(1/4295) The disulfide-bonded loop of chromogranin B mediates membrane binding and directs sorting from the trans-Golgi network to secretory granules.

The disulfide-bonded loop of chromogranin B (CgB), a regulated secretory protein with widespread distribution in neuroendocrine cells, is known to be essential for the sorting of CgB from the trans-Golgi network (TGN) to immature secretory granules. Here we show that this loop, when fused to the constitutively secreted protein alpha1-antitrypsin (AT), is sufficient to direct the fusion protein to secretory granules. Importantly, the sorting efficiency of the AT reporter protein bearing two loops (E2/3-AT-E2/3) is much higher compared with that of AT with a single disulfide-bonded loop. In contrast to endogenous CgB, E2/3-AT-E2/3 does not undergo Ca2+/pH-dependent aggregation in the TGN. Furthermore, the disulfide-bonded loop of CgB mediates membrane binding in the TGN and does so with 5-fold higher efficiency if two loops are present on the reporter protein. The latter finding supports the concept that under physiological conditions, aggregates of CgB are the sorted units of cargo which have multiple loops on their surface leading to high membrane binding and sorting efficiency of CgB in the TGN.  (+info)

(2/4295) The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis.

Polarized secretion requires proper targeting of secretory vesicles to specific sites on the plasma membrane. Here we report that the exocyst complex plays a key role in vesicle targeting. Sec15p, an exocyst component, can associate with secretory vesicles and interact specifically with the rab GTPase, Sec4p, in its GTP-bound form. A chain of protein-protein interactions leads from Sec4p and Sec15p on the vesicle, through various subunits of the exocyst, to Sec3p, which marks the sites of exocytosis on the plasma membrane. Sec4p may control the assembly of the exocyst. The exocyst may therefore function as a rab effector system for targeted secretion.  (+info)

(3/4295) The optically determined size of exo/endo cycling vesicle pool correlates with the quantal content at the neuromuscular junction of Drosophila larvae.

According to the current theory of synaptic transmission, the amplitude of evoked synaptic potentials correlates with the number of synaptic vesicles released at the presynaptic terminals. Synaptic vesicles in presynaptic boutons constitute two distinct pools, namely, exo/endo cycling and reserve pools (). We defined the vesicles that were endocytosed and exocytosed during high K+ stimulation as the exo/endo cycling vesicle pool. To determine the role of exo/endo cycling vesicle pool in synaptic transmission, we estimated the quantal content electrophysiologically, whereas the pool size was determined optically using fluorescent dye FM1-43. We then manipulated the size of the pool with following treatments. First, to change the state of boutons of nerve terminals, motoneuronal axons were severed. With this treatment, the size of exo/endo cycling vesicle pool decreased together with the quantal content. Second, we promoted the FM1-43 uptake using cyclosporin A, which inhibits calcineurin activities and enhances endocytosis. Cyclosporin A increased the total uptake of FM1-43, but neither the size of exo/endo cycling vesicle pool nor the quantal content changed. Third, we increased the size of exo/endo cycling vesicle pool by forskolin, which enhances synaptic transmission. The forskolin treatment increased both the size of exo/endo cycling vesicle pool and the quantal content. Thus, we found that the quantal content was closely correlated with the size of exo/endo cycling vesicle pool but not necessarily with the total uptake of FM1-43 fluorescence by boutons. The results suggest that vesicles in the exo/endo cycling pool primarily participate in evoked exocytosis of vesicles.  (+info)

(4/4295) Human granulocytic ehrlichiosis agent and Ehrlichia chaffeensis reside in different cytoplasmic compartments in HL-60 cells.

The human granulocytic ehrlichiosis (HGE) agent resides and multiplies exclusively in cytoplasmic vacuoles of granulocytes. Double immunofluorescence labeling was used to characterize the nature of the HGE agent replicative inclusions and to compare them with inclusions containing the human monocytic ehrlichia, Ehrlichia chaffeensis, in HL-60 cells. Although both Ehrlichia spp. can coinfect HL-60 cells, they resided in separate inclusions. Inclusions of both Ehrlichia spp. were not labeled with either anti-lysosome-associated membrane protein 1 or anti-CD63. Accumulation of myeloperoxidase-positive granules were seen around HGE agent inclusions but not around E. chaffeensis inclusions. 3-(2, 4-Dinitroanilino)-3'-amino-N-methyldipropylamine and acridine orange were not localized to either inclusion type. Vacuolar-type H+-ATPase was not colocalized with HGE agent inclusions but was weakly colocalized with E. chaffeensis inclusions. E. chaffeensis inclusions were labeled with the transferrin receptor, early endosomal antigen 1, and rab5, but HGE agent inclusions were not. Some HGE agent and E. chaffeensis inclusions colocalized with major histocompatibility complex class I and II antigens. These two inclusions were not labeled for annexins I, II, IV, and VI; alpha-adaptin; clathrin heavy chain; or beta-coatomer protein. Vesicle-associated membrane protein 2 colocalized to both inclusions. The cation-independent mannose 6-phosphate receptor was not colocalized with either inclusion type. Endogenously synthesized sphingomyelin, from C6-NBD-ceramide, was not incorporated into either inclusion type. Brefeldin A did not affect the growth of either Ehrlichia sp. in HL-60 cells. These results suggest that the HGE agent resides in inclusions which are neither early nor late endosomes and does not fuse with lysosomes or Golgi-derived vesicles, while E. chaffeensis resides in an early endosomal compartment which accumulates the transferrin receptor.  (+info)

(5/4295) gp49B1 inhibits IgE-initiated mast cell activation through both immunoreceptor tyrosine-based inhibitory motifs, recruitment of src homology 2 domain-containing phosphatase-1, and suppression of early and late calcium mobilization.

We define by molecular, pharmacologic, and physiologic approaches the proximal mechanism by which the immunoglobulin superfamily member gp49B1 inhibits mast cell activation mediated by the high affinity Fc receptor for IgE (FcepsilonRI). In rat basophilic leukemia-2H3 cells expressing transfected mouse gp49B1, mutation of tyrosine to phenylalanine in either of the two immunoreceptor tyrosine-based inhibitory motifs of the gp49B1 cytoplasmic domain partially suppressed gp49B1-mediated inhibition of exocytosis, whereas mutation of both abolished inhibitory capacity. Sodium pervanadate elicited tyrosine phosphorylation of native gp49B1 and association of the tyrosine phosphatases src homology 2 domain-containing phosphatase-1 (SHP-1) and SHP-2 in mouse bone marrow-derived mast cells (mBMMCs). SHP-1 associated transiently with gp49B1 within 1 min after coligation of gp49B1 with cross-linked FcepsilonRI in mBMMCs. SHP-1-deficient mBMMCs exhibited a partial loss of gp49B1-mediated inhibition of FcepsilonRI-induced exocytosis at concentrations of IgE providing optimal exocytosis, revealing a central, but not exclusive, SHP-1 requirement in the counter-regulatory pathway. Coligation of gp49B1 with cross-linked FcepsilonRI on mBMMCs inhibited early release of calcium from intracellular stores and subsequent influx of extracellular calcium, consistent with SHP-1 participation. Because exocytosis is complete within 2 min in mBMMCs, our studies establish a role for SHP-1 in the initial counter-regulatory cellular responses whereby gp49B1 immunoreceptor tyrosine-based inhibition motifs rapidly transmit inhibition of FcepsilonRI-mediated exocytosis.  (+info)

(6/4295) Augmentation is a potentiation of the exocytotic process.

Short-term synaptic enhancement is caused by an increase in the probability with which synaptic terminals release transmitter in response to presynaptic action potentials. Since exocytosed vesicles are drawn from a readily releasable pool of packaged transmitter, enhancement must result either from an increase in the size of the pool or an elevation in the fraction of releasable vesicles that undergoes exocytosis with each action potential. We show here that at least one major component of enhancement, augmentation, is not caused by an increase in the size of the readily releasable pool but is instead associated with an increase in the efficiency with which action potentials induce the exocytosis of readily releasable vesicles.  (+info)

(7/4295) Role of mitochondrial dysfunction in the Ca2+-induced decline of transmitter release at K+-depolarized motor neuron terminals.

The present study tested whether a Ca2+-induced disruption of mitochondrial function was responsible for the decline in miniature endplate current (MEPC) frequency that occurs with nerve-muscle preparations maintained in a 35 mM potassium propionate (35 mM KP) solution containing elevated calcium. When the 35 mM KP contained control Ca2+ (1 mM), the MEPC frequency increased and remained elevated for many hours, and the mitochondria within twitch motor neuron terminals were similar in appearance to those in unstimulated terminals. All nerve terminals accumulated FM1-43 when the dye was present for the final 6 min of a 300-min exposure to 35 mM KP with control Ca2+. In contrast, when Ca2+ was increased to 3.6 mM in the 35 mM KP solution, the MEPC frequency initially reached frequencies >350 s-1 but then gradually fell approaching frequencies <50 s-1. A progressive swelling and eventual distortion of mitochondria within the twitch motor neuron terminals occurred during prolonged exposure to 35 mM KP with elevated Ca2+. After approximately 300 min in 35 mM KP with elevated Ca2+, only 58% of the twitch terminals accumulated FM1-43. The decline in MEPC frequency in 35 mM KP with elevated Ca2+ was less when 15 mM glucose was present or when preparations were pretreated with 10 microM oligomycin and then bathed in the 35 mM KP with glucose. When glucose was present, with or without oligomycin pretreatment, a greater percentage of twitch terminals accumulated FM1-43. However, the mitochondria in these preparations were still greatly swollen and distorted. We propose that prolonged depolarization of twitch motor neuron terminals by 35 mM KP with elevated Ca2+ produced a Ca2+-induced decrease in mitochondrial ATP production. Under these conditions, the cytosolic ATP/ADP ratio was decreased thereby compromising both transmitter release and refilling of recycled synaptic vesicles. The addition of glucose stimulated glycolysis which contributed to the maintenance of required ATP levels.  (+info)

(8/4295) Diffusion barriers limit the effect of mobile calcium buffers on exocytosis of large dense cored vesicles.

Fast exocytosis in melanotropic cells, activated by calcium entry through voltage-gated calcium channels, is very sensitive to mobile calcium buffers (complete block at 800 microM ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA)). This indicates that calcium diffuses a substantial distance from the channel to the vesicle. Surprisingly, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), having a similar KD for calcium as EGTA but a approximately 100 times faster binding rate, blocked exocytosis only twice as effectively as EGTA. Using computer simulations, we demonstrate that this result cannot be explained by free diffusion and buffer binding rates. We hypothesized that local saturation of calcium buffers is involved. A diffusion barrier for both calcium and buffer molecules, located 50-300 nm from the membrane and reducing diffusion 1000 to 10,000 times, generated similar calcium concentrations for specific concentrations of EGTA and BAPTA. With such barriers, calcium rise phase kinetics upon short step depolarizations (2-20 ms) were faster for EGTA than for BAPTA, implying that short depolarizations should allow exocytosis with 50 microM EGTA but not with 25 microM BAPTA. This prediction was confirmed experimentally with capacitance measurements. Coupling exocytosis to calcium dynamics in the model, we found that a barrier with a approximately 3000 times reduced diffusion at approximately 130 nm beneath the membrane best explains the experimentally observed effects of EGTA and BAPTA on block and kinetics of release.  (+info)