PI(3,5)P2'nun mitoz boyunca analizi

Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) is a low abundant phosphatidylinositol derivative involved in several signaling and regulatory pathways such as membrane fusion/fission, Ca+2 signaling, vacuolar acidification, selective and bulk autophagy, stress response, Multi vesicular body (MVB) pathway and RNA granule transportation . Previous genetic screens in budding yeast identified upstream regulators of PI(3,5)P2 which are VAC7, VAC14 and FAB1 among genes necessary for growth of mitotic exit defective cells, which suggest a novel role for PI(3,5)P2 in mitosis, specifically during mitotic exit. In this thesis we aimed investigating the interplay between mitosis and PI(3,5)P2 .We first asked whether PI(3,5)P2 synthesis is regulated by the cell cycle. To this end, we analyzed localization and levels of Atg18, a known PI(3,5)P2 effector, throughout the cell cycle by using fluorescence live cell microscopy. Atg18-GFP localized to the vacuole membrane dependent on PI(3,5)P2 and Vac7. We showed that Atg18-GFP predominantly localized at the periphery of the daughter vacuole rather than mother vacuole during mitosis. We next asked which proteins PI(3,5)P2 interacts with during mitosis. We employed a pull-down approach using mitotic cell extracts and PI(3,5)P2 coated beads. Net1, a nucleolar protein that binds the mitotic exit triggering phosphatase Cdc14, was pulled down with PI(3,5)P2. In addition, several ribosomal and rRNA related proteins such as; RPS11B, RPL24A, RPL5, NSR1, RVB2 came out as hits of our assay. Taken together, daughter specific localization of Atg18 and thus PI(3,5)P2 may indicate a daughter-specific role in the asymmetric cell division of budding yeast. In addition, potential PI(3,5)P2 interactors may be the link to the functions of PI(3,5)P2 in mitotic exit, rRNA granule transportation and translation. Further studies such as using Western Blotting approaches, lipid strips, colocalization assays and growth assays will reveal the functional significance of the asymmetric PI(3,5)P2 synthesis as well as the specificity and significance of identified novel PI(3,5)P2 interactors.