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In most eukaryotes, phosphoinositides (PIs) have crucial roles in multiple cellular functions. Although the cellular levels of phosphatidylinositol 5-phosphate (PI5P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) are extremely low relative to some other PIs, emerging evidence demonstrates that both lipids are crucial for the endocytic pathway, intracellular signaling, and adaptation to stress. Mutations that causes defects in the biosynthesis of PI5P and PI(3,5)P2 are linked to human diseases including neurodegenerative disorders. Here, we review recent findings on cellular roles of PI5P and PI(3,5)P2, as well as the pathophysiological importance of these lipids.Key words: Phosphoinositides, Membrane trafficking, Endocytosis, Vacuoles/Lysosomes, Fab1/PIKfyve

Significance Defects in biosynthesis of the signaling lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P ₂] are associated with profound neurodegeneration and early mortality in both humans and mice. However, surprisingly little is known about the functions of this lipid in cells, including neurons, where its loss has the most dramatic impact. Prompted by the striking localization of mammalian homolog of yeast vacuole segregation mutant (Vac14), part of the PI(3,5)P ₂ synthesis complex, to excitatory synapses, we developed new tools to measure and manipulate PI(3,5)P ₂ synthesis in hippocampal neurons. We find that dynamic changes in PI(3,5)P ₂ synthesis impose bidirectional changes on synaptic strength by regulating AMPA-type glutamate receptor trafficking and that activity-dependent regulation of this lipid is crucial for enduring forms of synaptic depression, findings that implicate PI(3,5)P ₂-dependent signaling as a critical synaptic regulatory pathway. Dynamic regulation of phosphoinositide lipids (PIPs) is crucial for diverse cellular functions, and, in neurons, PIPs regulate membrane trafficking events that control synapse function. Neurons are particularly sensitive to the levels of the low abundant PIP, phosphatidylinositol 3,5-bisphosphate [PI(3,5)P ₂], because mutations in PI(3,5)P ₂-related genes are implicated in multiple neurological disorders, including epilepsy, severe neuropathy, and neurodegeneration. Despite the importance of PI(3,5)P ₂ for neural function, surprisingly little is known about this signaling lipid in neurons, or any cell type. Notably, the mammalian homolog of yeast vacuole segregation mutant (Vac14), a scaffold for the PI(3,5)P ₂ synthesis complex, is concentrated at excitatory synapses, suggesting a potential role for PI(3,5)P ₂ in controlling synapse function and/or plasticity. PI(3,5)P ₂ is generated from phosphatidylinositol 3-phosphate (PI3P) by the lipid kinase PI3P 5-kinase (PIKfyve). Here, we present methods to measure and control PI(3,5)P ₂ synthesis in hippocampal neurons and show that changes in neural activity dynamically regulate the levels of multiple PIPs, with PI(3,5)P ₂ being among the most dynamic. The levels of PI(3,5)P ₂ in neurons increased during two distinct forms of synaptic depression, and inhibition of PIKfyve activity prevented or reversed induction of synaptic weakening. Moreover, altering neuronal PI(3,5)P ₂ levels was sufficient to regulate synaptic strength bidirectionally, with enhanced synaptic function accompanying loss of PI(3,5)P ₂ and reduced synaptic strength following increased PI(3,5)P ₂ levels. Finally, inhibiting PI(3,5)P ₂ synthesis alters endocytosis and recycling of AMPA-type glutamate receptors (AMPARs), implicating PI(3,5)P ₂ dynamics in AMPAR trafficking. Together, these data identify PI(3,5)P ₂-dependent signaling as a regulatory pathway that is critical for activity-dependent changes in synapse strength.

The multiprotein Fab1p/PIKfyve-complex regulating the abundance of the phospholipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P 2 ) is highly conserved among eukaryotes. In yeast/mammals, it is composed of the phosphatidylinositol 3-phosphate 5-kinase Fab1p/PIKfyve, the PtdIns(3,5)P 2 phosphatase Fig4p/Sac3 and the scaffolding subunit Vac14p/ArPIKfyve. The complex is located to vacuolar membranes in yeast and to endosomal membranes in mammals, where it controls the synthesis and turnover of PtdIns(3,5)P 2 . In this study, we analyzed the role and function of the Fab1p/PIKfyve-complex scaffold protein SmVAC14 in the filamentous ascomycete Sordaria macrospora (Sm). We generated the Smvac14 deletion strain ∆vac14 and performed phenotypic analysis of the mutant. Furthermore, we conducted fluorescence microscopic localization studies of fluorescently labeled SmVAC14 with vacuolar and late endosomal marker proteins. Our results revealed that SmVAC14 is important for maintaining vacuolar size and appearance as well as proper sexual development in S. macrospora . In addition, SmVAC14 plays an important role in starvation stress response. Accordingly, our results propose that the turnover of PtdIns(3,5)P 2 is of great significance for developmental processes in filamentous fungi.

The signaling lipid, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂), likely functions in multiple signaling pathways. Here, we report the characterization of a mouse mutant lacking Vac14, a regulator of PI(3,5)P₂ synthesis. The mutant mice exhibit massive neurodegeneration, particularly in the midbrain and in peripheral sensory neurons. Cell bodies of affected neurons are vacuolated, and apparently empty spaces are present in areas where neurons should be present. Similar vacuoles are found in cultured neurons and fibroblasts. Selective membrane trafficking pathways, especially endosome-to-TGN retrograde trafficking, are defective. This report, along with a recent report on a mouse with a null mutation in Fig4, presents the unexpected finding that the housekeeping lipid, PI(3,5)P₂, is critical for the survival of neural cells.

Lysosomes have an important role in cellular protein and organelle quality control, metabolism, and signaling. On the surface of lysosomes, the PIKfyve/Fab1 complex generates phosphatidylinositol 3,5-bisphosphate, PI-3,5-P₂, which is critical for lysosomal membrane homeostasis during acute osmotic stress and for lysosomal signaling. Here, we identify the inverted BAR protein Ivy1 as an inhibitor of the Fab1 complex with a direct influence on PI-3,5-P₂ levels and vacuole homeostasis. Ivy1 requires Ypt7 binding for its function, binds PI-3,5-P₂, and interacts with the Fab1 kinase. Colocalization of Ivy1 and Fab1 is lost during osmotic stress. In agreement with Ivy1’s role as a Fab1 regulator, its overexpression blocks Fab1 activity during osmotic shock and vacuole fragmentation. Conversely, loss of Ivy1, or lateral relocalization of Ivy1 on vacuoles away from Fab1, results in vacuole fragmentation and poor growth. Our data suggest that Ivy1 modulates Fab1-mediated PI-3,5-P₂ synthesis during membrane stress and may allow adjustment of the vacuole membrane environment.

In yeast and animal cells, phosphatidylinositol‐3‐monophosphate 5‐kinases produce phosphatidylinositol (3,5)‐bisphosphate (PtdIns(3,5)P₂) and have been implicated in endomembrane trafficking and pH control in the vacuole. In plants, PtdIns(3,5)P₂ is synthesized by the Fab1 family, four orthologs of which exist in Arabidopsis: FAB1A and FAB1B, both from the PIKfyve/Fab1 family; FAB1C and FAB1D, both without a PIKfyve domain and of unclear role. Using a reverse genetics and cell biology approach, we investigated the function of the Arabidopsis genes encoding FAB1B and FAB1D, both highly expressed in pollen. Pollen viability, germination and tube morphology were not significantly affected in homozygous mutant plants. In vivo, mutant pollen fertilized ovules leading to normal seeds and siliques. The same result was obtained when mutant ovules were fertilized with wild‐type pollen. Double mutant pollen for the two genes was able to fertilize and develop plants no different from the wild‐type. At the cellular level, fab1b and fab1d pollen tubes were found to exhibit perturbations in membrane recycling, vacuolar acidification and decreased production of reactive oxygen species (ROS). Subcellular imaging of FAB1B‐GFP revealed that the protein localized to the endomembrane compartment, whereas FAB1D‐GFP localized mostly to the cytosol and sperm cells. These results were discussed considering possible complementary roles of FAB1B and FAB1D.