Rab GTPases associated with insulin containing secretory granules are key in targeting, docking and assembly of molecular complexes governing pancreatic β-cell exocytosis. Four Rab3 isoforms along with Rab27A are associated with insulin granules, yet elucidation of the distinct roles of these Rab families on exocytosis remains unclear. To define specific actions of these Rab families we employ Rab3GAP and/or EPI64A GTPase activating protein overexpression in β-cells from wild-type or Ashen mice to selectively transit the entire Rab3 family or Rab27A to a GDP-bound state. Ashen mice carry a spontaneous mutation that eliminates Rab27A expression. Using membrane capacitance measurements we find that GTP/GDP nucleotide cycling of Rab27A is essential for generation of the functionally defined immediately releasable pool and central to regulating the size of the readily releasable pool. By comparison, nucleotide cycling of Rab3 GTPases, but not of Rab27A, is essential for a kinetically rapid filling of the readily releasable pool with secretory granules. Aside from these distinct functions, Rab3 and Rab27A GTPases demonstrate considerable functional overlap in building the readily releasable granule pool. Hence, while Rab3 and Rab27A cooperate to generate release-ready secretory granules in β-cells, they also direct unique kinetic and functional properties of the exocytotic pathway.

FIZ1 (Flt-3 Interacting Zinc-finger) interacts and co-purifies with the rod-specific transcription factor NRL (Neural Retina Leucine zipper). We hypothesize that FIZ1 is part of an interface between cell-specific factors, like NRL, and more ubiquitous regulatory networks that vary the absolute expression levels of some rod-specific genes (i.e. Rhodopsin). As part of an ongoing exploration of FIZ1's role in neural retina, in vivo, we have taken the first look at FIZ1 expression in the developing mouse retina during the retinal maturation period. Using the normal C57/B6 mouse as a model, multiple approaches were used including: immunoblotting, immunohistochemistry, and quantitative real-time PCR. Functional implications of FIZ1/NRL interaction, on NRL-and CRX-mediated activation of the Rhodopsin (Rho) and cGMPphosphodiesterase β-subunit gene (PDE6B) promoters, were examined by co-transfection assays. Immunoblot analysis revealed that FIZ1 protein levels were lowest in immature mouse neural retina (P0). FIZ1 concentration increased at least ten-fold as the neural retina matured to the adult state (P21 and later). Immunohistochemical comparison of immature post-natal and mature adult retina revealed increasing FIZ1 protein in photoreceptors, the inner plexiform layer, and the ganglion cell layer. Total retinal Fiz1 mRNA content increased as the neural retina matured. The expected increase in Rho mRNA level was also monitored as a genetic marker of photoreceptor maturation. In transient co-transfection assays of CV1 cells, FIZ1 synergized with NRL to activate transcription from the Rho and PDE6B gene promoters with some differences. In the case of the Rho promoter, FIZ1 synergized when both NRL and CRX were present. With the PDE6B promoter, FIZ1 synergized with NRL alone, and the inclusion of CRX decreased this synergy.Conclusions-These findings support previous evidence that FIZ1 is present in rodphotoreceptors. (Co-immunoprecipitation from nuclear-protein extracts with rod-specific NRL) FIZ1 expression increases in the neural retina during the retinal maturation period. Additionally, in vitro experiments demonstrate that FIZ1 has the potential to significantly increase the NRLmediated activation of photoreceptor-specific promoters. While CRX is not a strong activator of the PDE6B promoter, alone or with NRL, CRX decreased the synergy of NRL with FIZ1.

We have developed an improved procedure for isolating and transfecting a chromaffin cellenriched population of primary cells from adult mouse adrenal glands. Significantly, the parameters of a novel electroporation transfection technique were optimized to achieve an average transfection efficiency of 45 % on the small number of cells derived from the mouse glands. Such transfection efficiency was previously unachievable with the electroporation protocols conventionally used with bovine chromaffin cells, even with use of large cell numbers. Our small scale technique now makes feasible the use of genetically homogenous inbred mouse models for investigations on the exocytotic pathway without the time, expense, and cellular changes associated with viral approaches. High fidelity co-expression of multiple plasmids in individual cells is a further advantage of the procedure. To assess whether the biophysical characteristics of mouse adrenal chromaffin cells were altered by this process, we examined structural integrity using immunocytochemistry and functional response to stimuli using calcium imaging, amperometry, and whole-cell capacitance and current clamp recordings. We conclude these parameters are minimally affected. Finally, we demonstrate that high transfection efficiency makes possible the use of primary mouse adrenal chromaffin cells, rather than a cell line, in human growth hormone secretion assays for high throughput evaluation of secretion.

protein Von Willebrand factor (VWF), however, under specific conditions WPBs also contain a cocktail of small pro-inflammatory cytokines. WPB exocytosis is driven by an increase in intracellular free calcium ion concentration ([Ca 2þ ] i ); the majority of WPB fusion events result in complete discharge of cargo components, however, in a small fraction of cases cytokines are selectively released indicating that the fusion pore may act as a molecular size filter. Carbon fibre amperometry has been used to characterize fusion pore behavior in a number of cell types, but to date this approach has not been applied to WPBs. Therefore, we used this technique in combination with simultaneous optical imaging of fluorescent WPB exocytosis and changes in [Ca 2þ ] i and can report, for the first time, the kinetic properties of WPB fusion pore formation and expansion in human cultured endothelial cells. A clear delay (mean ~50 ms) is seen between the onset of the current spike and the increase in intra-WPB EGFP fluorescence indicating that WPB alkalinsation is delayed by the strong proton buffering capacity of the WPB lumen (55 mM/pH unit). Analysis of current spike parameters reveal a mean 25-75% rise time, peak amplitude and decay time of 1.63 ms, 50 pA and 6.63 ms respectively. Approximately 50% of current spikes were preceded by a foot signal of mean duration 6.34 ms. Occasional low amplitude, prolonged current increases, reminiscent of stand alone foot signals, were observed in conjunction with morphological rounding of WPBs, possibly reflecting kiss-and-run or lingering kiss fusion events. Following characterization of the WPB fusion pore under control conditions the impact of changing cellular parameters, including cholesterol levels, was also assessed.

Three kinetic components of exocytosis have been described in retinal bipolar neurons. They are thought to reflect the fusion of a docked pool of ribbon-tethered synaptic vesicles termed the rapid pool, the releasable pool of ribbon-tethered vesicles, and a cytoplasmic reserve pool. In neurons, assembly of SNARE proteins facilitates exocytosis. We asked whether these pools could be distinguished on the basis of SNARE complex formation. Syntaxin3B is a t-SNARE in ribbon synapses. We generated a fluorescent peptide based on the syntaxin3B SNARE binding motif from goldfish. The peptide was dialyzed into isolated synaptic terminals of goldfish retinal bipolar cells via a whole-terminal recording electrode. A scrambled peptide served as control. Exocytosis was monitored with membrane capacitance measurements. Beginning one minute after breakin, a 1s stimulation, sufficient to deplete the releasable pool, was given every 60 seconds. The first exocytotic response was not significantly altered by the syn-taxin3B peptide. However, by the fourth pulse, the exocytotic response in terminals dialyzed with the syntaxin3B peptide was reduced by 89% relative to the first, whereas that with the control peptide was reduced by only 45% (p < 0.04). This effect was not due to a reduction in calcium influx. Next, we implemented a pulse train protocol that captures the three components of release. Control terminals showed both depletion and replenishment of the pools. Terminals dialyzed with the syntaxin3B peptide showed: 1) immediate loss of the exocytotic component attributed to the reserve pool 2) decreased refilling of the rapid and releasable pools. The results demonstrate that reserve pool vesicles are unlike those in the rapid and releasable pools. It is likely that pool refilling is inhibited by the syntaxin3B peptide because reserve vesicles establish new SNARE complexes when they join a fusion-competent vesicle pool.

Chronic manipulation of synaptic activity drives bidirectional alterations in synaptic efficacy. Compensatory tuning of presynaptic neurotransmitter release maintains levels of synaptic activity within an optimal range for efficient information processing. This homeostatic form of plasticity is apparent in cultured hippocampal neurons and appears to involve the ubiquitinproteasome system (UPS). Examination of presynaptic mechanisms underlying homeostatic plasticity has identified multiple positive regulators of exocytosis subject to proteasomal degradation. However, homeostatic compensation can involve up-or down-regulation of exocytosis and substantially less is known of the negative regulation of vesicle release. Tomosyn, a presynaptically active SNARE protein, is unique in that it is cytosolic and serves to potently inhibit vesicle release at central synapses. Proteomic analysis of tomosyn revealed an interaction with the E3 ubiquitin-ligase HRD1. Here we aim to test the hypothesis that the UPS serves as an activity-dependent mechanism to precisely regulate tomosyn proteostasis, and in turn manipulates exocytosis. Consistent with this hypothesis, endogenous tomosyn levels in cultured rat hippocampal neurons (18-25 DIV) increase following application of the proteasome inhibitors MG132 (50mM, 4h) and lactacystin (10mM, 4h). Moreover, our data indicate that the tomosyn-HRD1 interaction is activitydependent, as chronic AMPAR blockade (CNQX, 24h) results in an increase in HRD1 co-immunoprecipitation with tomosyn and consequentially a decrease in overall tomosyn protein level. To assess tomosyn's role homeostatic plasticity we use an optical reporter of exocytosis, vGlut1-pHluorin. Results show that chronic activity blockade via CNQX (40mM, 24h) enhances vesicle release in response to a stimulus train (10Hz, 10s) as compared to nonstimulated controls. This effect is dependent upon tomosyn, as shRNAmediated tomosyn knock-down mitigates the compensatory enhancement of exocytosis. These data strongly implicate tomosyn as a key presynaptic molecular target which is subject to regulation by the UPS and facilitates activitydependent homeostatic plasticity.