Wang, Juan. / "July 2010." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 58-66). / Abstracts in English and Chinese. / Thesis/ Assessment Committee --- p.ii / Statement --- p.iii / Abstract --- p.iv / 摘要 --- p.vi / Acknowledgements --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter CHAPTER1 --- General Introduction --- p.1 / Chapter 1.1 --- The Plant Secretory Pathway --- p.1 / Chapter 1.2 --- Exocytosis --- p.4 / Chapter 1.3 --- Exocyst complex --- p.5 / Chapter 1.3.1 --- Exocyst complex in plants --- p.6 / Chapter 1.4 --- Project Objectives --- p.6 / Chapter CHAPTER 2 --- Materials and Methods --- p.8 / Chapter 2.1 --- Plasmid construction --- p.8 / Chapter 2.2 --- Expression and Purification of Recombinant Protein --- p.11 / Chapter 2.3 --- Transient Expression of Arabidopsis Protoplasts --- p.13 / Chapter 2.4 --- Transformation of Tobacco BY-2 and Arabidopsis Cells --- p.14 / Chapter 2.5 --- Antibodies --- p.16 / Chapter 2.6 --- Immunofluorescent Studies --- p.19 / Chapter 2.7 --- FM4-64 Uptake Study --- p.20 / Chapter 2.8 --- Drug Treatments --- p.21 / Chapter 2.9 --- Electron Microscope of Resin-Embedded Cells --- p.21 / Chapter 2.10 --- Sucrose Starvation --- p.23 / Chapter 2.11 --- Chemical Screening --- p.23 / Chapter CHAPTER 3 --- Results --- p.25 / Chapter 3.1 --- Expression of (X)FP-tagged AtExo70E2 in Arabidopsis protoplasts --- p.25 / Chapter 3.2 --- AtExo70E2 labels the PM and organelles which do not lie on the secretory or endocytic pathways --- p.27 / Chapter 3.3 --- "Transgenic Arabidopsis and tobacco BY-2 cell lines expressing AtExo70E2, and AtExo70E2-specific antibodies help in identifying Exocyst-positive organelles (EXPO)" --- p.31 / Chapter 3.4 --- EXPO do not label with FM4-64 --- p.36 / Chapter 3.5 --- EXPO are exocytic double membraned organelles --- p.39 / Chapter 3.6 --- EXPO are true organelles in the cytoplasm that are not induced by sucrose starvation --- p.44 / Chapter 3.7 --- EXPO can be specifically affected by one chemical compound --- p.48 / Chapter CHAPTER 4 --- Discussion and Future Perspectives --- p.51 / Chapter 4.1 --- Discussion --- p.51 / Chapter 4.1.1 --- Exocyst and EXPO --- p.51 / Chapter 4.1.2 --- Are EXPO related to Autophagosomes? --- p.54 / Chapter 4.1.3 --- EXPO and exosomes: agents of unconventional secretion --- p.55 / Chapter 4.2 --- Future perspectives --- p.56 / References --- p.58

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