3. SUMMARY Background: The origin and genesis of the autophagosomal membrane is still a matter of debate. There are indications that the phospholipid phosphatidylinositol 3-phosphate (PI3P) may play a pivotal role in early formative stages, by acting as a recruiter of secondary proteins containing PI3P binding FYVE domains (including DFCP1). This project seeks to investigate the role of PI3P in autophagy; where it is located, when it is needed, and how autophagy (as measured by readouts of the autophagosome marker protein LC3) can be regulated by varying levels of PI3P. Methods: Confocal fluorescence microscopy, Live Cell Imaging, Fixed cell imaging/ Immunofluorescence(IF), standard biochemical assays, Western blots etc. Cloning methods are used to attach fluorescent markers (GFP, RFP etc) to proteins containing FYVE domains (2XFYVE, DFCP1 etc) and LC3. Their movement and localisation, colocalisation etc. is tracked by live imaging and fixed cell immunofluorescence (IF) using Zeiss Confocal 710 and 780 microscopes, as well as wide field Deltavision microscopy. Autophagy in cells (mainly hTERT-RPE1) is induced by incubation in salt solution (EBSS) for approx. 2h. Site-spec ific, inducible depletion of PI3P at the ER-membrane is achieved by using an ER-targeted phosphatase (MTMR1) in a Rapalogue-system. Quantification of autophagic activity is measured by ImageJ on Western Blots against LC3 (main autophagic marker), and by quantifying LC3 spot formation using manual counting and automated ScanR analysis. Results: -Little colocalization is observed between 2XFYVE and LC3 -Starvation induces spot formation of DFCP1 in U2OS and RPE1 cells -DFCP1 and 2XFYVE label distinct PI3P-pools -Overexpression of truncated DFCP1 inhibits autophagy Conclusions and future implications 1) 2XFYVE does not colocalize with autophagy related PI3P-pools 2) DFCP1 and 2XFYVE recognise distinct PI3P pools. 3) Blocking of ER-localised PI3P by overexpression of truncated DFCP1 can reduce autophagy. By using a targeted Rapalogue system, one future aim is to study the different pools of PI3P, i.e. endosomal pool vs ER pool, and find out which pool(s) is necessary for autophagy, and to what extent. If one could find ways to inhibit autophagy by specifically altering autophagic PI3P, this could have implications ranging from cancer therapy to optimal industrial production of recombinant proteins.