Autophagy in Health, Disease and Longevity
Autophagy is an evolutionary conserved eukaryotic bulk degradation pathway that results in regulated cellular clearance and secures cellular survival. Autophagic dysfunction has been found to be associated with various human diseases including cancer and neurodegeneration. Aiming to develop therapeutics capable to cure age-related human diseases, the molecular details of autophagy are now becoming understood.
We identified the human WIPI gene family (WIPI-1, -2, -3, and -4) and have begun to establish that WIPI genes are aberrantly expressed in a variety of human cancers (Proikas-Cezanne et al., Oncogene 2004). Using biochemical techniques coupled with confocal and electron microscopy we have demonstrated that WIPI-1 (Atg18 in S. cerevisiae) specifically binds PI(3)P at the onset of autophagy and is essential for autophagosome formation. Upon binding to PI(3)P, WIPI-1 protein accumulation at autophagosomal membranes (WIPI-1 puncta-formation) can be visualized by fluorescent microscopy, representing a novel monitoring system for mammalian autophagy.
We have started to employ the WIPI-1 puncta-formation assay for automated high content screening purposes and found that this assay is suitable for drug and siRNA screening approaches, as well as for high through-put analyses of mammalian autophagy. Currently, we are assigning a biological function to all of the different human WIPI family members and wish to unravel the signaling network regulating WIPI-meditaed autophagy as well as the WIPI interactome. We aim to target the autophagic activity in human diseases by modulating WIPI gene expression and WIPI protein activity.
Established methods and competences
WIPI puncta-formation assay, mammalian autophagy assays, longevity assays, fluorescence-based automated high content and high through-put image aqusition and analyses, confocal laser microscopy, in vivo cell imaging, protein-lipid binding assay, yeast 2-hybrid screening, no-hybrids screening, 2-dimensional phospho-peptide mapping and phosphoamino acid analyses, immunoprecipitation, in vitro protein kinase assays, protein purification, cDNA library generation, site-directed mutagenesis, gene silencing.
Primary mouse and human cells, human tumor cell lines, C. elegans, M. musculus (in cooperations), S. cerevisiae (in cooperations), human iPS (in cooperation).