Bulk endocytosis in central nerve terminals is activated by strong stimulation; however, the speed at which it is initiated and for how long it persists is still a matter of debate. To resolve this issue, we performed a characterization of bulk endocytic retrieval using action potential trains of increasing intensity. Bulk endocytosis was monitored by the loading of the fluorescent dyes FM2-10 and FM1-43, uptake of tetramethylrhodamine-dextran (40 kDa), or morphological analysis of uptake of the fluid-phase marker horseradish peroxidase. When neuronal cultures were subjected to mild stimulation (200 action potentials at 10 Hz), bulk endocytosis was not observed using any of our assay systems. However, when more intense trains of action potentials (400 or 800 action potentials at 40 and 80 Hz, respectively) were applied to neurons, bulk endocytosis was activated immediately, with the majority of bulk endocytosis complete by the end of stimulation. This contrasts with single synaptic vesicle endocytosis, the majority of which occurred after stimulation was terminated. Thus, bulk endocytosis is a fast event that is triggered during strong stimulation and provides the nerve terminal with an appropriate mechanism to meet the demands of synaptic vesicle retrieval during periods of intense synaptic vesicle exocytosis.

Protein phosphorylation by protein kinase C (PKC) has been implicated in the control of neurotransmitter release and various forms of synaptic plasticity. The PKC substrates responsible for phosphorylation-dependent changes in regulated exocytosis in vivo have not been identified. Munc18a is essential for neurotransmitter release by exocytosis and can be phosphorylated by PKC in vitro on Ser-306 and Ser-313. We demonstrate that it is phosphorylated on Ser-313 in response to phorbol ester treatment in adrenal chromaffin cells. Mutation of both phosphorylation sites to glutamate reduces its affinity for syntaxin and so acts as a phosphomimetic mutation. Unlike phorbol ester treatment, expression of Munc18 with this phosphomimetic mutation in PKC phosphorylation sites did not affect the number of exocytotic events. The mutant did, however, produce changes in single vesicle release kinetics, assayed by amperometry, which were identical to those caused by phorbol ester treatment. Furthermore, the effects of phorbol ester treatment on release kinetics were occluded in cells expressing phosphomimetic Munc18. These results suggest that the dynamics of vesicle release events during exocytosis are controlled by PKC directly through phosphorylation of Munc18 on Ser-313. Phosphorylation of Munc18 by PKC may provide a mechanism for the control of exocytosis and thereby synaptic plasticity.Protein phosphorylation has been long known as an important mechanism for the regulation of exocytosis although, with only a few exceptions such as the synapsins (1), the targets for regulation by phosphorylation in vivo are unknown. Treatment with phorbol esters modifies regulated exocytosis in many different neuronal and non-neuronal (2, 3) cell types leading to increased vesicle recruitment into the ready releasable pool (4 -6), acceleration of fusion pore expansion (7), or changes in the kinetics of exocytosis (8,9). PKC also has a key role in synaptic plasticity (10). The effects of phorbol ester were originally attributed to activation of PKC 1 although the PKC substrates responsible had not been identified, and it is not known if the same target regulates all of the parameters modified by phorbol esters. The SNARE proteins, syntaxin 1, SNAP-25, and VAMP play key roles in exocytosis (11-13), and formation of the SNARE complex has been suggested to be a driving force for membrane fusion (14). The syntaxin-binding protein Munc18a (15) (29) and synaptotagmin I (25). In no case has the functional consequences of these phosphorylation events for exocytosis been established. Indeed, the phosphorylation of SNAP-25 by PKC in PC12 cells lagged well behind the effects of phorbol ester on the extent of exocytosis (29). In that study, it was also shown that the phorbol ester effects had both a PKC-dependent and a PKC-independent component. The synaptic protein Munc13 has been identified as an alternative phorbol ester-binding protein (30, 31), and recently it has been suggested that the effects of phorbol ester on synaptic transmission are mediat...

BackgroundAlthough the ultrastructure of the schistosome esophageal gland was described >35 years ago, its role in the processing of ingested blood has never been established. The current study was prompted by our identification of MEG-4.1 expression in the gland and the observation of erythrocyte uncoating in the posterior esophagus.Methodology/Principal FindingsThe salient feature of the posterior esophagus, characterized by confocal and electron microscopy, is the enormous increase in membrane surface area provided by the plate-like extensions and basal invaginations of the lining syncytium, with unique crystalloid vesicles releasing their contents between the plates. The feeding process was shown by video microscopy to be divided into two phases, blood first accumulating in the anterior lumen before passing as a bolus to the posterior. There it streamed around a plug of material revealed by confocal microscopy as tethered leucocytes. These were present in far larger numbers than predicted from the volume of the lumen, and in varying states of damage and destruction. Intact erythrocytes were detected in the anterior esophagus but not observed thereafter, implying that their lysis occurred rapidly as they enter the posterior. Two further genes, MEGs 4.2 and 14, were shown to be expressed exclusively in the esophageal gland. Bioinformatics predicted that MEGs 4.1 and 4.2 possessed a common hydrophobic region with a shared motif, while antibodies to SjMEG-4.1 showed it was bound to leucocytes in the esophageal lumen. It was also predicted that MEGs 4.1 and 14 were heavily O-glycosylated and this was confirmed for the former by 2D-electrophoresis and Western blotting.Conclusions/SignificanceThe esophageal gland and its products play a central role in the processing of ingested blood. The binding of host antibodies in the esophageal lumen shows that some constituents are antibody targets and could provide a new source of vaccine candidates.

The stimulated dephosphorylation of the dephosphin group of endocytic proteins by calcineurin and their subsequent rephosphorylation by cyclin-dependent kinase 5 (cdk5) is required for synaptic vesicle (SV) retrieval in central nerve terminals. However, the specific endocytic pathway(s) controlled by these enzymes is unknown. To address this issue, we combined functional and morphological assays of endocytosis in primary neuronal cultures with pharmacological and molecular ablation of calcineurin and cdk5 activity. During strong stimulation, inhibition of calcineurin or cdk5 blocked uptake of the activity-dependent membrane marker FM1-43, but not the more hydrophilic FM2-10. However, FM2-10 uptake-measured poststimulation was sensitive to cdk5 and calcineurin inhibition, indicating that a slow form of endocytosis persists after termination of stimulation. In parallel EM studies, inhibition of cdk5 during strong stimulation greatly reduced horseradish peroxidase labeling of plasma membrane-derived nerve terminal endosomes, but not SVs. Furthermore, during mild stimulation, FM1-43 uptake was unaffected by cdk5 inhibition and the SV membrane was exclusively retrieved via a single SV route, suggesting that recruitment of the endosomal route of membrane retrieval is activity dependent. Thus, we propose that the calcineurin/cdk5-dependent phosphorylation cycle of the dephosphins specifically controls a slow endocytic pathway that proceeds via endosomal intermediates and is activated by strong physiological stimulation in central nerve terminals.

Cyclic AMP-dependent protein kinase (PKA) enhances regulated exocytosis in neurons and most other secretory cells. To explore the molecular basis of this effect, known exocytotic proteins were screened for PKA substrates. Both cysteine string protein (CSP) and soluble NSF attachment protein-␣ (␣-SNAP) were phosphorylated by PKA in vitro, but immunoprecipitation of cellular ␣-SNAP failed to detect 32 P incorporation. In contrast, endogenous CSP was phosphorylated in synaptosomes, PC12 cells, and chromaffin cells. In-gel kinase assays confirmed PKA to be a cellular CSP kinase, with phosphorylation occurring on Ser 10 . PKA phosphorylation of CSP reduced its binding to syntaxin by 10-fold but had little effect on its interaction with HSC70 or G-protein subunits. Furthermore, an in vivo role for Ser 10 phosphorylation at a late stage of exocytosis is suggested by analysis of chromaffin cells transfected with wild type or non-phosphorylatable mutant CSP. We propose that PKA phosphorylation of CSP could modulate the exocytotic machinery, by selectively altering its availability for protein-protein interactions.Exocytosis is the final stage of the secretory pathway and involves the fusion of secretory vesicles with the plasma membrane in a constitutive or regulated manner (1). In regulated exocytosis, vesicles accumulate in the cytoplasm and only fuse with the plasma membrane upon receipt of an appropriate stimulus (usually, but not always, an increase in intracellular free Ca 2ϩ ). As regulated exocytosis is the basis of chemical transmission in the brain, much research has been devoted to uncovering its molecular mechanism. This has revealed the involvement of a large number of proteins (2, 3), which can be classified into three groups. The first group, proteins involved in vesicle fusion events in all eukaryotes, includes the SNAP 1 receptors, SNAPs, RABs, and the Sec1 family. The second group comprises proteins involved in regulated exocytosis in various cell types and diverse organisms but absent in yeast. This group includes the synaptotagmins and cysteine string proteins (CSP). The third class can be defined as proteins whose role in regulated exocytosis is cell type-specific. An example from this group is the synapsins, which are important modulators of the synaptic vesicle cycle in neurons (4). The complex interactions between the numerous proteins of these classes presumably enables sophisticated fine-tuning of exocytosis to suit the particular physiological needs of each cell type.In addition to the cell type-specific repertoire of exocytotic proteins expressed, further control over the exocytotic mechanism can be exerted post-translationally (5). Indeed, a large number of studies have implicated protein kinases in the modulation of regulated exocytosis from many cell types by using cellpermeable inhibitors or activators, including Ca 2ϩ /calmodulindependent protein kinase II (6, 7), mitogen-activated protein kinase (8), cGMP-dependent protein kinase (9), and tyrosine kinases (8). However, one shortfall of t...