Both structural and immunogold EM studies have also been carried to identify the storage PVCs in developing tobacco seeds and mungbean cotyledons. Biochemically, storage PVCs in both developing tobacco seeds and mungbean cotyledons were labeled by VSRAt-1, S2 (globulin-like proteins), BiP and DIP antibodies. Structurally, storage PVCs in developing tobacco seeds, sized about 200 nm diameter, contain wavy limiting membrane with electron-dense core and periphery translucent outer layer with internal vesicles. In contrast, storage PVCs in mungbean cotyledon, sized about 400 nm diameter, contain electron-dense and translucent area located adjacent to each other. / Further drug treatments studies demonstrated that the lytic PVCs/MVBs in tobacco BY-2 cells were distinct from the storage PVCs in seed cells. BFA and wortmannin treatments respectively caused the lytic PVCs in tobacco BY-2 cells to become aggregate and vacuolated, whereas the storage PVCs in seed cells remained unchanged in response to treatments of these drugs. Therefore, the storage PVCs in developing seeds are biochemically distinct from the lytic PVCs in tobacco BY-2 cells. / Plant cells contain both lytic vacuole and protein storage vacuole. Prevacuolar compartments (PVCs) are membrane-bounded organelles mediating protein trafficking between the Golgi apparatus and vacuoles in the plant secretory pathways. Multivesicular bodies (MVBs) have recently identified as the lytic PVCs in tobacco BY-2 cells. However, little is known about the dynamics of the lytic PVCs. In addition, the existence and identity of PVCs for protein storage vacuole (termed storage PVCs in this study) remain unknown. / This thesis research addressed two important biological questions: the dynamics of the lytic PVCs and the identity of the storage PVCs in plant cells. Towards this goal, I have demonstrated that the Golgi apparatus and the lytic PVCs, marked by YFP fusion reporters in transgenic tobacco BY-2 cells, have different sensitivity to brefeldin A (BFA) treatments. BFA at high concentrations (50 to 100 microg/mL) caused both YFP-marked Golgi stacks and lytic PVCs to form aggregates in a dosage-dependent and time-dependent manner. Confocal immunofluorescence and immunogold EM studies with specific organelle antibody markers further demonstrated that BFA-induced aggregates derived from the lytic PVCs were distinct from but physically associated with the Golgi aggregates. Thus, the BFA effects on the secretory organelles have been extended to the lytic PVCs. / Tse, Yu Chung. / "September 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4521. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 156-164). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Lo Sze Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 108-115). / Abstracts in English and Chinese. / Thesis committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract (in English) --- p.vi / Abstract (in Chinese) --- p.viii / Table of content --- p.x / List of tables --- p.xv / List of figures --- p.xvi / List of abbreviations --- p.xix / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- The secretory pathway --- p.2 / Chapter 1.1.1 --- Endoplasmic reticulum --- p.2 / Chapter 1.1.2 --- Golgi complex --- p.3 / Chapter 1.1.3 --- Vacuoles --- p.3 / Chapter 1.1.4 --- Prevacuolar compartment --- p.4 / Chapter 1.2 --- The secretory pathway in plant cells --- p.5 / Chapter 1.2.1 --- The secretory pathway in yeast and mammalian cells --- p.7 / Chapter 1.2.2 --- The lytic pathway in plant cells --- p.8 / Chapter 1.2.3 --- The protein storage vacuole pathway in plant cells --- p.10 / Chapter 1.3 --- Dynamic studies between organelles --- p.12 / Chapter 1.4 --- Objectives of this thesis research --- p.13 / Chapter Chapter 2 --- Development of Transgenic Cell Lines Expressing PVC and Golgi Markers --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.1.1 --- Putative PVC marker --- p.16 / Chapter 2.1.2 --- Golgi marker --- p.17 / Chapter 2.1.3 --- Dynamic studies --- p.18 / Chapter 2.1.4 --- Cell culture study --- p.18 / Chapter 2.2 --- Materials and Methods --- p.21 / Chapter 2.2.1 --- Plant material --- p.21 / Chapter 2.2.2 --- Construction of fusion reporters --- p.22 / Chapter 2.2.2.1 --- Cloning materials --- p.22 / Chapter 2.2.2.2 --- Vector preparation --- p.22 / Chapter 2.2.2.3 --- Cloning of pGFP-BP-80K and pGFP-BP-80H --- p.24 / Chapter 2.2.2.4 --- Cloning of pGFP-α-TIPH --- p.28 / Chapter 2.2.3 --- Transformation of tobacco BY-2 cells --- p.30 / Chapter 2.2.3.1 --- Agrobacterium transformation --- p.30 / Chapter 2.2.3.2 --- BY-2 cell transformation --- p.30 / Chapter 2.2.4 --- Screening of transgenic BY-2 cells --- p.31 / Chapter 2.2.4.1 --- Killing curve study --- p.31 / Chapter 2.2.4.2 --- Antibiotic selection --- p.32 / Chapter 2.2.4.3 --- Fluorescence microscopy screening (For single-construct cell lines) --- p.33 / Chapter 2.2.4.4 --- Confocal laser scanning microscopy (CLSM) screening (For double-construct cell lines) --- p.33 / Chapter 2.2.5 --- Detection of fluorescent protein expression --- p.35 / Chapter 2.2.5.1 --- Confocal imaging --- p.35 / Chapter 2.2.5.2 --- Protein extraction and subcellular fractionation --- p.36 / Chapter 2.2.5.3 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.36 / Chapter 2.2.5.4 --- Western blot analysis --- p.37 / Chapter 2.2.5.5 --- Cell culture study --- p.37 / Chapter 2.3 --- Results --- p.39 / Chapter 2.3.1 --- Hygromycin concentration at 50 mg/L was optimal for selection --- p.39 / Chapter 2.3.2 --- Lower transformation efficiency for double-construct cell lines --- p.40 / Chapter 2.3.3 --- Screening of transgenic cell lines --- p.41 / Chapter 2.3.4 --- Both pGFP-BP-80K and pGFP- a -TIPH expressed as punctate signals in single-construct cell lines --- p.45 / Chapter 2.3.5 --- Weak punctate or diffuse signals were detected from PVC markers in double-construct cell lines --- p.47 / Chapter 2.3.6 --- GFP reporters were successfully transformed into BY-2 cells --- p.51 / Chapter 2.3.7 --- Profiles of fluorescent signals in transgenic cells during cell culture --- p.53 / Chapter 2.4 --- Discussion --- p.59 / Chapter 2.4.1 --- Abnormal cell growth might be due to high selection pressure --- p.59 / Chapter 2.4.2 --- Double-construct cell lines developed were not yet suitable for further study --- p.60 / Chapter 2.4.3 --- Single-construct cell lines expressing putative PVC markers were developed --- p.62 / Chapter 2.4.4 --- 2- to 3-day-old cells were more suitable for subsequent studies --- p.63 / Chapter Chapter 3 --- Characterization of Transgenic Tobacco BY-2 Cell Expressing Reporters for Distinct Prevacuolar Compartments --- p.66 / Chapter 3.1 --- Introduction --- p.67 / Chapter 3.1.1 --- Wortmannin --- p.69 / Chapter 3.1.2 --- Brefeldin A --- p.70 / Chapter 3.1.3 --- FM4-64 --- p.71 / Chapter 3.2 --- Materials and Methods --- p.73 / Chapter 3.2.1 --- Plant material --- p.73 / Chapter 3.2.2 --- Confocal immunofluorescence studies --- p.73 / Chapter 3.2.3 --- Drug treatment studies --- p.74 / Chapter 3.2.3.1 --- Wortmannin treatment --- p.74 / Chapter 3.2.3.2 --- BFA treatment --- p.75 / Chapter 3.2.4 --- FM4-64 uptake study --- p.76 / Chapter 3.3 --- Results --- p.78 / Chapter 3.3.1 --- Organelles marked by GFP- a -TIP CT reporters did not localize at Golgi compartment --- p.78 / Chapter 3.3.2 --- Wortmannin induced GFP- a -TIP marked organelles to vacuolated --- p.81 / Chapter 3.3.3 --- GFP- a -TIP CT reporters partially colocalized with VSRin wortmannin-treated cells --- p.83 / Chapter 3.3.4 --- BFA induced GFP- a -TIP marked organelles to form BFA- induced compartments --- p.88 / Chapter 3.3.5 --- GFP-α -TIP CT reporter colocalized with internalized FM4-64 --- p.91 / Chapter 3.4 --- Discussion --- p.94 / Chapter 3.4.1 --- GFP- α -TIP CT reporter was a putative PVC marker --- p.94 / Chapter 3.4.2 --- GFP- a -TIP marked organelles behaved differently from lytic PVCs --- p.95 / Chapter 3.4.3 --- GFP- a -TIP marked organelles were not lytic PVCs --- p.96 / Chapter 3.4.4 --- FM4-64 uptake study reveals a new PVC marker --- p.98 / Chapter Chapter 4 --- Summary and Future Prospects --- p.100 / Chapter 4.1 --- Summary --- p.101 / Chapter 4.1.1 --- Hypothesis --- p.101 / Chapter 4.1.2 --- Development of transgenic cell lines --- p.102 / Chapter 4.1.3 --- Characterization of organelles marked by GFP- a -TIP CT reporter --- p.103 / Chapter 4.2 --- Future prospects --- p.106 / Reference --- p.108

Osteoclasts are cells that degrade bone, by forming a ruffled border (RB) membrane, contained within an actin-rich attachment site (the sealing zone; SZ). Lysosomal vesicles fuse to the RB, and release their contents into the extracellular space to degrade bone matrix. LC3, a marker of autophagosomes, localises to the RB, implying that either canonical autophagy (i.e. autophagosomes) or non-canonical autophagy (a process where LC3 localises to non-autophagic membrane) is involved in the resorptive function of osteoclasts. To examine this in detail, this study used a model with reduced canonical autophagy (FIP200 conditional knockout mouse), and two non-canonical autophagy deficient models (Rubicon knockdown in RAW 264.7-cell derived osteoclasts and an Atg16L1 WD40 domain knockout mouse). Using advanced imaging and molecular techniques I examined whether impairing either process affected LC3 RB localisation and resorption. Reducing canonical autophagy through FIP200 deficiency did not significantly affect GFP-LC3 RB localisation or bone homeostasis. However, impairing non-canonical autophagy resulted in a trend towards increased resorption in vitro. In Atg16L1 WD40 domain-deficient osteoclasts, this may be due to the significantly larger SZs formed in the mutants, which were often stable and contained LysoTracker-positive acidic vesicles within them, putatively signalling increased resorption. As LC3 was frequently observed at the RB, I then examined the LC3-interacting lysosomal adaptor protein, PLEKHM1. I showed that in the PLEKHM1 functional knockout mouse model (R714 STOP), osteoclasts still form RBs but have impaired resorption in vitro. Detailed analysis of multiple aspects of resorption in PLEKHM1 deficient osteoclasts, was required to uncover these defects which may underlie the osteopetrotic phenotype observed in PLEKHM1 deficient mice. Overall this work reveals the potential role of non-canonical autophagy in osteoclast function. Additional dissection of this pathway in osteoclasts may uncover further new insights regarding the regulation of bone resorption and defects underlying bone disorders.

No description available.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

No description available.

The tonoplast H$ sp+$-ATPase of Beta vulgaris L. was partially purified by Triton X-100 solubilization and Sepharose 4B chromatography resulting in the enrichment of two polypeptides (57 and 67 kDa). Kinetic analysis of ($ alpha$-$ sp{32}$P) BzATP labeling identified the 57 kDa polypeptide as a nucleotide-binding subunit with a possible regulatory function. In addition, ($ sp{14}$C) DCCD-labeling identified a 16 kDa polypeptide as a putative transmembrane proton channel. It is concluded that the tonoplast H$ sp+$-ATPase is a multimer composed of at least three polypeptides. / Anti-57 and anti-67 kDa sera reacted with polypeptides of the corresponding size in bovine chromaffin granules, bovine clathrin-coated vesicles, and yeast vacuolar membranes, suggesting common structural features and common ancestry for endomembrane H$ sp+$-ATPases of different organelles and different phyla. Anti-57 serum was used to isolate a cDNA encoding the corresponding subunit from Arabidopsis. Protein sequence analysis revealed homologies between endomembrane, F$ sb0$F$ sb1$ and archaebacterial ATPases, suggesting that these different classes of ATPases have evolved from a common ancestor.

Thesis (Ph. D.)--Carleton University, 1992. / Also available in electronic format on the Internet.

No description available.

在真核细胞中，细胞自噬是一个将细胞物质吞噬到自噬体降解的保守的代谢过程。自噬体起始于自噬前体结构(PAS) 并由其逐渐扩展和延伸形成其最后的双膜结构，最后和溶酶体（lysosome）或液泡（vacuole）融合得以降解。在酵母和动物细胞中，研究已发现一系列自噬体相关基因（ATG）蛋白参与调控自噬体的形成。自噬体形成的相关研究存在两个主要的未解决的问题，它们包括自噬体的膜来源和膜变形机制。而在植物中，相当一部分关键的自噬体同源蛋白的缺失导致其分子机制研究仍处于初步阶段。在本研究中，我主要通过利用SH3P2，一个N 端含有BAR (Bin-Amphiphysin-Rvs) 结构域及C 端含有SH3（Src homology 3）结构域的蛋白作为探针, 在拟南芥中研究自噬体的形成. 在进一步的研究中，借助了免疫细胞化学技术（抗体），分子技术（萤光蛋白标记），基因技术（RNAi 干扰）以及蛋白作用（酵母双杂交和免疫共沉淀）等不同的生化以及细胞生物学手段，我发现在植物细胞中也存在保守的自噬体的形成模式，而在其过程中, SH3P2 起着重要的调控作用。通过研究我发现：1）在拟南芥植物中，绿色荧光蛋白标记的SH3P2-GFP 蛋白具有对自噬诱导的应答反应；；2）在拟南芥转基因植物和PSBD 悬浮细胞中，SH3P2-GFP 蛋白与自噬体标记蛋白共定位； 3） 在自噬途径中，SH3P2-GFP 活跃地参与在自噬体膜变形过程中并且定位在自噬前体结构包括其扩展结构的膜上； 4）基因敲低SH3P2 在拟南芥植物中是致死的并且抑制自噬体的形成过程；5) SH3P2 能通过它的BAR 结构域互相聚合; 6）SH3P2 可以结合磷脂酰肌醇-3-磷酸（PI3P）并且与磷脂酰肌醇-3-激酶复合体存在联系；7）SH3P2 通过它的SH3 结构域直接与ATG8 结合。综上所述，此项研究发掘了一个新型的膜相关蛋白SH3P2 参与在拟南芥植物自噬途径中，而其与ATG8 的直接相互结合同时也揭示了一个新的自噬形成调控机制。 / In eukaryotic cells, autophagy is a conserved catabolic mechanism by engulfing the cytoplasmic cargoes into a structure termed autophagosome. In general, autophagosome is initiated from a site named PAS (phagophore assembly site preautophagosome structure), which then expands and elongates to form a double membrane structure. Ultimately, the outer membrane of autophagosome will fuse with the lysosome or vacuole membrane and deliver the cargoes for degradation or recycling. In yeast and animal cells, a number of ATGs (autophagy related genes) have been identified to regulate the autophagosome formation. Studies of the autophagosome formation involve two main unsolved questions: the membrane origin and the membrane deformation mechanism. In plants, several key players responsible for autophagosome biogenesis are missing and the molecular mechanisms for the autophagosome formation remain elusive. In this study, I have used SH3P2, which contains a N-terminus BAR (Bin-Amphiphysin-Rvs) domain and C-terminus SH3 (Src homology 3) domain, as a probe, to study the autophagosome formation in plants. Using a combination of immunocytochemical (antibodies), molecular (GFP fusions), genetic (RNAi) and interaction (Yeast two-hybrid and Co-IP) approaches, I have shown that a conserved autophagosome formation model exists in plant cells and SH3P2 plays an essential role in the autophagy pathway in Arabidopsis thaliana. I have found that 1) SH3P2-GFP fusion proteins response to autophagic induction in transgenic Arabidopsis plants; 2) SH3P2-GFP colocalize with the known autophagosome markers in both transgenic Arabidopsis plant and PSBD cells; 3) SH3P2-GFP localizes on the PAS membrane and actively participates in membrane deformation events during autophagosome formation throughout its expansion process via the dynamic and ultra structural analysis; 4) Knock-down of SH3P2 is developmental lethal and suppresses the autophagosome formation and autophagic flux; 5) SH3P2 has a self-interaction via its BAR domain; 6) SH3P2 binds to PI3P (Phosphatidylinositol-3-Phosphate) and associates with the PI3K (Phosphatidylinositol-3-Phosphate Kinase) complex; 7) SH3P2 directly interacts with ATG8 via its SH3 domain. Taken together, this thesis research has identified a novel membrane-associated protein and demonstrated its essential role in autophagy in plant. The demonstration for the direct association between SH3P2 and the ATG8 complex may provide an insightful mechanism for autophagosome regulation in Arabidopsis thaliana. / Detailed summary in vernacular field only. / Zhuang, Xiaohong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 97-104). / Abstracts also in Chinese. / Statement --- p.I / Abstract --- p.II / 摘要 --- p.IV / Acknowledgements --- p.VI / Table of Contents --- p.VIII / List of Tables --- p.X / List of Figures --- p.XI / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Introduction of Autophagy --- p.2 / Chapter 1.2 --- Molecular Machinery for Autophagy --- p.5 / Chapter 1.3 --- Membrane Origins of Autophagosome --- p.8 / Chapter 1.4 --- Membrane Sensors for Autophagosome Formation --- p.10 / Chapter 1.4.1 --- ATG14 --- p.10 / Chapter 1.4.2 --- Bif1 (Bax-interacting factor 1) --- p.11 / Chapter 1.5 --- Autophagy in Plants --- p.12 / Chapter 1.6 --- Research Objectives --- p.13 / Chapter Chapter 2 --- SH3P2 Defines a Conserved Autohagosome Formation Process in Arabidopsis --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.2 --- Materials and Methods --- p.19 / Chapter 2.2.1 --- Plasmid Construction --- p.19 / Chapter 2.2.2 --- Plant Materials, Growth and Treatment Conditions --- p.24 / Chapter 2.2.3 --- Transient Expression in Protoplasts and Confocal Imaging --- p.24 / Chapter 2.2.4 --- Antibody Generation, Protein Extraction and Western Blot Analysis --- p.25 / Chapter 2.2.5 --- Immunofluorescence Confocal Study --- p.26 / Chapter 2.2.6 --- Electron Microscopy (EM) Study --- p.26 / Chapter 2.2.7 --- Accession Numbers --- p.27 / Chapter 2.3. --- Results --- p.28 / Chapter 2.3.1. --- SH3P2-GFP Fusion Proteins Response to Autophagic Induction in Arabidopsis --- p.28 / Chapter 2.3.2 --- The SH3P2-GFP Positive Compartments are Overlapped with Autophagosome Markers --- p.36 / Chapter 2.3.3 --- Dynamic Analysis of SH3P2-GFP Positive Compartments in Arabidopsis Transgenic Plants upon Autophagic Induction --- p.42 / Chapter 2.3.4 --- EM Analysis of the subcellular localization of SH3P2 after autophagic induction --- p.44 / Chapter 2.4 --- Discussion --- p.52 / Chapter Chapter 3 --- SH3P2 is Essential for Plant Development and Autophagic Pathway in Arabidopsis --- p.54 / Chapter 3.1 --- Introduction. --- p.55 / Chapter 3.2.1 --- Plasmid Construction --- p.57 / Chapter 3.2.2 --- Plant Materials, Growth and Treatment Conditions --- p.57 / Chapter 3.2.3 --- Transient Expression in Protoplasts and Confocal Imaging --- p.58 / Chapter 3.2.4 --- Protein Extraction and Immunoblot Analysis --- p.58 / Chapter 3.2.5 --- RT-PCR --- p.59 / Chapter 3.3 --- Results --- p.60 / Chapter 3.3.1 --- RNAi Knockdown of SH3P2 is Developmental Lethal --- p.60 / Chapter 3.3.2 --- RNAi Knockdown of SH3P2 Suppresses the Autophagosome Formation and Autophagic Flux --- p.63 / Chapter 3.4 --- Discussion --- p.71 / Chapter Chapter 4 --- SH3P2 is Associated with the ATG Machinery --- p.73 / Chapter 4.1 --- Introduction --- p.74 / Chapter 4.2 --- Materials and Methods --- p.76 / Chapter 4.2.1 --- Plasmid Construction --- p.76 / Chapter 4.2.2 --- Plant Materials, Growth and Treatment Conditions --- p.76 / Chapter 4.2.3 --- Recombinant Protein Expression --- p.77 / Chapter 4.2.4 --- In Vitro Lipid Binding Assay --- p.77 / Chapter 4.2.5 --- Yeast-two Hybrid Analysis --- p.78 / Chapter 4.2.5 --- Immunoprecipitation Analysis --- p.78 / Chapter 4.3 --- Results --- p.80 / Chapter 4.3.1 --- SH3P2 Binds to PI3P --- p.80 / Chapter 4.3.2 --- SH3P2 Has a Strong Self-interaction via the BAR Domain --- p.82 / Chapter 4.3.3 --- SH3P2 is Associated with the PI3K Complex and Interacts with ATG8 --- p.84 / Chapter 4.4 --- Discussion --- p.86 / Chapter Chapter 5 --- Discussions and Perspectives --- p.87 / Chapter 5.1 --- Discussions --- p.88 / Chapter 5.1.1 --- Autophagosome Formation is Conserved in Arabidopsis thaliana --- p.88 / Chapter 5.1.2 --- SH3P2 Interacts with the ATG8 Complex and is Required for the Autophagic Pathway in Arabidopsis thaliana --- p.90 / Chapter 5.1.3 --- A Novel Membrane-associated Regulator for Autophagosome formaiton in Arabidopsis thaliana --- p.92 / Chapter 5.2 --- Working Model of SH3P2 during Autophagosome Formation in Arabidopsis --- p.93 / Chapter 5.3 --- Future Perspectives --- p.96 / References --- p.97 / List of Publications --- p.104