拟南芥ARA7(AtRabF2b), 是植物Rab5 小分子GTP结合蛋白家族中的一员。研究显示，ARA7定位于液泡前体（PVC）或者多囊泡体（MVB），并在液泡运输的过程中起到重要作用。光学显微镜研究显示，GTP酶缺陷突变体ARA7Q69L会引起MVB迅速扩大。然而扩大的MVB的膜来源，超微结构，和动态学研究还未被涉及。在本论文研究中，我在转基因拟南芥悬浮细胞中表达了绿色荧光蛋白标记的ARA7Q69L, 通过共聚焦荧光显微镜，活细胞成像，电子显微镜，和免疫电镜研究，我发现MVB的扩大是由MVB的同源融合引起的。这个独特现象使ARA7Q69L成为研究MVB蛋白定位的重要工具。 / 此外，我还研究了花粉管顶端透明区聚集小泡的囊泡运输。在极性生长的花粉管顶端，聚集了很多运输小泡。为满足花粉管顶端快速生长的需要，这些聚集的小泡会在严格调控下进行活跃的胞吞和胞吐活动。但是由于小泡体积小，运动快，它们的活动情况难以用传统方法进行研究。研究显示SCAMP蛋白位于花粉管顶端小泡和细胞膜。因此顶端小泡的活动情况可以通过SCAMP荧光蛋白标记进行研究。通过活细胞成像，荧光漂白恢复和药物处理，我发现这些小泡至少包括了内吞小泡和分泌小泡两类，分泌小泡抵达花粉管亚顶端区后与细胞膜融合，将物质释放到细胞外，而内吞小泡则是由尖端的细胞质膜内陷包裹而形成，并经由花粉管中央区域转运至细胞内。 / Arabidopsis ARA7 (AtRabF2b), a member of plant Rab5 small GTPase that functions in vacuolar transporting pathway, is known to localize to late endosome of prevacuolar compartments (PVCs), identified as multivesicular bodies (MVBs) in plant cells. The constitutively active mutant of ARA7 (ARA7Q69L) had been shown to induce the formation of enlarged MVBs at the light microscope level. However, the membrane origins, ultrastructures and dynamics of ARA7Q69L-induced MVB enlargement remain elusive. In this study, I have first generated transgenic Arabidopsis cell lines expressing GFP-ARA7Q69L under the control of an inducible promoter. Using a combination of confocal immunofluorescence, live-cell imaging and transmission electron microscopy (TEM) analysis, I have demonstrated that the formation of GFP-ARA7Q69L-induced enlarged MVBs was probably derived from homotypic fusion of MVBs. This unique character makes ARA7Q69L a useful and reliable tool for studying relative localizations of different proteins on MVBs. / In addition, another project I carried out is about dynamics of tip region vesicles in growing tobacco pollen tubes. During polarized growth of pollen tubes, the tip region is enriched with transporting vesicles, where endocytosis and exocytosis are highly active. Due to the small size and fast movements of tip region vesicles, it is difficult to study their trafficking and dynamics using conventional tools. The Arabidopsis secretory carrier membrane protein 4 (AtSCAMP4), which labels the plasma membrane (PM) and tip region vesicles in tobacco (Nicotiana tabacum) pollen tubes, was used as a marker to study vesicle dynamics and transportation at the tip region. Using a combination of live-cell imaging, fluorescence recovery after photo-bleaching (FRAP) analysis and drug treatments, I have demonstrated that the AtSCAMP4-labeled vesicles in tip region are composed of both endocytic and exocytic vesicles. They are probably secreted to the PM at apical flank region, recovered through endocytosis at the apex and then driven back through the centre of pollen tubes. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Jia, Tianran. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 56-61). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Thesis/Assessment Committee --- p.i / Statement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Acknowledgements --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xi / List of Figures --- p.xi / List of Abbreviations --- p.xiv / Chapter Chapter 1 --- p.1 / Dynamics and Ultrastructure of Enlarged Multivesicular Bodies in Transgenic Arabidopsis Cells --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Material and Methods --- p.5 / Chapter 1.2.1 --- Plasmid construction --- p.5 / Chapter 1.2.2 --- Transient expression --- p.5 / Chapter 1.2.3 --- Transformation of Arabidopsis cells --- p.5 / Chapter 1.2.4 --- Confocal immunofluorescence studies --- p.6 / Chapter 1.2.5 --- Dynamic study of GFP fusions in transgenic cells using spinning disk confocal microscopy --- p.7 / Chapter 1.2.6 --- Electron microscopy study --- p.7 / Chapter 1.3 --- Results --- p.8 / Chapter 1.3.1 --- GFP-ARA7Q69L-induced enlarged MVBs colocalize with the MVB markers in Arabidopsis protoplasts --- p.8 / Chapter 1.3.2 --- Generation and characterization of transgenic Arabidopsis PSBD cell lines expressing GFP-ARA7Q69L under the control of a heat shock promoter --- p.12 / Chapter 1.3.3 --- GFP-ARA7Q69L colocalized with PVC/MVB marker anti-VSR on enlarged MVB but separated from both TGN and Golgi markers in transgenic cells --- p.16 / Chapter 1.3.4 --- Dynamics of membrane fusion of GFP-ARA7Q69L-labeled organelles in transgenic cells --- p.19 / Chapter 1.3.5 --- Ultrastructure of enlarged MVBs induced by GFP-ARA7Q69L --- p.23 / Chapter 1.4 --- Discussion --- p.34 / Chapter Chapter 2 --- p.37 / Dynamics of AtSCAMP4-labeled vesicles in the tip region of tobacco growing pollen tubes --- p.37 / Chapter 2.1 --- Introduction --- p.38 / Chapter 2.2 --- Material and Methods --- p.41 / Chapter 2.2.1 --- Plasmid construction --- p.41 / Chapter 2.2.2 --- Pollen particle bombardment --- p.41 / Chapter 2.2.3 --- Fluorescence recovery after photo-bleaching (FRAP) analysis --- p.41 / Chapter 2.2.4 --- Drug treatment and FM4-64 labeling --- p.42 / Chapter 2.3 --- Results and Discussion --- p.42 / Chapter 2.3.1 --- AtSCAMP4 colocalizes with both the endocytic marker FM4-64 and the exocytic marker AtRabA4d at the pollen tip region --- p.42 / Chapter 2.3.2 --- AtSCAMP4-labeled vesicles reach the apical flank region for exocytosis, then generated by endocytosis in the extreme apex --- p.46 / Chapter 2.3.3 --- Effects of drug treatments on GFP-AtSCAMP4-labeled vesicles --- p.49 / Chapter 2.4 --- Conclusion --- p.52 / Supplemental Figures --- p.54 / References --- p.56

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