Roles of phosphatidylinositol 4-phosphate in Drosophila larval secretory granule biogenesis. Cheng-I J Ma^1,2, Jason Burgess^2,3, Lauren M Del Bel^2,3, Barbara Barylko⁴, Gordon Polevoy², Janet Rollins⁵, Joseph P Albanesi⁴, Helmut Krämer⁶, Julie A Brill^1,2,3. 1) Institute of Medical Science, University of Toronto, Toronto, ON, Canada; 2) Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada; 3) Department of Molecular Genetics, University of Toronto, ON, Canada; 4) Department of Pharmacology, UT Southwestern Medical Center, Dallas, TX, USA; 5) Division of Natural Sciences, The College of Mount Saint Vincent, Riverdale, NY, USA; 6) Department of Neuroscience, UT Southwestern Medical Center, Dallas, TX, USA.

   Phosphatidylinositol 4-kinases (PI4Ks) are responsible for production of the lipid phosphatidylinositol 4-phosphate (PI4P), a crucial resident of Golgi membranes that regulates membrane trafficking events such as secretion. Interestingly, we have discovered that PI4KII but not fwd function is required for normal development of the Drosophila larval salivary gland. In PI4KII mutants, mucin-containing glue granules were considerably smaller than in wild type. Furthermore, PI4KII formed dynamic tubular networks along microtubules. Portions of these tubules colocalized with late endosomes and lysosomes marked with YFP-Rab7, suggesting these PI4KII positive tubules were dynamic endosomal structures. In PI4KII mutants, these tubular structures were absent and enlarged lysotracker positive endosomes were observed. These enlarged acidic endosomes contained mucin cargo proteins as well as the glue granule associated SNARE SNAP24. Our data thus far suggested that PI4KII function is required for the proper trafficking of granule proteins, where mistrafficked granule proteins accumulated in the late endosome. To further examine the role of PI4P in granule biogenesis, we are employing a reverse genetic screen to identify potential genetic interactions using publicly available transgenic RNAi lines. A selection of candidate genes including Rabs, SNAREs, and BAR domain containing proteins will be screened to identify players involved in the PI4KII mediated membrane tubulation, fusion and fission. We will also examine which of the known PI4P binding proteins play a role in glue granule biogenesis.