regulation PtdIns(3,5)P2 regulates endosomal operations (fission and fusion) that maintain endomembrane homeostasis and proper performance of the trafficking pathways emanating from or traversing endosomes. Decrease of PtdIns(3,5)P2 levels upon perturbations of cellular PIKfyve by heterologous expression of enzymatically inactive PIKfyve point mutants, siRNA-medicated silencing, pharmacological inhibition and PIKFYVE knockout all cause formation of multiple cytosolic vacuoles, which become larger over time. Importantly, the vacuolation induced by PIKfyve dysfunction and PtdIns(3,5)P2 depletion is reversible and could be selectively rescued by cytosolic microinjection of PtdIns(3,5)P2, overexpression of PIKfyve or wash-out of the PIKfyve inhibitor YM201636. Sac3 phosphatase activity in the PAS complex also plays an important role in regulating PtdIns(3,5)P2 levels and maintaining endomembrane homeostasis. Thus, cytoplasmic vacuolation induced by the dominant-negative PIKfyveK1831E mutant is suppressed upon co-expression of a Sac3 phosphatase-inactive point-mutant along with ArPIKfyve. In vitro reconstitution assays of endosome fusion and multivesicular body (MVB) formation/detachment (fission) suggest a positive role of PtdIns(3,5)P2 in MVB fission from maturing early endosomes and a negative role in endosome fusion. PtdIns(3,5)P2 is implicated in the microtubule-dependent retrograde transport from early/late endosomes to the trans Golgi network. Acute insulin treatment increases PtdIns(3,5)P2 levels in 3T3L1 adipocytes, both in isolated membranes and intact cells to promote insulin effect on GLUT4 cell surface translocation and glucose transport. These cells also show a marked PtdIns(3,5)P2 increase upon hyperosmotic shock. Other stimuli, including mitogenic signals such as IL-2 and UV light in lymphocytes, activation of protein kinase C by PMA in platelets and EGF stimulation of COS cells, also increase PtdIns(3,5)P2 levels. PtdIns(3,5)P2 plays a key role in growth and development as evidenced by the preimplantation lethality of the PIKfyve knockout mouse model. The fact that the heterozygous PIKfyve mice are ostensibly normal and live to late adulthood with only ~60% of the wild-type PtdIns(3,5)P2 levels suggests that PtdIns(3,5)P2 might normally be in excess. ArPIKfyve/Vac14 or Sac3/Fig4 knockout in mice results in a 30-50% decrease in PtdIns(3,5)P2 levels and cause similar massive central neurodegeneration and peripheral neuropathy. These studies suggest that reduced PtdIns(3,5)P2 levels, by a yet-to-be identified mechanism, mediate neuronal death. In contrast, MTMR2 phosphatase knockout, which also causes peripheral neuropathy, is accompanied by elevation in PtdIns(3,5)P2. Thus, whether and how the abnormal levels of PtdIns(3,5)P2 selectively affect peripheral neuronal functions remains unclear. Effectors Phosphoinositides are generally viewed as membrane-anchored signals recruiting specific cytosolic effector proteins. So far, several proteins have been proposed as potential PtdIns(3,5)P2 effectors.