BBB Seminar: Rainer Pepperkok
High throughput microscopy to study membrane traffic and disease mechanisms
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
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 characterized 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 such putative molecules involved 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, integrity of cellular organelles and function of vesicular coat complexes. In order to achieve the throughput that such analyses require we have developed a high content/throughput microscopy platform automating and integrating all steps in microscope-based experiments including sample preparation, image acquisition and automated analysis of complex cellular phenotypes.
The technology also allows automated performance of complex imaging protocols such as FRAP (fluorescence recovery after photobleaching) or FCCS (fluorescence cross-correlation spectroscopy) experiments at high throughput.
Our data reveal an unexpected relationship between secretory pathway function and genes involved in general metabolic integrity or signal transduction pathways. This provides the basis for an integrative understanding of the global cellular organization and the regulation of the secretory pathway and organelle biogenesis.
We have also used high throughput microscopy technology to investigate, if and how trafficking factors play a role in cholesterol homeostasis and/or the processing, transport and function of disease relevant cargo, such as the ion channels CFTR or ENaC which play a key role in cystic fibrosis.
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Host: Hans-Hermann Gerdes , Department of Biomedicine