High content/throughput microscopy identifies novel networks in membrane traffic and organelle biogenesis

The secretory pathway in mammalian cells has evolved to facilitate protein secretion and the transfer of a huge variety of cargo molecules to internal and cell surface membranes in a specific and timely manner. Extensive genetic and biochemical efforts over many years have identified and characterised much of the core machinery of this pathway. However, it is clear that regulatory components and their inter-dependence on other cellular metabolic events and pathways need to be elucidated to reach a more comprehensive understanding of the secretory pathway. In order to identify putative molecules involved in in the regulation of the secretory pathway and organelle biogenesis, we have developed and applied functional assays to assess the effect of knock-ins by cDNA over-expression and knock-downs by RNAi, on processes such as constitutive protein transport, Golgi integrity and function of vesicular coat complexes. In order to achieve the throughput that such analyses require we have developed a fully automated high content screening microscopy platform including sample preparation, image acquisition and automated analysis of complex cellular phenotypes. We have applied this technology to genome-wide siRNA screens to identify and characterize comprehensively the genes and their underlying functional networks involved in the regulation of secretory membrane traffic and organelle biogenesis. Our data reveal an unexpected relationship between secretory pathway function and genes involved in general metabolic integrity. This provides the basis for an integrative understanding of the global cellular organisation and the regulation of the secretory pathway.