The Golgi apparatus is lies at the heart of the secretory pathway and is found in all eukaryotic cells, where it plays an important role in the post-translational modification and sorting of membrane and secreted proteins and lipids. Although the Golgi has been extensively studied at the cellular level, there have been few studies examining its role in the physiology and development of multicellular organisms. The aim of this project is to investigate the importance of the Golgi apparatus in vivo, using zebrafish as a model, and to determine how defects in Golgi function leads to human disease. The focus of the project will be the golgin family of coiled-coil proteins that are thought to functionally link Golgi membranes together to maintain the structure of the organelle and facilitate efficient trafficking of cargo. Interestingly, mutation of two golgins, GMAP-210 and GORAB, causes disease in humans that specifically affect the skin, cartilage and bone, suggesting that golgins are required for optimum modification and trafficking of extracellular matrix proteins, a major secreted cargo in the body. Zebrafish are an excellent model in which to investigate the skin, bone and cartilage, and are amenable to genetic manipulation. We will therefore perform analysis of zebrafish embryos deficient in various golgin proteins. This will be done by morpholino ablation and using recently generated zebrafish mutant lines deficient in several golgins. The project will use a variety of established techniques including zebrafish husbandry, injection of embryos, in situ hybridisation, confocal and electron microscopy, histology, and live imaging of embryos. The results generated during the project will provide important insights into the importance of the golgins for development and physiology in vivo, and reveal how defects in these proteins can lead to human disease.

• Smits, P. et al, (2010). Lethal skeletal dysplasia in mice and humans lacking the golgin GMAP-210. N Engl J Med 362, 206-216.
• Hennies, H.C. et al, (2008). Gerodermia osteodysplastica is caused by mutations in SCYL1BP1, a Rab-6 interacting golgin. Nat Gen 40, 1410-1412.
• Ramirez, I.B. and Lowe, M. (2009). Golgins and GRASPs: holding the Golgi together. Sem Cell Dev Biol 20, 770-779.
• Lieschke, G.J. and Currie, P.D. (2007). Animal models of human disease: Zebrafish swim into view. Nat Rev Gen 8, 353-367.

Proteomics analysis of disease pathways offers a way to decipher the underlying pathogenesis leading to the development of novel drugs for the treatment of the disease. In this project we will exploit the power of proteomics to define the mechanisms responsible for the X-linked disorder Oculocerebrorenal Syndrome of Lowe (OCRL or Lowe syndrome), which is characterised by defects in the eyes, brain, and kidneys. Lowe syndrome is brought about by mutation of OCRL1, a lipid phosphatase that functions in membrane traffic and intracellular signalling. Our recent studies have indicated a role for OCRL1 in recycling within the endocytic pathway, and we would now like to determine the mechanisms involved and the extent to which OCRL1 regulates this process in different cell types. To achieve this aim, protein complexes containing OCRL1 from a variety of human tissues will be analyzed using mass spectrometry for identification of specific binding partners. This will be combined with comparative proteomics of wild-type zebrafish and a mutant lacking OCRL1, allowing us to map the disease pathway for Lowe syndrome. Proteins identified using these approaches will be characterized in detail to decipher how loss of OCRL1 leads to Lowe syndrome, and help design drugs for the treatment of the disorder. The project will use mass spectrometry based proteomics, cell culture, PCR and DNA cloning, and fluorescence and electron microscopy.

• Mann, M. (2006). Functional and quantitative proteomics using SILAC. Nat Rev Mol Cell Biol 12, 952-958.
• Noakes et al, (2011). The PH domain proteins IPIP27A and B link OCRL1 to receptor recycling in the endocytic pathway. Mol Biol Cell 22, 606-623.
• Pirruccello, M. and De Camilli, P. (2012). Inositol 5-phosphatases: insights from the Lowe syndrome protein OCRL. Trends Biochem Sci 37, 134-143.