Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] hydrolysis by phospholipase C (PLC) is a conserved mechanism of signalling. Given the low abundance of PI(4,5)P2, its hydrolysis needs to be coupled to resynthesis to ensure continued PLC activity; however, the mechanism by which depletion is coupled to resynthesis remains unknown. PI(4,5)P2 synthesis is catalyzed by the phosphorylation of phosphatidylinositol 4 phosphate (PI4P) by phosphatidylinositol 4 phosphate 5 kinase (PIP5K). In Drosophila photoreceptors, photon absorption is transduced into PLC activity and during this process, PI(4,5)P2 is resynthesized by a PIP5K. However, the mechanism by which PIP5K activity is coupled to PI(4,5)P2 hydrolysis is unknown. In this study, we identify a unique isoform dPIP5KL, that is both necessary and sufficient to mediate PI(4,5)P2 synthesis during phototransduction. Depletion of PNUT, a non-redundant subunit of the septin family, enhances dPIP5KL activity in vitro and PI(4,5)P2 resynthesis in vivo; co-depletion of dPIP5KL reverses the enhanced rate of PI(4,5)P2 resynthesis in vivo. Thus, our work defines a septin-mediated mechanism through which PIP5K activity is coupled to PLC-mediated PI(4,5)P2 hydrolysis.

Lowe syndrome is an X-linked recessive monogenic disorder resulting from mutations in the OCRL gene that encodes a phosphatidylinositol 4,5 bisphosphate 5-phosphatase. The disease affects three organs-the kidney, brain and eye and clinically manifests as proximal renal tubule dysfunction, neurodevelopmental delay and congenital cataract. Although Lowe syndrome is a monogenic disorder, there is considerable heterogeneity in clinical presentation; some individuals show primarily renal symptoms with minimal neurodevelopmental impact whereas others show neurodevelopmental defect with minimal renal symptoms. However, the molecular and cellular mechanisms underlying this clinical heterogeneity remain unknown. Here we analyze a Lowe syndrome family in whom affected members show clinical heterogeneity with respect to the neurodevelopmental phenotype despite carrying an identical mutation in the OCRL gene. Genome sequencing and variant analysis in this family identified a large number of damaging variants in each patient. Using novel analytical pipelines and segregation analysis we prioritize variants uniquely present in the patient with the severe neurodevelopmental phenotype compared to those with milder clinical features. The identity of genes carrying such variants underscore the role of additional gene products enriched in the brain or highly expressed during brain development that may be determinants of the neurodevelopmental phenotype in Lowe syndrome. We also identify a heterozygous variant in CEP290, previously implicated in ciliopathies that underscores the potential role of OCRL in regulating ciliary function that may impact brain development. More generally, our findings demonstrate analytic approaches to identify high-confidence genetic variants that could underpin the phenotypic heterogeneity observed in monogenic disorders.

The hydrolysis of phosphatidylinositol 4,5‐ bisphosphate [PI(4,5)P2] at the plasma membrane by receptor activated phospholipase C (PLC) activity is a conserved mechanism of signal transduction. Given the low abundance of PI(4,5)P2 at the plasma membrane, its hydrolysis needs to be coupled to lipid resynthesis to ensure continued PLC activity during receptor activation. However, the mechanism by which PI(4,5)P2depletion during signalling is coupled to its resynthesis remains unknown. PI(4,5)P2synthesis is catalyzed by lipid kinase activity and the phosphorylation of phosphatidylinositol 4 phosphate (PI4P) by phosphatidylinositol 4 phosphate 5 kinase (PIP5K) is the final step in this process. In Drosophila photoreceptors, sensory transduction of photon absorption is transduced into PLC activity leading to an electrical response to light. During this process, PI(4,5)P2is resynthesized by a PIP5K activity but the mechanism by which the activity of this enzyme is coupled to PLC signalling is not known. In this study, we identify a unique protein isoform of dPIP5K,dPIP5KL that is both necessary and sufficient to mediate PI(4,5)P2synthesis during phototransduction. The activity of dPIP5KLin vitrois enhanced by depletion of PNUT, a non‐redundant subunit of the septin family of GTP binding proteins and in vivo, depletion of dPIP5KL rescues the effect of pnut depletion on the light response and PI(4,5)P2resynthesis during PLC signalling. Thus, our work defines a septin 7 mediated mechanism through which PIP5K activity is coupled to ongoing PLC mediated PI(4,5)P2 depletion.

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The hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] at the plasma membrane by receptor activated phospholipase C (PLC) activity is a conserved mechanism of signal transduction. Given the low abundance of PI(4,5)P2 at the plasma membrane, its hydrolysis needs to be coupled to lipid resynthesis to ensure continued PLC activity during receptor activation. However, the mechanism by which PI(4,5)P2 depletion during signalling is coupled to its resynthesis remains unknown. PI(4,5)P2 synthesis is catalyzed by lipid kinase activity and the phosphorylation of phosphatidylinositol 4 phosphate (PI4P) by phosphatidylinositol 4 phosphate 5 kinase (PIP5K) is the final step in this process. In Drosophila photoreceptors, sensory transduction of photon absorption is transduced into PLC activity leading to an electrical response to light. During this process, PI(4,5)P2 is resynthesized by a PIP5K activity but the mechanism by which the activity of this enzyme is coupled to PLC signalling is not known. In this study, we identify a unique protein isoform of dPIP5K, dPIP5KL that is both necessary and sufficient to mediate PI(4,5)P2 synthesis during phototransduction. The activity of dPIP5KL in vitro is enhanced by depletion of PNUT, a non-redundant subunit of the septin family of GTP binding proteins and in vivo, depletion of pnut rescues the effect of dPIP5KL depletion on the light response and PI(4,5)P2 resynthesis during PLC signalling. Lastly we find that depletion of Septin Interacting Protein 1 (Sip1),previously shown to bind PNUT, phenocopies the effect of dPIP5KL depletion in vivo. Thus, our work defines a septin 7 and Sip1 mediated mechanism through which PIP5K activity is coupled to ongoing PLC mediated PI(4,5)P2 depletion.