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|Title:||The study Of the intra-Golgi trafficking and molecular organization of the Golgi||Authors:||Tie, Hieng Chiong||Keywords:||Science::Biological sciences::Molecular biology||Issue Date:||2020||Publisher:||Nanyang Technological University||Source:||Tie, H. C. (2020). The study Of the intra-Golgi trafficking and molecular organization of the Golgi. Doctoral thesis, Nanyang Technological University, Singapore||Abstract:||The mammalian Golgi complex adopts a complicated architecture which is unresolvable under a conventional light microscope. Based on a previously developed method, Golgi protein localizations by imaging centers of mass (GLIM), we quantitatively mapped the axial localization of dozens of Golgi proteins along the Golgi axis in nocodazole-induced Golgi mini-stacks. In this study, we explored the lateral localization of Golgi proteins. Using Airyscan microscopy, we identified and validated that GPP130 and several Golgins, including Golgin84, Giantin and GCC185 exclusively localized to Golgi cisternal rims. Hence, they were used to discern the orientations (i.e. side and en face views) of Golgi mini-stacks. By systematically screening Golgi enzymes and components of trafficking machinery, we found that the former preferentially localized to the cisternal interior, whereas the latter was mostly found at the periphery or the rim of the Golgi mini-stack. In addition, after averaging large number of en face and side view images, we generated quantitative molecular organization maps, which summarize the axial and lateral localizations of Golgi proteins in the Golgi mini-stack. For the first time, we provide imaging evidence on the spatial separation of the processing and transport domains to the cisternal interior and rim of the Golgi mini-stack, respectively. Notably, the TGN was observed to organize into a multi-tier architecture axially and demonstrate two types of molecular localization patterns, i.e. compact lump and scattered puncta, laterally. To investigate intra-Golgi trafficking of secretory cargos, their lateral localizations were analysed. Conventional or small secretory cargos (<115 kDa), including GFP, mCherry-GPI, CD59, E-cadherin, VSVG, CD8a-Furin, Lamp1, TfR and CD-MPR, were found to mainly localize in the cisternal interior. In contrast, bulky cargos (>20,000 kDa), including collagenX, FM4-CD8a and FM4-moxGFP aggregates, mainly localized at the cisternal rim. Collectively, our imaging data suggest a size-dependent lateral partitioning model for secretory cargos, where bulky and small cargos preferentially localize to the cisternal rim and interior, respectively. Intriguingly, the synchronous cargo waves of VSVG and TfR were observed to progressively transit from the cis- to the trans-side of the Golgi mini-stack. Furthermore, various secretory cargos were observed to exit the Golgi at either the trans-Golgi or the TGN. The cytosolic tail of Furin was found to be sufficient for VSVG-tso45-Furin to exit the Golgi at the TGN. Besides that, the tyrosine motif of Lamp1 could be important in guiding the protein to access and exit the Golgi at the TGN. Hence, we propose that both the trans-Golgi and TGN can function as Golgi exit sites. Cargos harbouring the TGN targeting signal(s) can access and exit the Golgi at the TGN in a signal-dependent manner. In contrast, cargos without such a signal constitutively exit at the trans-Golgi by default.||URI:||https://hdl.handle.net/10356/145735||DOI:||10.32657/10356/145735||Rights:||This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).||Fulltext Permission:||embargo_20220106||Fulltext Availability:||With Fulltext|
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