation by 3-isobutyl-1-methylxanthine was blocked by GTP, but not by ATP or cAMP. These results indicate that the rate of phagolysosome fusion in Acanthamoeba homogenates may be regulated by cyclic nucleotides, with enhancement of the fusion rate by cAMP and inhibition of the rate by cGMP. The extent of the reaction increased spontaneously and markedly during the first few hours after preparation of the homogenates. This activation appears to be because of a slow conversion of a significant fraction of the vacuole population from a fusion-incompetent to a fusion-competent, cyclic nucleotide-sensitive state.     

Endosomal WASH and exocyst complexes control exocytosis of MT1-MMP at invadopodia

Remodeling of the extracellular matrix by carcinoma cells during metastatic dissemination requires formation of actin-based protrusions of the plasma membrane called invadopodia, where the trans-membrane type 1 matrix metalloproteinase (MT1-MMP) accumulates. Here, we describe an interaction between the exocyst complex and the endosomal Arp2/3 activator Wiskott-Aldrich syndrome protein and Scar homolog (WASH) on MT1-MMP–containing late endosomes in invasive breast carcinoma cells. We found that WASH and exocyst are required for matrix degradation by an exocytic mechanism that involves tubular connections between MT1-MMP–positive late endosomes and the plasma membrane in contact with the matrix. This ensures focal delivery of MT1-MMP and supports pericellular matrix degradation and tumor cell invasion into different pathologically relevant matrix environments. Our data suggest a general mechanism used by tumor cells to breach the basement membrane and for invasive migration through fibrous collagen-enriched tissues surrounding the tumor.     

Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling

Synaptic vesicles have been proposed to form through two mechanisms: one directly from the plasma membrane involving clathrin-dependent endocytosis and the adaptor protein AP2, and the other from an endosomal intermediate mediated by the adaptor AP3. However, the relative role of these two mechanisms in synaptic vesicle recycling has remained unclear. We now find that vesicular glutamate transporter VGLUT1 interacts directly with endophilin, a component of the clathrin-dependent endocytic machinery. In the absence of its interaction with endophilin, VGLUT1 recycles more slowly during prolonged, high-frequency stimulation. Inhibition of the AP3 pathway with brefeldin A rescues the rate of recycling, suggesting a competition between AP2 and -3 pathways, with endophilin recruiting VGLUT1 toward the faster AP2 pathway. After stimulation, however, inhibition of the AP3 pathway prevents the full recovery of VGLUT1 by endocytosis, implicating the AP3 pathway specifically in compensatory endocytosis.     

In search of the fusion pore of exocytosis

Research on calcium-triggered exocytosis has converged on the fusion pore as a critical kinetic intermediate. Using sensitive biophysical methods to record signals from living cells in the act of releasing neurotransmitter or hormone has provided clues about the structure and composition of fusion pores. The dynamics of fusion pore opening, closing, and dilating has revealed how specific proteins transduce a calcium binding signal to catalyze membrane fusion. The fusion pore determines how rapidly neurotransmitter is expelled from a vesicle into the synaptic cleft. This rate places constraints on the form of a synaptic response during different modes of release.     

Regulation of the fusion pore conductance during exocytosis by cyclin-dependent kinase 5

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase involved in synaptogenesis and brain development, and its enzymatic activity is essential for slow forms of synaptic vesicle endocytosis. Recent work also has implicated Cdk5 in exocytosis and synaptic plasticity. Pharmacological inhibition of Cdk5 modifies secretion in neuroendocrine cells, synaptosomes, and brain slices; however, the specific mechanisms involved remain unclear. Here we demonstrate that dominant-negative inhibition of Cdk5 increases quantal size and broadens the kinetics of individual exocytotic events measured by amperometry in adrenal chromaffin cells. Conversely, Cdk5 overexpression narrows the kinetics of fusion, consistent with an increase in the extent of kiss-and-run exocytosis. Cdk5 inhibition also increases the total charge and current of catecholamine released during the amperometric foot, representing a modification of the conductance of the initial fusion pore connecting the granule and plasma membrane. We suggest that these effects are not attributable to an alteration in catecholamine content of secretory granules and therefore represent an effect on the fusion mechanism itself. Finally, mutational silencing of the Cdk5 phosphorylation site in Munc18, an essential protein of the late stages of vesicle fusion, has identical effects on amperometric spikes as dominant-negative Cdk5 but does not affect the amperometric feet. Cells expressing Munc18 T574A have increased quantal size and broader kinetics of fusion. These results suggest that Cdk5 could, in part, control the kinetics of exocytosis through phosphorylation of Munc18, but Cdk5 also must have Munc18-independent effects that modify fusion pore conductance, which may underlie a role of Cdk5 in synaptic plasticity.     

The rate and extent of calcium bioavailability from two oral dosage forms in rats

The purpose of the present work was to compare oral bioavailability of calcium from two calcium preparations, Calcium Sandoz forte 500 mg and Calcium Spofa effervescens. The pharmacokinetic study was carried out on rats, and plasma levels of 45Ca after administration of labelled calcium solutions were determined. Appropriate equations describing the two-compartment open model and the one-compartment model with first order absorption were fitted to the observed i.v. and oral data, respectively, using weighted nonlinear least-squares regression analysis. The extent and the time profile of the rate of 45Ca systemic bioavailability were assessed. Both parameters suggested identical bioavailability of calcium from the two dosage forms compared.     

Revisiting the role of SNAREs in exocytosis and membrane fusion

For over a decade SNARE hypotheses have been proposed to explain the mechanism of membrane fusion, yet the field still lacks sufficient evidence to conclusively identify the minimal components of native fusion. Consequently, debate concerning the postulated role(s) of SNAREs in membrane fusion continues. The focus of this review is to revisit original literature with a current perspective. Our analysis begins with the earliest studies of clostridial toxins, leading to various cellular and molecular approaches that have been used to test for the roles of SNAREs in exocytosis. We place much emphasis on distinguishing between specific effects on membrane fusion and effects on other critical steps in exocytosis. Although many systems can be used to study exocytosis, few permit selective access to specific steps in the pathway, such as membrane fusion. Thus, while SNARE proteins are essential to the physiology of exocytosis, assay limitations often prevent definitive conclusions concerning the molecular mechanism of membrane fusion. In all, the SNAREs are more likely to function upstream as modulators or priming factors of fusion.     

Signaling complexes control the chemotaxis kinase by altering its apparent rate constant of autophosphorylation

Autophosphorylating histidine kinase CheA is central to signaling in bacterial chemotaxis. The kinase donates its phosphoryl group to two response regulators, CheY that controls flagellar rotation and thus motility and CheB, crucial for sensory adaptation. As measured by coupled CheY phosphorylation, incorporation into signaling complexes activates the kinase ～1000‐fold and places it under control of chemoreceptors. By the same assay, receptors modulate kinase activity ～100‐fold as a function of receptor ligand occupancy and adaptational modification. These changes are the essence of chemotactic signaling. Yet, the enzymatic properties affected by incorporation into signaling complexes, by chemoreceptor ligand binding or by receptor adaptational modification are largely undefined. To investigate, we performed steady‐state kinetic analysis of autophosphorylation using a liberated kinase phosphoryl‐accepting domain, characterizing kinase alone, in isolated core signaling complexes and in small arrays of core complexes assembled in vitro with receptors contained in isolated native membranes. Autophosphorylation in signaling complexes was measured as a function of ligand occupancy and adaptational modification. Activation by incorporation into signaling complexes and modulation in complexes by ligand occupancy and adaptational modification occurred largely via changes in the apparent catalytic rate constant (k_cat). Changes in the autophosphorylation k_cat accounted for most of the ～1000‐fold kinase activation in signaling complexes observed for coupled CheY phosphorylation, and the ～100‐fold inhibition by ligand occupancy or modulation by adaptational modification. Our results indicate no more than a minor role in kinase control for simple sequestration of the autophosphorylation substrate. Instead they indicate direct effects on the active site.     

Amisyn regulates exocytosis and fusion pore stability by both syntaxin-dependent and syntaxin-independent mechanisms

Amisyn and tomosyn are related by the possession of a C-terminal vesicle-associated membrane protein-like domain that allows them to bind to syntaxin 1 and assemble into SNARE complexes. The formation of inactive complexes may sequester syntaxin and allow tomosyn and amisyn to act as inhibitors of exocytosis. We aimed to use adrenal chromaffin and PC12 cells to probe this possible mode of action of amisyn and tomosyn in dense core granule exocytosis. Although tomosyn is expressed by adrenal chromaffin and PC12 cells, amisyn expression could not be detected allowing examination of the effect of introduction of amisyn expression onto a neuronal-like background. Overexpression of m-tomosyn1 and expression of amisyn both inhibited Ca2+-induced exocytosis in transfected PC12 cells. Surprisingly, this inhibition was not removed when amisyn and tomosyn constructs were used in which key residues required for efficient binding to syntaxin1 were mutated. The effect of amisyn was further characterized using carbon fiber amperometry in chromaffin cells. Expression of amisyn had no effect on the basic characteristics of the amperometric spikes but reduced the number of spikes elicited. This inhibitory action on the extent of exocytosis was also seen with the amisyn mutant deficient in syntaxin1 binding. In addition, expression of amisyn resulted in an increase in the lifetime of the prespike foot, and this effect was abolished by the mutations. These results show that tomosyn and amisyn can negatively regulate exocytosis independently of syntaxin and also that amisyn can regulate the stability of the fusion pore.     

Different conformational switches underlie the calmodulin-dependent modulation of calcium pumps and channels

We have used fluorescence spectroscopy to investigate the structure of calmodulin (CaM) bound with CaM-binding sequences of either the plasma membrane Ca-ATPase or the skeletal muscle ryanodine receptor (RyR1) calcium release channel. Following derivatization with N-(1-pyrene)maleimide at engineered sites (T34C and T110C) within the N- and C-domains of CaM, contact interactions between these opposing domains of CaM resulted in excimer fluorescence that permits us to monitor conformational states of bound CaM. Complementary measurements take advantage of the unique conserved Trp within CaM-binding sequences that functions as a hydrophobic anchor in CaM binding and permits measurements of both a local and global peptide structure. We find that CaM binds with high affinity in a collapsed structure to the CaM-binding sequences of both the Ca-ATPase and RyR1, resulting in excimer formation that is indicative of contact interactions between the N- and the C-domains of CaM in complex with these CaM-binding peptides. There is a 4-fold larger amount of excimer formation for CaM bound to the CaM-binding sequence of the Ca-ATPase in comparison to RyR1, indicating a closer structural coupling between CaM domains in this complex. Prior to CaM association, the CaM-binding sequences of the Ca-ATPase and RyR1 are conformationally disordered. Upon CaM association, the CaM-binding sequence of the Ca-ATPase assumes a highly ordered structure. In comparison, the CaM-binding sequence of RyR1 remains conformationally disordered irrespective of CaM binding. These results suggest an important role for interdomain contact interactions between the opposing domains of CaM in stabilizing the structure of the peptide complex. The substantially different structural responses associated with CaM binding to Ca-ATPase and RyR1 indicates a plasticity in their respective binding mechanisms that accomplishes different physical mechanisms of allosteric regulation, involving either the dissociation of a C-terminal regulatory domain necessary for pump activation or the modulation of intersubunit interactions to diminish RyR1 channel activity.     

Inhibition of trichocyst exocytosis and calcium influx in Paramecium by amiloride and divalent cations

Summary— Regulated exocytosis of defensive secretory organelles, the trichocysts, as well as a transient Ca²⁺-influx can be induced in Paramecium by aminoethyldextran (Kerb?uf and Cohen, J Cell Biol (1990) 111, 2527). Knoll et al (Febs Lett (1992) 304, 265) reported that veratridine was also a secretagogue for Paramecium. Here we show that, like aminoethyldextran, veratridine induces a transient Ca²⁺-influx. Both aminoethyldextran-and veratridine-induced exocytosis and associated Ca²⁺-influx were: i) blocked in the nd12 thermosensitive mutant at the non-permissive temperature; and ii) inhibited by amiloride and four divalent cations, Ba²⁺, Mg²⁺, Sr²⁺ and Co²⁺. This suggests that, although of different chemical nature, aminoethyldextran and veratridine act through the same physiological pathway. In addition, the inhibitory doses are comparable to the ones found to inhibit a hyperpolarization-sensitive Ca²⁺-current described in Paramecium (Preston et al (1992) J Gen Physiol 100, 233). The possibility that the activation of this Ca²⁺-current by the secretagogue represents an early step in the regulation of trichocyst exocytosis is discussed.     

Integrin-supported fast rate intracellular delivery of plasmid DNA by extracellular matrix protein embedded calcium phosphate complexes

Extracellular matrix (ECM) proteins, such as collagen and fibronectin, play vital roles in development and maintenance of hard tissue (bone or tooth) and are, consequently, thoroughly investigated for construction of biomimetic scaffolds in combination with calcium phosphate (CaP) material (the major component of hard tissue) for bone or dental tissue engineering. Realizing the natural affinity of ECM components toward inorganic constituents of hard tissue, we successfully constructed the nanohybrids of DNA/CaP particles with either collagen 1 or fibronectin, which finally possessed the capability of specific recognition of integrin receptor for being swiftly internalized across the plasma membrane, leading to remarkably high transgene expression in mammalian cells. This new approach with precise receptor-specific delivery as well as 10- to 50-fold enhanced efficiency level compared to the classical one, has immediate applications for basic research and large scale production of recombinant therapeutic proteins and looks promising for gene therapy.     

Direct control of exocytosis by receptor-mediated activation of the heterotrimeric GTPases Gi and G(o) or by the expression of their active G alpha subunits.

The exocytotic release of potent hormones is a tightly controlled process. Its direct regulation without the involvement of second messengers would ensure rapid signal processing. In streptolysin O-permeabilized insulin-secreting cells, a preparation allowing dialysis of cytosolic macromolecules, activation of alpha 2-adrenergic receptors caused pertussis toxin-sensitive inhibition of calcium-induced exocytosis. This inhibition was mimicked very efficiently by the use of specific receptor-mimetic peptides, indicating the involvement of Gi and, to a lesser extent, of G(o). The regulation was exerted beyond the ATP-dependent step of exocytosis. In addition, low nanomolar amounts of pre-activated Gi/G(o) directly inhibited exocytosis. As transient overexpression of constitutively active mutants of G alpha i1, G alpha i2, G alpha i3 and G alpha o2 but not of G alpha o1 reproduced this regulation, the G alpha subunit alone is sufficient to induce inhibition. These results define exocytosis as an effector for heterotrimeric G-proteins and delineate the properties of the transduction pathway.     

Complexin regulates the closure of the fusion pore during regulated vesicle exocytosis

Membrane fusion during exocytosis and throughout the cell is believed to involve members of the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) family of proteins. The assembly of these proteins into a four-helix bundle may be part of the driving force for bilayer fusion. Regulated exocytosis in neurons and related cell types is specialized to be fast and Ca(2+)-dependent suggesting the involvement of other regulatory proteins specific for regulated exocytosis. Among these are the complexins, two closely related proteins that bind only to the assembled SNARE complex. We have investigated the function of complexin by analysis of single vesicle release events in adrenal chromaffin cells using carbon fiber amperometry. These cells express complexin II, and overexpression of this protein modified the kinetics of vesicle release events so that their time course was shortened. This effect depended on complexin interaction with the SNARE complex as introduction of a mutation of Arg-59, a residue that interacts with synaptobrevin in the SNARE complex, abolished its effects. The data are consistent with a function for complexin in stabilizing an intermediate of the SNARE complex to allow kiss-and-run recycling of the exocytosed vesicle.     

Regulation of sodium and calcium channels by signaling complexes

Membrane depolarization and intracellular calcium transients generated by activation of voltage-gated sodium and calcium channels are local signals, which initiate physiological processes such as action potential conduction, synaptic transmission, and excitation-contraction coupling. Targeting of effector proteins and regulatory proteins to ion channels is an important mechanism to ensure speed, specificity, and precise regulation of signaling events in response to local stimuli. In this article, we review recent experimental results showing that sodium and calcium channels form local signaling complexes, in which effector proteins, anchoring proteins, and regulatory proteins interact directly with ion channels. The intracellular domains of these channels serve as signaling platforms, mediating their participation in intracellular signaling processes. These protein-protein interactions are important for efficient synaptic transmission and for regulation of ion channels by neurotransmitters and intracellular second messengers. These localized signaling complexes are essential for normal function and regulation of electrical excitability, synaptic transmission, and excitation-contraction coupling.