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by Tse Yu Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 133-138). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vi / Table of Contents --- p.vii / List of Tables --- p.xi / List of Figures --- p.xii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1. --- The Plant secretory pathway --- p.2 / An overview on the secretory pathway --- p.2 / Vesicular pathways and transport vesicles --- p.4 / Chapter 2. --- Vacuolar sorting receptors --- p.6 / BP-80 and its homologues --- p.6 / RMR proteins --- p.7 / Chapter 3. --- Prevacuolar compartments --- p.8 / PVCs in mammalian and yeast cells --- p.8 / PVCs for seed protein storage vacuoles --- p.9 / PVCs for lytic vacuoles --- p.11 / Chapter Chapter 2 --- Development of Transgenic Tobacco BY-2 Cell Lines Expressing Fluorescent Markers for Golgi and Prevacuolar Compartments --- p.15 / Chapter 1. --- Introduction --- p.16 / Chapter 1.1 --- Fluorescent proteins are useful tools in studying protein trafficking and subcellular localization in living cells --- p.16 / Chapter 1.2 --- Tobacco BY-2 cells --- p.18 / Chapter 1.3 --- Plant prevacuolar compartments --- p.19 / Chapter 2. --- Materials and Methods --- p.21 / Chapter 2.1 --- Construction of RFP-BP-80 and RFP-α-TIP reporters --- p.21 / Chapter 2.2 --- Construction of YFP-BP-80 and YFP-α-TIP reporters --- p.27 / Chapter 2.3 --- Construction of YFP markers for Golgi organelles --- p.32 / Chapter 2.4 --- Agrobacterium electroporation --- p.33 / Chapter 2.5 --- Transformation of tobacco BY-2 cells --- p.34 / Chapter 2.6 --- Screening of transgenic BY-2 cells expressing RFP markers --- p.35 / Chapter 2.8 --- Production of anti-BP-80 CT antibody --- p.43 / Chapter 2.9 --- Chemicals --- p.45 / Chapter 2.10 --- Primers --- p.45 / Chapter 2.11 --- Bacterial strain --- p.46 / Chapter 3. --- Results --- p.47 / Chapter 3.1 --- Generation and characterization of transgenic BY-2 cell lines expressing RFP reporters --- p.47 / Chapter 3.2 --- Generation and preliminary characterization of transgenic BY-2 cell lines expressing YFP reporters --- p.55 / Chapter 3.3 --- Confocal detection ofYFP reporters in transgenic cell lines --- p.64 / Chapter 3.4 --- Characterization of anti-BP-80 CT antibody --- p.66 / Chapter 4. --- Discussion --- p.68 / Chapter Chapter 3 --- Dynamic of Plant Prevacuolar Compartments in Transgenic Tobacco BY-2 Cells --- p.72 / Chapter 1. --- Introduction --- p.73 / Chapter 1.1 --- The plant secretory pathway --- p.73 / Chapter 1.2 --- Organelle markers in plant secretory pathway --- p.74 / Chapter 1.3 --- Markers for Lytic PVCs --- p.75 / Chapter 2. --- Materials and Methods --- p.77 / Chapter 2.1 --- Confocal immunofluorescence studies --- p.77 / Chapter 2.2 --- FM4-64 uptake study --- p.79 / Chapter 2.3 --- Brefeldin A treatment --- p.79 / Chapter 2.4 --- Wortmannin treatment --- p.80 / Chapter 2.5 --- Movement study of YFP-marked PVC --- p.82 / Chapter 3. --- Results --- p.83 / Chapter 3.1 --- Different internal organelles were labeled by two different YFP reporters --- p.83 / Chapter 3.2 --- The YFP-BP-80 reporter localized with endogenous VSR proteins --- p.86 / Chapter 3.3 --- Brefeldin A enlarged PVC organelles --- p.89 / Chapter 3.4 --- Identity of PVC-derived BFA-induced compartments --- p.99 / Chapter 3.5 --- Wortmannin induced PVCs to form small vacuoles --- p.102 / Chapter 3.6 --- PVCs are mobile organelles in living cells --- p.112 / Chapter 4. --- Discussion --- p.114 / Chapter Chapter 4 --- Summary and Future Perspectives --- p.123 / Chapter 1. --- Summary --- p.124 / The hypothesis --- p.124 / Development of three transgenic cell lines --- p.125 / Distinct organelles were marked by two different YFP reporters --- p.126 / The YFP-BP-80 reporter defined the lytic PVCs --- p.126 / Response of YFP-marked PVCs to Brefeldin A treatment --- p.127 / Response of YFP-marked PVCs to Wortmannin treatment --- p.127 / PVCs are mobile organelles in living cells --- p.129 / Chapter 2. --- Future perspectives --- p.130 / References --- p.133

Further in vivo and in vitro studies using the truncated OsRMR1 proteins from the culture media of transgenic BY-2 cells demonstrated that OsRMR1 functioned as a receptor in transporting vicilin-like storage proteins via specific interaction with their vacuolar sorting determinants. Taken together, the OsRMR1 is a sorting receptor for the PSV pathway that defines a novel organelle as PVC for PSV in rice. / Receptor-mediated protein sorting is one of the mechanisms for transporting soluble proteins to the protein storage vacuoles (PSVs) in plant cells. Members of vacuolar sorting receptor (VSR) family proteins and receptor homology region-transmembrane domain-RING-H2 (RMR) family proteins have been shown to function in mediating the transport of storage proteins to PSVs in plants. However, no prevacuolar compartment (PVC) for the PSV pathway has been identified. In this study, I used a rice RMR protein (OsRMR1) as a probe to study the PSV pathway in rice. Using confocal immunofluorescent and immunogold electron microscopy (EM) with specific OsRMR1 antibodies, I have identified a novel organelle as a PVC for the PSV pathway, because OsRMR1 antibodies labeled the Golgi apparatus, trans-Golgi network (TGN) and the novel organelle in both rice cultured cells and developing rice seeds, as well as the protein body Type II (PBII) in developing rice seeds. This novel organelle is morphologically distinct from the lytic PVC or multivesicular body (MVB). / Shen, Yun. / "May 2008." / Adviser: Liwen Jiang. / Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1428. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 124-139). / 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.

p38 mitogen-activated protein kinases (MAPKs) control the endocytic trafficking of various growth-related cell surface receptors and transporters. Herein, I demonstrate that p38 MAPKs also regulate autophagy, or the process of self-cannibalism. In my studies, inhibition of p38 MAPKs triggered rapid formation of autophagosomes in prostate cancer cells, even under nutrient-rich conditions, and remarkably, the autophagosomal membranes emanated from endoplasmic reticulum exit sites via the concerted actions of the small GTPases, ARF1 and SAR1. Once formed, the autophagosomes fused with late endosomes and/or lysosomes, in a Rab7-dependent manner, to form “hybrid organelles” that were co-labeled with ER, autophagic, late endosomal, and lysosomal markers. Unlike other inducers of autophagy, however, inhibition of p38 MAPKs suppressed the fission of hybrid organelles, resulting in a profound but reversible accumulation of large cytoplasmic vacuoles. Thus, in addition to their previously reported roles in endocytosis, p38 MAPKs appear to coordinately regulate autophagy and the downstream biogenesis and fission of hybrid organelles. / text

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 135-155). Also available in print.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 186-198).

The aberrant accumulation of aggregated proteins is a pathologic hallmark across adult-onset neurodegenerative diseases, the majority of which have no effective treatment. Although the relative importance of these structures to pathogenesis has been proposed in several diseases, there is little understanding of how we might accelerate the turnover of aggregated proteins, and in turn, a lack of consensus about whether targeting them would provide any therapeutic benefit. The overarching goal of my dissertation is to address both of these questions by focusing on how the pathway macroautophagy might handle protein aggregates in the adult brain. Aggregation-prone proteins are preferentially degraded through the lysosome-mediated degradation pathway macroautophagy (referred to hereafter as autophagy) (Ravikumar 2002; Iwata 2005; Yamamoto 2006). Although studies suggest that aggregates are degraded in bulk by autophagy (Ravikumar 2002; Iwata 2005), studies show that they are more likely degraded in an adaptor-protein dependent manner (Lemasters, 2005; Kraft, 2008; Hanna, 2012; Isakon, 2012; Filimonenko, 2010). In the Yamamoto lab, we have found that the adaptor, the Autophagy-linked FYVE protein (Alfy/WDFY3), is required for the degradation of detergent-insoluble aggregated proteins through selective autophagy in cell-based systems and the adult brain (Simonsen, 2004; Eenjes, 2016; Filimonenko, 2010; Fox, 2020). Through immunohistochemical and loss-of-function studies, Alfy has been implicated in the turnover of disease-relevant protein aggregates including mHtt, α-synuclein, SOD1, and TDP-43, as well as protein complexes such as the midbody ring (Filimonenko, 2010; Clausen, 2010; Han, 2014; Hocking, 2010; Isakson, 2013; Kadir, 2016). Here, I present a potential strategy to suppress disease progression across neurodegenerative disorders by increasing the levels, and thereby the function, of Alfy. I hypothesized that genetically augmenting Alfy levels in the brain will be sufficient to alleviate aggregate burden and delay the onset of proteotoxic stress in different mouse models of neurodegeneration. Using biochemical and genetic approaches, I conducted an extensive in vivo study, demonstrating that augmenting Alfy expression levels in mice can be neuroprotective, and that Alfy may be an influential genetic modifier of neurodegenerative disease. Using two independent genetic approaches that upregulate Alfy expression, I found that they both dramatically delay the onset of disease phenotypes in mouse models of Huntington’s disease, synucleinopathy and TDP-43 proteinopathy. First, I found that ectopic overexpression of Alfy has a pronounced, neuroprotective effect on reducing aggregation, improving motor function, and extending survival in disease models. In parallel, I used mouse genetics to verify the potency of a rare Alfy variant identified in a large Venezuelan cohort of Huntington’s disease that correlated with delayed onset in Huntington’s disease by 10-20 years. Excitingly, in support of our hypothesis, I found that the presence of this single nucleic acid polymorphism led to an increase in steady state levels of Alfy in both humans and in mice, and it was sufficient to recapitulate the benefits of ectopic Alfy overexpression. Taken together, these studies demonstrate that increasing Alfy levels in the brain are sufficient to augment the turnover of aggregated proteins, and may be an effective therapeutic strategy that can be beneficial across neurodegenerative diseases.

Vacuolar sorting receptors (VSRs) are type I integral membrane family proteins that mediate protein transport from late Golgi or trans-Golgi network (TGN) to vacuole via prevacuolar compartment (PVC) in plant cells. The N-terminus of a VSR is believed to be important for cargo binding while its transmembrane domain (TMD) and cytoplasmic tail (CT) are essential for its correct subcellular localization. In this study, I first developed and tested an expression system using transgenic tobacco BY-2 cells to produce truncated VSR proteins (VSRNT) lacking the TMD/CT into the cultured media. The secreted VSRs bind specifically to the vacuolar sorting determinants (VSDs) of known vacuolar proteins and such binding is calcium dependent in vitro. Thus, VSR cargo proteins are likely secreted into the cultured media along with the truncated VSRs, which enable the identification of various VSR cargo proteins from the cultured media of transgenic cells. I then identified these putative VSR cargo proteins through liquid-chromatography with tandem mass spectrometry (LC-MS/MS) and Fourier transform mass spectrometry (FT-MS) using transgenic Arabidopsis cell suspension cultures PSB-D expressing these truncated VSRs. Among the 17 unique proteins found in the cultured media of transgenic Arabidopsis PSB-D cell line expressing VSRNT, an Arabidopsis glycosyl hydrolase family 3 protein At5g10560 (GH3) was chosen for further study on VSR-cargo protein interaction. GFP-tagged GH3 fusion protein was found to co-localize with VSR-RFP marker protein in PVC, whereas GH3 was also shown to interact with a VSR protein BP-80. Loss-of-function analysis demonstrated that the GH3 contained a vacuolar sorting determinant (VSD) for PVC targeting. / Suen, Pui Kit. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 77-84). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.