Types Of Exocytosis Research Articles

Regulatory Mechanism of SNAP23 in Phagosome Formation and Maturation.

Synaptosomal associated protein of 23 kDa (SNAP23), a plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), is a ubiquitously expressed protein that is generally involved in fusion of the plasma membrane and secretory or endosomal recycling vesicles during several types of exocytosis. SNAP23 is expressed in phagocytes, such as neutrophils, macrophages, and dendritic cells, and functions in both exocytosis and phagocytosis. This review focuses on the function of SNAP23 in immunoglobulin G Fc receptor-mediated phagocytosis by macrophages. SNAP23 and its partner SNAREs mediate fusion of the plasma membrane with intracellular organelles or vesicles to form phagosomes as well as the fusion of phagosomes with endosomes or lysosomes to induce phagosome maturation, characterized by reactive oxygen species production and acidification. During these processes, SNAP23 function is regulated by phosphorylation. In addition, microtubule-associated protein 1A/1B light chain 3 (LC3)-associated phagocytosis, which tightly promotes or suppresses phagosome maturation depending on the foreign target, requires SNAP23 function. SNAP23 that is enriched on the phagosome membrane during LC3-associated phagocytosis may be phosphorylated or dephosphorylated, thereby enhancing or inhibiting subsequent phagosome maturation, respectively. These findings have increased our understanding of the SNAP23-associated membrane trafficking mechanism in phagocytes, which has important implications for microbial pathogenesis and innate and adaptive immune responses.

Analysis of Kinetically Distinguished Synaptic Subtypes in Hippocampal Neurons

Neural transmission and information processing requires not only quantitative modulation of transmitter release but also temporal coordination of the information flow. Here we describe that the onset of synaptic exocytosis in specialized individual synapses can be delayed by several seconds after the start of electrical stimulation. Hippocampal neurons from newborn rats were cultured in vitro until they obtained functionally mature synapses. Synaptic vesicles were fluorescently labeled using 600 pulses at 30 Hz. After thorough wash-out synapses were destained in two consecutive stimulation cycles, where the first served as internal control. Neurons were either treated with 1 µM fluoxetine or control solution during the second cycle and kinetic parameters from both cycles were compared. Mean exocytosis of FM followed roughly a single exponential decay curve during the stimulation period. Detailed analysis, however, revealed that the general synaptic population was composed of at least 4 kinetically distinguished subtypes. FM-exocytosis kinetics were significantly altered by short term application of fluoxetine during the second stimulation cycle, which decreased the number of delayed synapses by more than 50 % and increased the speed of synaptic exocytosis significantly by rising the percentage of fast two-component synapses. Synapses in control experiments showed the opposite effects when they were stimulated in a second stimulation cycle. Exocytosis of synapses, expressing synaptopHlourin, was never delayed after start of stimulation. Our results suggest that the mode of exocytosis is switched between full vesicle fusion and “kiss-and-run” within the same synapse. The delay of FM release could be explained by “kiss-and-run” type of exocytosis since the dye couldn’t leave a vesicle through a narrow fusion pore. Both, H+ ions and neurotransmitter molecules, however, can leave the vesicle lumen through a pore, and therefore allow synaptopHlourin to monitor exocytosis.

Action potential-triggered somatic exocytosis in mesencephalic trigeminal nucleus neurons in rat brain slices

The neurons in the mesencephalic trigeminal nucleus (MeV) play essential roles in proprioceptive sensation of the face and oral cavity. The somata of MeV neurons are generally assumed to carry out neuronal functions but not to play a direct role in synaptic transmission. Using whole-cell recording and membrane capacitance (C(m)) measurements, we found that the somata of MeV neurons underwent robust exocytosis (C(m) jumps) upon depolarization and with the normal firing of action potentials in brain slices. Both removing [Ca(2+)](o) and buffering [Ca(2+)](i) with BAPTA blocked this exocytosis, indicating that it was completely Ca(2+) dependent. In addition, an electron microscopic study showed synaptic-like vesicles approximated to the plasma membrane in somata. There was a single Ca(2+)-dependent releasable vesicle pool with a peak release rate of 1912 fF s(-1). Importantly, following depolarization-induced somatic exocytosis, GABA-mediated postsynaptic currents were transiently reduced by 31%, suggesting that the somatic vesicular release had a retrograde effect on afferent GABAergic transmission. These results provide strong evidence that the somata of MeV neurons undergo robust somatic secretion and may play a crucial role in bidirectional communication between somata and their synaptic inputs in the central nervous system.

Role of VAMP8/endobrevin in Constitutive Exocytotic Pathway in HeLa Cells

To evaluate the role of VAMP8/endobrevin in constitutive exocytosis, we have examined the exocytotic pathways of VAMP8 and human growth hormone, both GFP-tagged, by total internal reflection fluorescence microscopy (TIRF-M). Human GH-GFP and VAMP8-GFP were similarly expressed in small round vesicles and elongated tubular vesicles in HeLa cells, and were mostly exocytosed at the peripheral area of the cells. VAMP8-GFP gave 2 types of exocytotic images: a burst type and a non-burst type. The burst type showed a sharp transient increase in the peak fluorescence intensity and a much slower decrease in the average intensity in the active windows, where exocytosis took place, as observed in the "full-fusion" type of exocytosis. The non-burst type showed a relatively long-lasting fusion to the plasma membrane with little transfer of VAMP8-GFP to the plasma membrane, as observed in the so-called "kiss-and-run" type of exocytosis. Endogenous VAMP8 and hGH-GFP were colocalized on the same vesicles at least in part. However, the constitutive exocytosis of hGH-GFP and CLuc, a secreted luciferase from Cypridina noctiluca, was normal, even when siRNAs for VAMP8 and VAMP3 robustly decreased their proteins. These results suggest that VAMP8 is not essential for constitutive exocytosis, although it can be involved in the exocytosis.

Lipid droplets highjack SNAREs

Most intracellular organelles are surrounded by a lipid bilayer and require specialized machinery for membrane fusion. Remarkably, lipid droplets, which contain a hydrophobic core and a monolayer of membrane lipids, can sequester distinct components of this fusion machinery and thereby downregulate certain types of exocytosis.

A Novel Ubiquitous Form of Munc-18 Interacts with Multiple Syntaxins.: USE OF THE YEAST TWO-HYBRID SYSTEM TO STUDY INTERACTIONS BETWEEN PROTEINS INVOLVED IN MEMBRANE TRAFFIC

Munc-18-1 is a 67-kDa neuronal protein that binds tightly to syntaxin 1 and functions in synaptic vesicle exocytosis (Hata, Y., Slaughter, C.A., and Südhof, T.C. (1993a) Nature 366, 347-351). We have now characterized a new Munc-18 isoform, Munc-18-2, that exhibits 63% amino acid sequence identity with Munc-18-1. Munc-18-2 is expressed in most tissues, whereas Munc-18-1 is primarily expressed in brain. Using recombinant Munc-18-1 and Munc-18-2 produced in COS cells, we show that both forms of Munc-18 bind tightly to syntaxins 1A, 2, and 3 but not to syntaxin 4. In an independent approach to study the binding specificities of Munc-18-1 and Munc-18-2, we used the yeast two-hybrid system. This assay system depends on protein-protein interactions in the cell nucleus. We validated its utility for studying membrane trafficking proteins by testing well characterized interactions between cytosolic proteins that are known to be physiologically important in exocytosis. Strong interactions, such as the binding of syntaxins 1-4 with SNAP-25, were effectively detected by the yeast two-hybrid assay, but weak binding, such as the binding of syntaxins to synaptotagmin or of synaptotagmin to neurexins, was not. Studies on full-length and truncated forms of Munc-18s by the yeast two-hybrid system confirmed their interactions with syntaxins. Both the N and the C terminus of Munc-18 were essential for binding. Munc-18-1 and Munc-18-2 bind only to syntaxins 1A, 2, and 3 but not 4 and 5 by yeast-two hybrid system assays. Our studies demonstrate that neural and non-neural tissues have distinct forms of Munc-18, which may function in different types of exocytosis. The lack of specificity of the interactions between syntaxins and Munc-18s indicates that specificity of membrane trafficking reactions is not dependent on this interaction.

Regulation of Membrane Fusion during Exocytosis

Publisher Summary This chapter describes the regulation of membrane fusion during exocytosis. Exocytosis involves the fusion of a secretory organelle with the cell membrane. It is important to discriminate between regulated (triggered) exocytosis and constitutive (untriggered) exocytosis. Specific signals and signal-transfer mechanisms occur only with regulated exocytosis. The mechanisms of membrane-fusion regulation are different from these two types of exocytosis. Exocytosis is of the PF–PF fusion type; plasmatic membrane faces fuse. It always follows the focal fusion scheme: fusion is restricted to a small area of ≤ 10 nm, before the exocytotic opening expands to release the secretory contents. Adenosine triphosphate (ATP) is required for intracellular transport of secretory organelles; it might also be required for a rearrangement of microfilamentous elements on the cell periphery during secretion. The protein phosphorylation, another phenomenon generally occurring during secretory activity, clearly requires ATP. The hydrolysis of membrane-bound ATP has been proposed to be a fusogenic process. Guanosine triphosphate (GTP) analogs stimulate membrane fusion in some systems; they might act by various mechanisms.

The fine structure of tomes' process of rat incisor ameloblasts and its relationship to the elaboration of enamel

The shape of Tomes' process and its relationship to enamel precursors, the growing enamel, and the apical terminal bars were studied with light and electron microscopes in the enamel organs of the lower incisors of adult rats. It was found that the proximal part of Tomes' process has a complex cross section which is described here as consisting of head, body, and large and small foot processes. The shape of the cross section is related to the direction of tooth eruption and the direction of the presumed sideways motion of the ameloblast. At the level of the apical terminal bars the ameloblast cross section is rectangular, with the long axis in the direction of the axis of the tooth. The major terminal bars have a large number of filaments showing little recognizable order, and an almost uninterrupted zonula adherens. The minor terminal bars have fewer filaments which often form well-defined bundles, and their zonula adherens is more frequently interrupted by membrane invaginations, maculae occludentes, and distensions of the extracellular space. Stippled material was seen first in irregularly shaped vesicles in the ameloblast apex just proximal to the terminal bar level. It appears to be secreted into the extracellular space at the level of the terminal bars and distal to them. It is incorporated into the interrod enamel in an erratic fashion and remains uncalcified for a period of time. The anomaly of its distribution may indicate that it is not an essential enamel component in the rat incisor. No new stippled material was seen to be secreted in rod enamel formation. The first enamel to be formed is a broad band of interrod enamel between Tomes' processes of the same row. It is formed in close proximity to, and thus presumably to a large extent by, Tomes' processes of the neighboring (in apical direction) row. A thin band of interrod enamel between rows is then deposited by ameloblasts of the adjacent rows, while the enamel rod is formed by one ameloblast. At the enamel growth fronts, the three enamel components (crystallites, pericrystal membrane, background matrix) appear almost simultaneously in the enamel space. Secretion granules seem to participate in rod enamel formation by a type of exocytosis.