Secretion of appropriate amounts of insulin from pancreatic β-cells is crucial for glucose homeostasis. The β-cells release insulin in response to glucose and other nutrients, hormones and neurotransmitters, which trigger intracellular signaling cascades, that result in exocytotic fusion of insulin-containing vesicles with the plasma membrane. Increases of the intracellular concentration of calcium ions ([Ca2+]i) trigger exocytosis, whereas the messenger cyclic adenosine monophosphate (cAMP) amplifies various steps of the secretion process. The protein Epac2 mediates some effects of cAMP, but little is known about its regulation in β-cells. In this study, the spatio-temporal dynamics of Epac2 was investigated in insulin-secreting MIN6-cells and primary β-cells using various cell signaling biosensors and live-cell fluorescence microscopy approaches. Increases in the cAMP concentration triggered translocation of Epac2 from the cytoplasm to the plasma membrane. Oscillations of cAMP induced by glucose and the insulin-releasing hormone GLP-1 were associated with cyclic translocation of Epac2. Analyses of Epac2 mutants showed that the high-affinity cyclic nucleotide-binding domain and Ras-association domains were crucial for the translocation, whereas neither the DEP domain, nor the low-affinity cAMP-binding domain were required for membrane binding. However, the latter domain targeted Epac2 to insulin granules at the plasma membrane, which promoted their priming for exocytosis. Depolarization-induced elevations of [Ca2+]i also stimulated Epac2 translocation, but the effects were complex and in the presence of high cAMP concentrations, [Ca2+]i increases often reduced membrane binding. The stimulatory effect of Ca2+ was mediated by increased Ras activity, while the inhibitory effect reflected reduced concentrations of the membrane phospholipid PtdIns(4,5)P2. Anti-diabetic drugs of the sulfonylurea class, suggested to directly activate Epac2, induced translocation indirectly by depolarizing β-cells to increase [Ca2+]i. Epac2 is an activator of Rap GTPases, and its translocation increased Rap activity at the plasma membrane. It is concluded that the subcellular localization of Epac2 is controlled by a complex interplay between cAMP, Ca2+ and PtdIns(4,5)P2 and that the protein controls insulin release by binding to the exocytosis machinery. These results provide new insights into the regulation of β-cell function and may facilitate the development of new anti-diabetic drugs that amplify insulin secretion.

Tomosyn is a protein belonging to the Lgl family and conserved across the animal and plant kingdom. Tomosyn is composed of N-terminal domain containing WD40 motif and Cterminal domain, where the R-SNARE motif is located at the end of the C-terminal domain. This motif is classified as homologous to R-SNARE motif of synaptobrevine, which is a protein located on the surface of the vesicles and participating in the formation of SNARE complex and subsequent fusion of the vesicles with the plasma membrane. Thus the role of this tomosyn is mainly the regulation of exocytosis. Apart from the animal tomosyn, its yeast homolog Sro7/Sro77 is also examined, however during the evolution it has lost its R-SNARE motif and therefore the plant tomosyns remain unexplored. The aim of this diploma thesis was to characterize both of the plant tomosyns: AtTYN1 and AtTYN2 in Arabidopsis thaliana plant. The experimental thesis included the bioinformatic analysis, the DNA construct creation, a search for interactors by yeast two-hybrid system and monitoring the localization using the confocal microscope. The bioinformatic analysis results, including the creation of phylogenetic tree, not only revealed the conservation of tomosyns across the different classes, but also the division of both tomosyns into different clusters. There was...

胞吐作用定義為囊運小泡將物質運輸到質膜或細胞外空間的轉運過程。其中關鍵的一步發生在同源SNARE 蛋白介導的膜融合之前，即將胞吐囊泡瞄向並靶定在適當的質膜位點。先前在酵母和哺乳動物中的研究表明，一個名為exocyst 的蛋白質複合體在這一關鍵步驟發揮作用。exocyst 蛋白複合體最早在酵母發現，之後這個複合體也在哺乳動物中被發現。這個複合體包含8 個不同的亞基：SEC3，SEC5，SEC6，SEC8，Sec10，Sec15，Exo70 和Exo84。Exocyst 同源蛋白也已在植物中發現。相比酵母和動物，exocyst 在植物體內的功能還鮮為人知，尤其是在胞吐運輸過程中的作用 。通過瞬時表達熒光蛋白標記的擬南芥同源的exocyst 蛋白Exo70：AtExo70E2 以及使用這個同源物的特異抗體，我們在擬南芥和煙草BY－2 懸浮培養細胞中發現了一種新的細胞器，並命名為exocyst 陽性細胞器（EXPO）。這種細胞器分別位於質膜或是細胞質中。由於它未能與任何傳統的細胞器標記物重合，或是被布雷菲爾德菌素A，渥曼青黴素和刀豆素A 影響，以及不能與FM4-64 重合，我們判斷這些細胞器不定位於常規的分泌或胞吞途徑中。對於快速冷凍樣本進行的免疫電子顯微鏡顯示EXPO 的雙膜性質，同時也發現了陽性標記的位於質膜外的單膜囊泡的存在。與此同時，在野生型細胞中也發現了同樣結構的細胞器。EXPO和自噬體非常相似， 都有兩層膜。然而，EXPO 不能被的自噬標記物（AtAtg8e）所標記。同時，在營養脅迫條件下，EXPO 的數量也沒有增加。因此，EXPO 代表著植物所特有的一種非常規分泌形式。 / 此外，通過在擬南芥原生質體內進行瞬時表達，我進一步證實在AtExo70E2 存在的條件下， 一些exocyst 成員可以被招募到EXPO 。AtExo70E2 的旁系同源物AtExo70A1 是在這方面物法取代AtExo70E2 的作用。蛋白蛋白相互作用分析證實了AtSec10 或AtSec6 與AtExo70E2 之間的相互作用。 AtExo70E2，而不是它在酵母或是動物中的同源蛋白，可以誘導EXPO 在動物細胞中的形成。反之，人或是酵母Exo70 同源蛋白都不能誘導EXPO 在植物細胞中的形成。這些結果表明AtExo70E2 在EXPO 形成過程中的特定的以及至關重要的作用。 / Exocytosis defines the process in which vesicles transport substances to the plasma membrane (PM)/extracellular space of the cell. One key step of exocytosis is the targeting and docking of the exocytic vesicles to the appropriate PM sites, which is prior to membrane fusion mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE). Previously studies have demonstrated that a protein complex called exocyst complex is involved in this key step in yeast and mammals. The exocyst complex, containing eight different subunits: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84, was first identified in yeast and subsequently in mammals. Exocyst homologs have also been found in plants. In comparison to its yeast and animal counterparts, little is known about the function of exocyst proteins in plants especially in the process of exocytosis. By using both antibodies specific for one of the orthlogs of exocyst protein: AtExo70E2 as well as transiently-expressed fluorescently-tagged constructs for this exocyst subunit, a novel organelle termed exocyst-positive organelle (EXPO) was identified in suspension cultured Arabidopsis and tobacco BY-2 cells. These organelles were located to both the plasma membrane and cytosol. Based on their failure to overlap with any conventional organelle markers or response to brefeldin A (BFA), wortmannin or concanamycin A (ConcA) treatments, as well as their inability to take up the endocytic dye FM4-64, these organelles were thus not lie on the conventional secretory or endocytic pathways of plant cells. Immunogold electron microscopy (EM) of cryofixed samples revealed the double membrane nature of EXPO and also produced labeling of large single-membrane bound vesicles outside of the PM. These structures were also identified in wild type cells. EXPO and autophagosomes are similar in that both have two boundary membranes. However, EXPO did not label positively with YFP-AtAtg8e, a standard marker for autophagosomes, nor did the number of EXPO increase when the cells were subjected to nutrient stress. Therefore, EXPO represents a form of unconventional secretion unique to plants. / Further studies demonstrated that a number of exocyst subunits can be positively recruited to EXPO in the presence of AtExo70E2 by performing transient expression in Arabidopsis protoplasts. The paralog AtExo70A1 is unable to substitute for AtExo70E2 in this regard. Protein-protein interaction assay have confirmed the interaction between AtExo70E2 and AtSec6 and AtSec10. AtExo70E2, but not its yeast counterpart, is also capable of inducing EXPO formation in animal cells. Inversely, neither human nor yeast Exo70 homologs are able to cause the formation of EXPO in Arabidopsis protoplasts. These results point to a specific and crucial role for AtExo70E2 in EXPO formation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ding, Yu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 101-118). / Abstracts also in Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiv / Chapter CHAPTER 1 --- p.1 / General Introduction --- p.1 / Chapter 1.1 --- The secretory system in eukaryotic cells --- p.2 / Chapter 1.2 --- Exocytosis and exocyst complex --- p.6 / Chapter 1.3 --- Project Objectives --- p.7 / Chapter CHAPTER 2 --- p.9 / Exocyst-positive organelles (EXPOs) mediate unconventional protein secretion in plant cells --- p.9 / Chapter 2.1 --- Abstract --- p.10 / Chapter 2.2 --- Introduction --- p.11 / Chapter 2.3 --- Materials and Methods --- p.12 / Chapter 2.4 --- Results --- p.20 / Chapter 2.4.1 --- Expression pattern of different AtExo70 paralogs with fluorescent tag in Arabidopsis protoplasts --- p.20 / Chapter 2.4.2 --- The organelles labeled by AtExo70E2 are distinct from well known endomembrane markers --- p.23 / Chapter 2.4.3 --- The AtExo70E2 positive organelles do not lie on the secretory or endocytic pathways --- p.27 / Chapter 2.4.4 --- Arabidopsis Exo70E2-specific antibodies confirm identity of AtExo70E2-positive organelles --- p.31 / Chapter 2.4.5 --- AtExo70E2 positive organelles are true and novel double membrane organelles --- p.33 / Chapter 2.4.6 --- EXPO are not autophagosomes but sequester cytosolic proteins to release them into the apoplast --- p.41 / Chapter 2.5 --- Discussion --- p.53 / Chapter 2.5.1 --- EXPO: novel organelles labeled by exocyst --- p.53 / Chapter 2.5.2 --- EXPO and autophagosome: same or not? --- p.55 / Chapter 2.5.3 --- EXPO: the evidence of unconventional secretion in plant cells --- p.56 / Chapter 2.6 --- Perspectives --- p.56 / Chapter CHATER 3 --- p.58 / AtExo70E2 is essential for exocyst subunit recruitment and for EXPO formation in both plants and animals --- p.58 / Chapter 3.1 --- Abstract --- p.59 / Chapter 3.2 --- Introduction --- p.60 / Chapter 3.3 --- Materials and Methods --- p.62 / Chapter 3.4 --- Results --- p.70 / Chapter 3.4.1 --- AtExo70E2 is required for the membrane recruitment of a number of exocyst subunits --- p.70 / Chapter 3.4.2 --- AtExo70E2 is required for the recruitment of some other, but not all, AtExo70 subunits --- p.74 / Chapter 3.4.3 --- AtExo70A1 is unable to recruit other exocyst subunits --- p.74 / Chapter 3.4.4 --- FRET and BiFC confirm interactions between AtExo70E2 and other exocyst subunits --- p.80 / Chapter 3.4.5 --- Arabidopsis Exo70E2 can also induce EXPO formation in animal cells --- p.84 / Chapter 3.4.6 --- Neither human nor yeast Exo70 can induce EXPO in plant protoplasts --- p.84 / Chapter 3.4.7 --- EXPO induced by AtExo70-GFP expression in HEK cells do not colocalize with standard organelle markers --- p.87 / Chapter 3.4.8 --- Electron microscopy confirms the presence of EXPO-like, double membrane structures in HEK cells after expression of AtExo70E2-GFP --- p.87 / Chapter 3.5 --- Discussion --- p.91 / Chapter 3.5.1 --- Plant exocyst and the discovery of EXPO --- p.91 / Chapter 3.5.2 --- AtExo70E2 is a key player in exocyst recruitment onto EXPO --- p.93 / Chapter 3.5.3 --- AtExo70E2 expression as a signal for EXPO formation --- p.96 / Chapter 3.6 --- Perspectives --- p.100 / References: --- p.101 / Chapter List of publications derived from this Ph.D. thesis research --- p.119

Distinct membrane fusion events in the polarized pancreatic acinar cell involve highly specific interactions between distinct sets of SNARE proteins forming exocytotic complexes, whose assembly is modulated by distinct Munc18 proteins. The Munc18 isoform responsible for these exocytotic events in the acinar cell is unknown. Here, I postulate Munc18b to regulate apical exocytosis in the acinar cell. Current dogma for the pathogenesis of acute pancreatitis, including alcoholic pancreatitis, is mis-targeting and deregulated fusion of zymogen granules with lysosomal bodies in the acinar cells. This derangement results in premature activation of proteolytic zymogens and autophagic digestion of cellular contents. I have hypothesized an alternate mechanism, which is pathologic exocytosis occurring at the basolateral plasma membrane, and further propose Munc18c to mediate this process in alcoholic pancreatitis. The aims of this thesis are to demonstrate the roles of Munc18b and Munc18c in regulated apical exocytosis and pathologic basolateral exocytosis underling alcoholic pancreatitis, respectively. In Chapter Three, using both real-time and static imaging techniques and biochemical tools, I demonstrated that Munc18c is dissociated from the acinar basal plasma membrane (BPM) when stimulated with postprandial CCK8 preceding preincubation of acini with postprandial 20-50mM ethanol concentrations. This activated Syntaxin (Syn)-4 and SNAP-23 on the BPM to complex with VAMP proteins on the granule to form the exocytotic SNARE complex that triggered basolateral exocytosis. This molecular mechanism of pathologic basolateral exocytosis was recapitulated in an Ethanol-diet rat model of pancreatitis. In Chapter Four, I determined Munc18b to be in the apical pole of the acinar cell to appropriately bind cognate Syn-2 and Syn-3 in the apical PM and ZGs. Here, I examined the structure-function of Munc18b on amylase secretion by employing Munc18b mutants with distinct affinities to Syn-2 and Syn-3. In Chapter Five, I discovered a novel EF-hand Ca2+-binding protein called Cab45b, which binds Munc18b to regulate its membrane targeting and interactions with Syntaxins in the acinar cell in a manner that influenced Ca2+-induced amylase release. Taken together, these studies clarify our understanding of the role of Munc18 proteins involved in regulated and pathologic membrane fusion events underlying physiologic digestive enzyme secretion and clinical alcoholic pancreatitis.

Distinct membrane fusion events in the polarized pancreatic acinar cell involve highly specific interactions between distinct sets of SNARE proteins forming exocytotic complexes, whose assembly is modulated by distinct Munc18 proteins. The Munc18 isoform responsible for these exocytotic events in the acinar cell is unknown. Here, I postulate Munc18b to regulate apical exocytosis in the acinar cell. Current dogma for the pathogenesis of acute pancreatitis, including alcoholic pancreatitis, is mis-targeting and deregulated fusion of zymogen granules with lysosomal bodies in the acinar cells. This derangement results in premature activation of proteolytic zymogens and autophagic digestion of cellular contents. I have hypothesized an alternate mechanism, which is pathologic exocytosis occurring at the basolateral plasma membrane, and further propose Munc18c to mediate this process in alcoholic pancreatitis. The aims of this thesis are to demonstrate the roles of Munc18b and Munc18c in regulated apical exocytosis and pathologic basolateral exocytosis underling alcoholic pancreatitis, respectively. In Chapter Three, using both real-time and static imaging techniques and biochemical tools, I demonstrated that Munc18c is dissociated from the acinar basal plasma membrane (BPM) when stimulated with postprandial CCK8 preceding preincubation of acini with postprandial 20-50mM ethanol concentrations. This activated Syntaxin (Syn)-4 and SNAP-23 on the BPM to complex with VAMP proteins on the granule to form the exocytotic SNARE complex that triggered basolateral exocytosis. This molecular mechanism of pathologic basolateral exocytosis was recapitulated in an Ethanol-diet rat model of pancreatitis. In Chapter Four, I determined Munc18b to be in the apical pole of the acinar cell to appropriately bind cognate Syn-2 and Syn-3 in the apical PM and ZGs. Here, I examined the structure-function of Munc18b on amylase secretion by employing Munc18b mutants with distinct affinities to Syn-2 and Syn-3. In Chapter Five, I discovered a novel EF-hand Ca2+-binding protein called Cab45b, which binds Munc18b to regulate its membrane targeting and interactions with Syntaxins in the acinar cell in a manner that influenced Ca2+-induced amylase release. Taken together, these studies clarify our understanding of the role of Munc18 proteins involved in regulated and pathologic membrane fusion events underlying physiologic digestive enzyme secretion and clinical alcoholic pancreatitis.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 149-157). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 149-157). Also available on the Internet.

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Septins are an evolutionarily conserved family of proteins that have been implicated in a multitude of cellular processes. Septin 5 is mainly expressed in the nervous system and it has been linked to regulated secretion through its binding to the SNARE protein syntaxin 1. However, the exact mechanism of septin 5 function in localized exocytosis remains unknown. Over-expression of septin 5 is known to lead to lower levels of secretion in HIT-T15 cells. Interestingly, in the current study, the knock-down of septin 5 also results in reduced levels of regulated secretion in PC12 cells, suggesting a more complex role of septin 5 that includes both negative and positive effects on exocytosis. Septin 5 knock-down data point to a possibility of septin 5 facilitating formation of a tether between the vesicles and their site of secretion.