Gross anatomy of the female genital organs of the domestic donkey (Equus asinus Linné, 1758).

Although donkeys play an important role as companion or pack and draught animals, theriogenological studies and anatomical data on the genital organs of the jenny are sparse. To provide anatomical descriptions and morphometric data, the organa genitalia feminina, their arteries and the ligamentum latum uteri of 10 adult but maiden jennies were examined by means of gross anatomical and morphometric techniques. In comparison with anatomical data of horses obtained from literature the genital organs of jennies appear to be more voluminous in relation to the body mass and the position of the ovaries is slightly further cranial than in mares. In asses, the ovaries contain large follicles reaching a diameter of up to 40 mm. The mesosalpinx is much wider than in the horse forming a considerably spacious bursa ovarica. The asinine ligamentum teres uteri reveals a very prominent cranial end, the 'appendix'. Tortuous mucosal folds occur in the wall of the jenny's cervical channel. The vascularization of the female genital organs of asses is very similar to that of horses. One of the examined specimens reveals a large mucosal fold dividing the cranial part of the vagina into a left and right compartment.

Synaptotagmin C2A loop 2 mediates Ca2+-dependent SNARE interactions essential for Ca2+-triggered vesicle exocytosis.

Synaptotagmins contain tandem C2 domains and function as Ca(2+) sensors for vesicle exocytosis but the mechanism for coupling Ca(2+) rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca(2+)-triggered vesicle exocytosis has not been directly assessed. We identified sites on synaptotagmin-1 that mediate Ca(2+)-dependent SNAP25 binding by zero-length cross-linking. Mutation of these sites in C2A and C2B eliminated Ca(2+)-dependent synaptotagmin-1 binding to SNAREs without affecting Ca(2+)-dependent membrane binding. The mutants failed to confer Ca(2+) regulation on SNARE-dependent liposome fusion and failed to restore Ca(2+)-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca(2+)-dependent SNARE binding by synaptotagmin is essential for Ca(2+)-triggered vesicle exocytosis and that Ca(2+)-dependent membrane binding by itself is insufficient to trigger fusion. A structure-based model of the SNARE-binding surface of C2A provided a new view of how Ca(2+)-dependent SNARE and membrane binding occur simultaneously.

PC12 cells as a model for studies of regulated secretion in neuronal and endocrine cells.

Pheochromocytoma-derived cell lines such as PC12 cells maintain a differentiated neuroendocrine phenotype and have been widely used as a convenient model system for a wide variety of cell biological studies on neurotrophin action, monoamine biogenesis, protein trafficking, and secretory vesicle dynamics. This chapter reviews a number of methods that are useful for studies of the regulated dense core vesicle secretory pathway. This includes protocols for maintaining cells and preserving their phenotype. A variety of assays are discussed for monitoring secretion in intact or permeable cells and in transfected cells. Specific methods for immunocytochemical studies in permeable cells are discussed. Finally, protocols for high-efficiency PC12 cell transfections and the isolation of stably transfected cell lines are provided.

Prime movers of synaptic vesicle exocytosis.

Chemical synapses contain specialized pre- and postsynaptic structures that underlie rapid synaptic transmission and its modulation. Studies of postsynaptic organization have revealed a network of interacting proteins that enable rapid synaptic responses and their modulation. Recent genetic and electrophysiological studies on two active zone proteins-RIM and Munc13-reveal important roles in priming vesicles for Ca(2+)-triggered fusion and in mediating the regulation of this process. This work sheds new light on how presynaptic structure provides speed and plasticity to synaptic transmission.

Synaptotagmin modulation of fusion pore kinetics in regulated exocytosis of dense-core vesicles.

In the exocytosis of neurotransmitter, fusion pore opening represents the first instant of fluid contact between the vesicle lumen and extracellular space. The existence of the fusion pore has been established by electrical measurements, but its molecular composition is unknown. The possibility that synaptotagmin regulates fusion pores was investigated with amperometry to monitor exocytosis of single dense-core vesicles. Overexpression of synaptotagmin I prolonged the time from fusion pore opening to dilation, whereas synaptotagmin IV shortened this time. Both synaptotagmin isoforms reduced norepinephrine flux through open fusion pores. Thus, synaptotagmin interacts with fusion pores, possibly by associating with a core complex of membrane proteins and/or lipid.

PIP kinase Igamma is the major PI(4,5)P(2) synthesizing enzyme at the synapse.

Disruption of the presynaptically enriched polyphosphoinositide phosphatase synaptojanin 1 leads to an increase of clathrin-coated intermediates and of polymerized actin at endocytic zones of nerve terminals. These changes correlate with elevated levels of PI(4,5)P(2) in neurons. We report that phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma), a major brain PI(4)P 5-kinase, is concentrated at synapses. Synaptojanin 1 and PIPKIgamma antagonize each other in the recruitment of clathrin coats to lipid membranes. Like synaptojanin 1 and other proteins involved in endocytosis, PIPKIgamma undergoes stimulation-dependent dephosphorylation. These results implicate PIPKIgamma in the synthesis of a PI(4,5)P(2) pool that acts as a positive regulator of clathrin coat recruitment and actin function at the synapse.

Drosophila CAPS is an essential gene that regulates dense-core vesicle release and synaptic vesicle fusion.

Calcium-activated protein for secretion (CAPS) is proposed to play an essential role in Ca2+-regulated dense-core vesicle exocytosis in vertebrate neuroendocrine cells. Here we report the cloning, mutation, and characterization of the Drosophila ortholog (dCAPS). Null dCAPS mutants display locomotory deficits and complete embryonic lethality. The mutant NMJ reveals a 50% loss in evoked glutamatergic transmission, and an accumulation of synaptic vesicles at active zones. Importantly, dCAPS mutants display a highly specific 3-fold accumulation of dense-core vesicles in synaptic terminals, which was not observed in mutants that completely arrest synaptic vesicle exocytosis. Targeted transgenic CAPS expression in identified motoneurons fails to rescue dCAPS neurotransmission defects, demonstrating a cell nonautonomous role in synaptic vesicle fusion. We conclude that dCAPS is required for dense-core vesicle release and that a dCAPS-dependent mechanism modulates synaptic vesicle release at glutamatergic synapses.

PI(4,5)P(2) regulation of surface membrane traffic.

Phosphatidylinositol 4,5-biphosphate (PI[4,5]P(2)) has emerged as an important signaling molecule in the membrane for regulating vesicle exo- and endocytosis and the accompanying actin cytoskeletal rearrangements. Localization studies with GFP-tagged binding domains and antibodies provide new views of the non-uniform, dynamic distribution of PI(4,5)P(2) in membranes and its organization in raft-like domains. The targeting of phosphoinositide kinases by GTPases can coordinate the reactions of membrane fusion and fission with cytoskeletal assembly, providing a basis for membrane movement.

G protein betagamma subunit-mediated presynaptic inhibition: regulation of exocytotic fusion downstream of Ca2+ entry.

The nervous system can modulate neurotransmitter release by neurotransmitter activation of heterotrimeric GTP-binding protein (G protein)-coupled receptors. We found that microinjection of G protein betagamma subunits (Gbetagamma) mimics serotonin's inhibitory effect on neurotransmission. Release of free Gbetagamma was critical for this effect because a Gbetagamma scavenger blocked serotonin's effect. Gbetagamma had no effect on fast, action potential-evoked intracellular Ca2+ release that triggered neurotransmission. Inhibition of neurotransmitter release by serotonin was still seen after blockade of all classical Gbetagamma effector pathways. Thus, Gbetagamma blocked neurotransmitter release downstream of Ca2+ entry and may directly target the exocytotic fusion machinery at the presynaptic terminal.

The C2B domain of synaptotagmin is a Ca(2+)-sensing module essential for exocytosis.

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca(2+) sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca(2+) ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca(2+)-sensing module. Here, we report that Ca(2+) drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca(2)+ are mediated by a set of conserved acidic Ca(2)+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca(2)+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.

Priming in exocytosis: attaining fusion-competence after vesicle docking.

Membrane contact established by tethering or docking mechanisms is not a sufficient condition for membrane fusion. In neural and neuroendocrine cells, only a small fraction of secretory vesicles docked at the plasma membrane are fusion-competent and undergo rapid ATP-independent fusion in response to Ca(2+) elevations. Additional biochemical events termed 'priming' are essential to render vesicles competent for Ca(2+)-triggered fusion. The priming of vesicles is ATP-dependent and a number of ATP-dependent priming reactions have been characterized in permeable neuroendocrine cells. These involve NSF-mediated priming of SNARE protein complexes, the ATP-dependent synthesis of phosphoinositides, and protein kinase-mediated protein phosphorylation. In addition, munc13 is an important protein involved in priming synaptic vesicles. An emphasis in this review is on recent work indicating that priming events identified in the pathways of regulated exocytosis share many features with pre-fusion processes characterized in constitutive fusion pathways.

Rapid regulated dense-core vesicle exocytosis requires the CAPS protein.

Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their precise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca(2+)-dependent activator protein for secretion), a protein required for Ca(2+)-dependent exocytosis of dense-core vesicles, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. Flash photolysis of caged Ca(2+) elicited biphasic capacitance increases consisting of rapid and slow components with distinct Ca(2+) dependencies. A threshold of approximately 10 microM Ca(2+) was required to trigger the slow component, while the rapid capacitance increase was recorded already at a intracellular Ca(2+) activity < 10 microM. Both kinetic membrane capacitance components were abolished by botulinum neurotoxin B or E treatment, suggesting involvement of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent vesicle fusion. The rapid but not the slow component was inhibited by CAPS antibody. These results were further clarified by immunocytochemical studies that revealed that CAPS was present on only a subset of dense-core vesicles. Overall, the results indicate that dense-core vesicle exocytosis in melanotrophs occurs by two parallel pathways. The faster pathway exhibits high sensitivity to Ca(2+) and requires the presence of CAPS, which appears to act at a late stage in the secretory pathway.

The C terminus of SNAP25 is essential for Ca(2+)-dependent binding of synaptotagmin to SNARE complexes.

The plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) and the vesicle SNARE protein vesicle-associated membrane protein (VAMP) are essential for a late Ca(2+)-dependent step in regulated exocytosis, but their precise roles and regulation by Ca(2+) are poorly understood. Botulinum neurotoxin (BoNT) E, a protease that cleaves SNAP25 at Arg(180)-Ile(181), completely inhibits this late step in PC12 cell membranes, whereas BoNT A, which cleaves SNAP25 at Gln(197)-Arg(198), is only partially inhibitory. The difference in toxin effectiveness was found to result from a reversal of BoNT A but not BoNT E inhibition by elevated Ca(2+) concentrations. BoNT A treatment essentially increased the Ca(2+) concentration required to activate exocytosis, which suggested a role for the C terminus of SNAP25 in the Ca(2+) regulation of exocytosis. Synaptotagmin, a proposed Ca(2+) sensor for exocytosis, was found to bind SNAP25 in a Ca(2+)-stimulated manner. Ca(2+)-dependent binding was abolished by BoNT E treatment, whereas BoNT A treatment increased the Ca(2+) concentration required for binding. The C terminus of SNAP25 was also essential for Ca(2+)-dependent synaptotagmin binding to SNAP25. syntaxin and SNAP25.syntaxin.VAMP SNARE complexes. These results clarify classical observations on the Ca(2+) reversal of BoNT A inhibition of neurosecretion, and they suggest that an essential role for the C terminus of SNAP25 in regulated exocytosis is to mediate Ca(2+)-dependent interactions between synaptotagmin and SNARE protein complexes.

Yeast Sec14p deficient in phosphatidylinositol transfer activity is functional in vivo.

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi secretory function. It is widely accepted, though unproven, that phosphatidylinositol transfer between membranes represents the physiological activity of phosphatidylinositol transfer proteins (PITPs). We report that Sec14pK66,239A is inactivated for phosphatidylinositol, but not phosphatidylcholine (PC), transfer activity. As expected, Sec14pK66,239A fails to meet established criteria for a PITP in vitro and fails to stimulate phosphoinositide production in vivo. However, its expression efficiently rescues the lethality and Golgi secretory defects associated with sec14-1ts and sec14 null mutations. This complementation requires neither phospholipase D activation nor the involvement of a novel class of minor yeast PITPs. These findings indicate that PI binding/transfer is remarkably dispensable for Sec14p function in vivo.

Ca(2+)-dependent activator protein for secretion is critical for the fusion of dense-core vesicles with the membrane in calf adrenal chromaffin cells.

Calcium-dependent activator protein for secretion (CAPS) is a neural/endocrine cell-specific protein that has been shown to function at the Ca(2+)-dependent triggering step of dense-core vesicle (DCV) exocytosis in permeabilized PC12 cells. To evaluate the function of CAPS under physiological conditions, we introduced affinity-purified anti-CAPS IgGs into calf adrenal chromaffin (AC) cells via a patch pipette and tested the kinetics of catecholamine secretion using both amperometric and membrane capacitance techniques. The antibodies reacted with a single major approximately 145 kDa protein in AC cells based on immunoblot analysis. AC cells stimulated with sequential trains of action potentials at 7 Hz resulted in successive secretory episodes of equivalent magnitude. When either of two different anti-CAPS IgGs or their Fab fragments were present, a rapid and progressive inhibition of catecholamine release ensued to a maximum of >80%. The effect was specific because preabsorption of IgGs with the respective antigens ablated the inhibitory effect, and the IgGs had no effect on Ca currents. CAPS immunoneutralization not only reduced the number of amperometric spikes but markedly altered the kinetic characteristics of the residual events. The remaining spikes were much smaller (by 85%) and broader (by approximately 3.5-fold) than those in control cells, suggesting that CAPS plays a role in determining release of vesicle contents via the fusion pore. Anti-CAPS IgGs also slowed the rate of the initial exocytotic capacitance burst, representing the docked-and-primed vesicle pool, by approximately 90% but had no effect on the kinetics of rapid endocytosis. These results suggest that CAPS is a key component regulating the fusion of DCVs to the plasma membrane, and possibly fusion pore dilation, in catecholamine secretion from AC cells.

Large dense-core vesicle exocytosis in PC12 cells.

A current major challenge in the study of regulated exocytosis is the identification of essential proteins that mediate the transit of secretory vesicles through trafficking stages such as recruitment, docking, and fusion. Defining the physiological roles and mechanisms of action of these essential proteins is paramount. The reconstitution of stages of regulated exocytosis in cell-free systems provides the opportunity to identify required proteins and establish their stage-specific mechanisms of action. PC12 cells, clonal cell lines of adrenal medullary origin, possess large dense-core vesicles that retain their competence for regulated exocytosis in a variety of permeable cell and isolated membrane preparations. We describe several cell-free systems for studies of regulated exocytosis derived from PC12 cells.

CAPS (mammalian UNC-31) protein localizes to membranes involved in dense-core vesicle exocytosis.

CAPS is a neural/endocrine-specific protein discovered as a cytosolic factor required for Ca2+-activated dense-core vesicle (DCV) exocytosis in permeable neuroendocrine cells. We report that CAPS is also a membrane-associated, peripherally bound protein in brain homogenates that localizes Selectively to plasma membranes and to DCVs but not to small clear synaptic vesicles (SVs). CAPS exhibits high affinity and saturable binding to DCVs by interaction with bilayer phospholipids. Specific CAPS antibodies inhibit Ca2+-activated norepinephrine release from lysed synaptosomes that contain membrane-associated CAPS, indicating that membrane-bound CAPS is essential for neural DCV exocytosis. CAPS is a functional component of the exocytotic machinery that localizes selectively to DCVs, and it may confer distinct regulatory features on neuropeptide and biogenic amine transmitter secretion.

Differential regulation of exocytosis by calcium and CAPS in semi-intact synaptosomes.

Using a novel approach to measure exocytosis in vitro from semi-intact synaptosomes, we establish that the Ca2+-dependent release of glutamate requires cytosolic factors for mobilization from the reserve pool. The cytosolic activity for glutamate release was not satisfied by CAPS, a soluble component required for norepinephrine (NE) release. Moreover, the CAPS-independent glutamate release from synaptic vesicles (SVs) was 200-fold less sensitive to Ca2+ than that required for dense core vesicles (DCVs). The differential regulation of exocytosis by CAPS, Ca2+, and potential novel cytosolic factor(s) suggests that the docking and fusion machinery controlling DCVs has diverged from that regulating glutamate-containing SVs.

Docking and fusion in neurosecretion.

A central issue in neurobiology concerns the mechanisms of membrane fusion that are essential for the rapid regulated delivery of neurotransmitters into the synapse. While many gene products are required for neurosecretion, recent research has focused on defining the core exocytotic machinery that is responsible for the docking of synaptic vesicles (SVs) and their fusion with the plasma membrane. N-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment protein (SNAP) and SNAP receptor (SNARE) proteins are essential for fusion but may not be critical for SV docking. Current evidence suggests that NSF functions during an ATP-dependent step after docking but before fusion. NSF may function to liberate SNARE proteins from complexes so that the proteins on apposed membranes align in a parallel fashion to bring SVs into close contact with the plasma membrane for fusion.