Genome-wide Quantitative Imaging of Membrane Traffic Pathways in Mammalian Cells

Abstract

In cells molecules are transported between membrane-bounded compartments by the process of membrane traffic. Due to the exquisite morphology and distribution of organelles in mammalian cells, imaging approaches are highly suited to the study of these transport pathways. Broadly, membrane traffic pathways con be categorised as secretory (endoplasmic reticulum to cell surface), endocytic (cell surface to endosomal-lysosomal system) and retrograde (endosome to endoplasmic reticulum). We are using a combination of genome-wide RNA interference and automated high content screening microscopy to systematically dissect each of these pathways, and quantify the relevance of all genes to them. Of particular interest is the identification of regulatory networks that span all three pathways. In addition, we are using the information from these screens to understand the mechanism by which synthetic nanoparticles interact with cells and then enter them. This information is fundamental to our design of next generation drug delivery vectors. Results from our screens indicate a surprising level of pathway complexity, and links to families of proteins never previously anticipated. Our work also highlights the power of quantitative analysis of organelle morphology, particularly using image texture features, as a tool to identify subtle regulatory machinery. We believe that our analyses will ultimately provide a complete systems-level view of how the endomembrane system is organised in mammalian cells. Stadler C, Rexhepaj E, Singan VR, Murphy RF, Pepperkok R, Uhlen M, Simpson JC & Lundberg E (2013). Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells. Nature Methods 10(4), 315-323. Simpson JC, Joggerst B, Laketa V, Verissimo F, Cetin C, Erfle H, Bexiga MG, Singan VR, Heriche J-K, Neumann B, Mateos A, Blake J, Bechtel S, Benes V, Wiemann S, Ellenberg J & Pepperkok R (2012). Genome-wide RNAi screening identifies human proteins with a function in the early secretory pathway. Nature Cell Biol. 14, 764-774.