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Molecular regulation of de novo Golgi biogenesis

Vegesna, Naga Venkata Gayathri

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The Golgi complex (GC) is a central organelle of the secretory pathway. It receives and distributes material from and to other cellular organelles and is thus involved in basic cellular processes such as differentiation, cell motility or signal transduction. In mammalian cells the GC acquires a highly dynamic and unique morphology that quickly disassembles before mitosis and reassembles thereafter. The molecular regulators involved in these processes however remain largely elusive. To understand the molecular mechanisms of Golgi biogenesis and its regulation in detail, I used a combinatory approach of RNAi and diffraction limited laser nanosurgery to deplete cells of their GC and monitor its de novo biogenesis in the karyoplasts by time-lapse and correlative light and electron microscopy. To first identify proteins that could play a role in Golgi biogenesis, I screened the Human Protein Atlas database and chose 31 proteins localized exclusively to the GC and classified them based on their behaviour upon Brefeldin A (BFA) treatment of cells. This showed that 13 proteins behaved like Golgi enzymes and relocated to the endoplasmic reticulum after BFA treatment. Another 8 proteins showed Golgi matrix-like behaviour and remained distributed throughout the cytoplasm as distinct Golgi remnants. The remaining 10 proteins showed a TGN/centrosome-like localisation after BFA treatment. Among the 18 Golgi matrix-like and TGN/centrosome-like proteins, I could validate the siRNA knockdown in 7 candidate proteins. Functional analysis of these 7 protein candidates by using laser nanosurgery to deplete the GC together with the target proteins and subsequent blocking their protein synthesis through RNAi showed an acceleration of the early phase of Golgi biogenesis upon depletion of GMAP210. Individual depletions of the 6 other Golgi proteins tested showed only a slight delay or no effect on Golgi biogenesis. However, double depletions of GRASP65 & 55 or GRASP65 & Giantin resulted in delays in the kinetics of the early phase of Golgi biogenesis for several hours. Ultra-structural analyses by correlative light and electron microscopy showed that the double depletion of GRASP65 & 55 affected the flattening of Golgi cisternae, an event that occurs in the later phases of Golgi biogenesis, and resulted in the accumulation of swollen cisternae even at the end of the experiments. In addition, the formation of complex and convoluted Golgi intermediates that usually occur in early phases during Golgi biogenesis was delayed or impaired in GRASP65 & 55 double depleted cells. The delay in the ability to form Golgi precursors in GRASP65 & 55 double depleted cells shows the important role of these two Golgi matrix proteins acting in concert in the initial stages of the process. In contrast to the existing literature data, I could not identify any evidence of the involvement of GRASP65/55 in Golgi stacking during de novo Golgi biogenesis from my experiments. The results of this PhD work further suggest that, most likely there is no single master regulator for the Golgi biogenesis and there is a significant degree of functional redundancy among Golgi matrix proteins involved in the process.

Document type: Dissertation
Supervisor: Pepperkok, Dr. Rainer
Date of thesis defense: 15 December 2016
Date Deposited: 02 Jan 2017 14:10
Date: 2017
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Service facilities > European Molecular Biology Laboratory (EMBL)
DDC-classification: 570 Life sciences
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