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One contentious issue regarding pancreatic acinar exocytosis concerns which SNAP25 isoform (SNAP23/29/47) mediates the various fusion events in this cell type. Based on dominant-negative over-expression studies, SNAP23 was hypothesized to be the putative isoform mediating apical exocytosis, basolateral exocytosis, and ZG-ZG fusion. Unfortunately, using a SNAP23 KD mouse model, 80% SNAP23 KD was insufficient to manifest any secretion phenotype. A novel syncollin-pHluorin exocytosis imaging technique initially meant to assess which fusion events are perturbed by SNAP23 KD was successfully developed and displayed improvements over previous imaging techniques. The syncollin-pHluorin imaging enabled visualization of apical and basolateral exocytosis as well as sequential ZG-ZG fusions. Combined with spinning disk microscopy, this assay allows 3D live exocytosis imaging with high temporal and spatial resolution. This novel imaging assay will be useful in visualizing apical, basolateral, sequential, and lateral fusion events for future acinar studies.

Our study aims to investigate the role of Syntaxin-3 in glucose stimulated insulin secretion (GSIS) and how it regulates the recruitment to plasma membrane and/or exocytotic fusion of insulin granules. We examined endogenous Syn-3 function by down-regulating its expression using siRNA/lenti-shRNA, which impaired GSIS. Although Syn-3 depleted cells showed no change in the number and fusion of docked granules, there was a reduction in newcomer granules and their subsequent exocytotic fusion. We then examined the effects of overexpressing Syn-3-WT, which enhanced biphasic GSIS. Since open conformation (OF) Syn-1A was reported to enhance exocytosis by promoting SNARE complex formation, we constructed OF Syn-3. Exogenous OF Syn-3 had no effect on secretion as it is unable to be trafficked to insulin granules. Taken together, we conclude that Syn-3 facilitates mobilization of newcomer insulin granules to the plasma membrane, to contribute to both first and second phase of GSIS in pancreatic beta cells.

One contentious issue regarding pancreatic acinar exocytosis concerns which SNAP25 isoform (SNAP23/29/47) mediates the various fusion events in this cell type. Based on dominant-negative over-expression studies, SNAP23 was hypothesized to be the putative isoform mediating apical exocytosis, basolateral exocytosis, and ZG-ZG fusion. Unfortunately, using a SNAP23 KD mouse model, 80% SNAP23 KD was insufficient to manifest any secretion phenotype. A novel syncollin-pHluorin exocytosis imaging technique initially meant to assess which fusion events are perturbed by SNAP23 KD was successfully developed and displayed improvements over previous imaging techniques. The syncollin-pHluorin imaging enabled visualization of apical and basolateral exocytosis as well as sequential ZG-ZG fusions. Combined with spinning disk microscopy, this assay allows 3D live exocytosis imaging with high temporal and spatial resolution. This novel imaging assay will be useful in visualizing apical, basolateral, sequential, and lateral fusion events for future acinar studies.

Our study aims to investigate the role of Syntaxin-3 in glucose stimulated insulin secretion (GSIS) and how it regulates the recruitment to plasma membrane and/or exocytotic fusion of insulin granules. We examined endogenous Syn-3 function by down-regulating its expression using siRNA/lenti-shRNA, which impaired GSIS. Although Syn-3 depleted cells showed no change in the number and fusion of docked granules, there was a reduction in newcomer granules and their subsequent exocytotic fusion. We then examined the effects of overexpressing Syn-3-WT, which enhanced biphasic GSIS. Since open conformation (OF) Syn-1A was reported to enhance exocytosis by promoting SNARE complex formation, we constructed OF Syn-3. Exogenous OF Syn-3 had no effect on secretion as it is unable to be trafficked to insulin granules. Taken together, we conclude that Syn-3 facilitates mobilization of newcomer insulin granules to the plasma membrane, to contribute to both first and second phase of GSIS in pancreatic beta cells.

Thesis (Ph.D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on March 24, 2009) Vita. Includes bibliographical references.

Exocyst positive organelle (EXPO) is a newly discovered double membrane organelle involved in exocytosis and likely other vesicle trafficking processes. EXPO is likely generated from the ER, fused with plasma membrane and released a single membrane vesicle to cell exterior. The Arabidopsis protein Exo70E2 was found to be associated with EXPO and therefore is considered as a marker of EXPO and might play a role in EXPO-mediated vesicle trafficking. Understanding the biological function of AtExo70E2 (abbreviated as E2 in this thesis) will be very helpful in unraveling the function of EXPO. The aim of this work was to use various molecular, genetic and physiological approaches to determine the possible role of Arabidopsis Exo70E2 in biological pathways. By using the Exo70E2pro:GUS line, the expression pattern of Exo70E2 was determined. Exo70E2 was expressed mainly in roots, especially in root tips and epidermal cells in the division and elongation zones of roots. Its expression level was induced when the seedlings were treated with Flg22, a peptide derived from bacterial flagillin protein that induces the plant defense response. The tissue subcellular localization of Exo70E2 was also studied using the 35S:Exo70E2-eYFP and Exo70E2pro:Exo70E2-GFP reporter lines. The GFP fusion protein was found primarily in the epidermal cells of roots even in the 35S:Exo70E2-eYFP lines. For phenotypic analysis resulting from mutations of the Exo70E2 gene, I obtained three T-DNA insertion mutant lines and generated its overexpression lines. The two mutant alleles, e2-2 and e2-3 are in the Columbia ecotype background and further characterized. e2-2 which has a T-DNA insertion in an exon is likely a knock out line as Exo70E2 gene transcript could not be detected. e2-3, which carries a T-DNA insertion in its promoter region, was found to accumulate a higher level of the transcript, suggesting that the insertion causes its enhanced expression of Exo70E2. There was no obvious difference between wild type and e2-2 in their phenotypes under different conditions tested in this study. However, e2-3 had a retarded growth phenotype when grown in soil or on MS medium. The seedlings of e2-3 on MS medium also had a yellowish color although such a phenotype was not obvious when they were grown in soil. When supplementing the MS medium with sucrose, glucose or mannitol, the growth of e2-3 was more reduced compared to wild type under these conditions. However, on the medium with NaCl or under phosphate deficiency, the yellowish phenotype of e2-3 was rescued and the mutant seedlings became relatively healthier than the seedlings under the regular MS medium. A proteomics approach was taken to compare protein secreted from the seedlings of wild type and the mutants. Proteins secreted by seedlings to the liquid medium were collected, concentrated and subjected to MS analysis. Comparison of the profiles of secreted proteins between the wild type and the mutants leaded to identification of candidate proteins whose secretion might be affected by the mutation. My study indicates that Exo70E2 and EXPO are involved in transporting proteins (likely also metabolites) to the exterior of cells and the rhizosphere and might play an important role in stress responses.

Dictyostelium discoideum is a simple model organism that can be used to study endocytic pathways such as phagocytosis and macropinocytosis because of its homology to cells of the mammalian innate immune system, namely macrophages and neutrophils. Consequently, Dictyostelium can also be used to study the process of exocytosis. In our laboratory, we generated Dictyostelium cells lacking superoxide dismutase SodC. Our data suggest that cells that lack SodC are defective in macropinocytosis and exocytosis when compared to wild type cells. In this study I describe a regulatory mechanism of macropinocytosis by SodC via regulation of RasG, which in turn controls PI3K activation and thus macropinocytosis. Our results show that proper metabolism of superoxide is critical for efficient particle uptake, for the proper trafficking of internalized particles, and a timely exocytosis of fluid uptake in Dictyostelium cells.

Although exocytosis is the major mechanism by which cells secrete products into their environment, little is known about the mechanism of this fundamental process. Previous studies on the regulation of luteinizing hormone (LH) exocytosis have used intact cells exclusively. It is not possible, however, to determine the precise requirements for exocytosis in intact cells since the cytosol is not directly accessible. Permeabilization of the plasma membrane allows experimental manipulation of the intracellular milieu while preserving the exocytic apparatus. The diameter of the atoxin pores (2-3 nm) allowed the exchange of small molecules such as ATP while larger cytosolic proteins such as lactate dehydrogenase were retained. Because of the slow exchange of small molecules through a-toxin pores a protocol was developed which combines prolonged pre-equilibration of the permeabilized cells at 0°C before stimulation with strong Ca²⁺ buffering. Under these conditions an increase in the [Ca²⁺]free stimulated a 15-20 fold increase in LH exocytosis (EC₅₀ pCa 5.5). After 12-15 minutes the rate of exocytosis declined and the cells became refractory to Ca²⁺. At resting [Ca²⁺]free (pea 7), cAMP stimulated a rapid, 2 - 3 fold, increase in LH exocytosis. cAMP caused a modest enhancement of Ca²⁺-stimulated LH exocytosis by causing a left shift in the EC₅₀ for Ca²⁺ from pCa 5.6 to pCa 5.9. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) synergistically enhanced cAMP-stimulated LH exocytosis, an effect which was further augmented by increasing the [Ca²⁺]free· Gonadotrophin-releasing hormone (GnRH) was found to stimulate cAMP production in intact pituitary cells. Since previous studies have shown that GnRH activates PKC and stimulates a rise in cytosolic [Ca²⁺]free, these results suggest that a synergistic interaction of the cAMP, PKC and Ca²⁺ second messenger systems is of importance in the mechanism of GnRH-stimulated LH exocytosis. When permeabilized cells were equilibrated for prolonged periods in the absence of MgATP, Ca²⁺-stimulated LH exocytosis declined. The time course of the decline closely followed the leakage of intracellular ¹⁴C-ATP. Addition of MgATP rapidly restored full Ca²⁺-stimulated LH exocytosis. Ca²⁺-, cAMP-, and PMA-stimulated LH exocytosis were all dependent on millimolar MgATP concentrations (EC₅₀ 1 .5-3 mM). It has been postulated that PKC is a mediator of Ca²⁺- stimulated exocytosis. Several findings in the present study argue against this hypothesis. Firstly, PMA and Ca²⁺ had additive effects on LH exocytosis at all [Ca²⁺]free· Secondly, PMA was able to stimulate further LH release from cells made refractory to high [Ca²⁺]free· Thirdly, the PKC inhibitor staurosporine did not inhibit Ca²⁺-stimulated LH exocytosis under conditions in which it inhibited PMAstimulated exocytosis. Fourthly, in cells desensitized to PMA by prolonged exposure to a high PMA concentrations, Ca²⁺-stimulated LH exocytosis was not inhibited. And finally, Ba²⁺+ was able to stimulate LH exocytosis to a maximal extent similar to Ca²⁺ despite the fact that Ba²⁺+ is an extremely poor activator of PKC. Since Ba²⁺+ is also a poor activator of calmodulin, this latter result implies that calmodulin does not mediate the effect of Ca²⁺. In agreement with this, the calmodulin inhibitor calmidazolium did not inhibit Ca²⁺-stimulated LH exocytosis. Since GTP-binding proteins have been implicated in regulated exocytosis in other cell systems, the effects of guanine nucleotides on LH exocytosis were examined. At resting cytosolic [Ca²⁺]free (pea 7), the GTP analogues GTPyS and GMPPNP stimulated LH exocytosis with similar potencies (EC₅₀ 20-50 μM). Additional experiments indicated that the effects of these GTP analogues could not be explained by activation of either PKC alone or cAMP-dependent protein kinase alone. In the presence of both PMA and cAMP, GMPPNP did not stimulate a further increase in the rate of LH exocytosis, suggesting that the stimulatory actions of guanine nucleotides may be mediated by the combined activation of PKC and generation of cAMP, as a result of activation of signal-transducing G proteins. In contrast, pretreatment of cells with GTPyS at low [Ca²⁺]free markedly inhibited subsequent responses to Ca²⁺, cAMP, PMA, and cAMP plus PMA. This inhibitory effect required lower GTPyS concentrations than the stimulatory effect (IC₅₀ 1-10 μM), and was not observed with GMPPNP. These findings indicate the involvement of a distinct guanine nucleotide-binding protein in exocytosis at a site distal to second messenger generation.

Cellular communication requires the transport of chemical messengers between intracellular compartments and from cell to cell. The regulated exocytosis of a secretory vesicle at the plasma membrane involves the merger of two bilayers, with markedly different lipid composition, within a millisecond time scale. The spatial and temporal control of the protein and lipid complement at these fusion sites is essential. A highly conserved family of proteins are known to drive this fusion event; SNAP-25 and syntaxin-1 (t-SNAREs) associate at the plasma membrane in a 1:1 stoichiometry to provide a binding site for the vesicle-membrane protein synaptobrevin (v-SNARE). The formation of this complex and subsequent fusion requires accessory proteins for efficient calcium-triggered exocytosis; which of these proteins facilitate the initial attachment of vesicle to the plasma membrane prior to fusion is still under debate. Specific sites for vesicle fusion have been proposed and the organisation of lipids and proteins at these fusion sites has been extensively investigated with limited spatial and temporal resolution; however the presence of raft-forming lipids at these sites as well as the arrangement of SNARE proteins at the molecular level is still under contention. The data presented within this thesis aims to elucidate the protein and lipid environment at the fusion site using super-resolution microscopy and advanced vesicle tracking. Under diffraction-limited microscopy the t-SNAREs are visualised as 200 nm homogenous clusters; however I have used single molecule localisation microscopy to reveal a more complex heterogeneous molecular arrangement. Quantification of lipid order exclusively at the plasma membrane provided insight into the influence of cholesterol-induced lipid arrangement on SNAP-25 localisation. In addition the t-SNARE interaction was investigated using TCSPC-FLIM identifying two lipid-order-dependent conformations in distinct clusters at the plasma membrane. Extensive vesicle tracking at optimum sampling rates demonstrated the ‘sampling’ behaviour of LDCVs and allowed characterisation of vesicle fusion sites. In summary I find that vesicles exhibit preference for residence and probably fusion at regions of plasma membrane with a low t-SNARE density; these proteins appear to exert control over exocytosis by adopting alternative conformations that are under cholesterol-induced regulation.