Abstract
Gene mutations in the phosphoinositide-metabolizing enzymes are linked to various human diseases. In mammals, PIKfyve synthesizes PtdIns(3,5)P(2) and PtdIns5P lipids that regulate endosomal trafficking and responses to extracellular stimuli. The consequence of pikfyve gene ablation in mammals is unknown. To clarify the importance of PIKfyve and PIKfyve lipid products, in this study, we have characterized the first mouse model with global deletion of the pikfyve gene using the Cre-loxP approach. We report that nearly all PIKfyve(KO/KO) mutant embryos died before the 32-64-cell stage. Cultured fibroblasts derived from PIKfyve(flox/flox) embryos and rendered pikfyve-null by Cre recombinase expression displayed severely reduced DNA synthesis, consistent with impaired cell division causing early embryo lethality. The heterozygous PIKfyve(WT/KO) mice were born at the expected Mendelian ratio and developed into adulthood. PIKfyve(WT/KO) mice were ostensibly normal by several other in vivo, ex vivo, and in vitro criteria despite the fact that their levels of the PIKfyve protein and in vitro enzymatic activity in cells and tissues were 50-55% lower than those of wild-type mice. Consistently, steady-state levels of the PIKfyve products PtdIns(3,5)P(2) and PtdIns5P selectively decreased, but this reduction (35-40%) was 10-15% less than that expected based on PIKfyve protein reduction. The nonlinear decrease of the PIKfyve protein versus PIKfyve lipid products, the potential mechanism(s) discussed herein, may explain how one functional allele in PIKfyve(WT/KO) mice is able to support the demands for PtdIns(3,5)P(2)/PtdIns5P synthesis during life. Our data also shed light on the known human disorder linked to PIKFYVE mutations.
Highlights
Gene mutations in the phosphoinositide-metabolizing enzymes are linked to various human diseases
We demonstrate here for the first time that whereas the PIKfyve-null embryos die during preimplantation development, the heterozygous mice appear without visible defects from birth to late adulthood despite the lack of translational compensation for the haploinsufficiency of the PIKfyve protein
Reduced Formation of PIKfyve-ArPIKfyve-Sac[3] (PAS) Regulatory Complexes in PIKfyveWT/KO—We have recently revealed that PIKfyve associates with the ArPIKfyve scaffold and Sac[3] phosphatase in a regulatory ternary assembly, called the PAS complex, that relays two opposing enzymatic activities, synthesis and breakdown of PtdIns(3,5)P2 (17, 18)
Summary
PIKfyve binds efficiently only to the ArPIKfyve-Sac[3] complex to form a triple heterooligomer in which Sac[3] fulfills a dual function (17, 18) Does it turn over PtdIns(3,5)P2 at the site of production, but together with ArPIKfyve, it secures robust PtdIns(3,5)P2 synthesis through activating PIKfyve (17, 18). Lethality of PIKfyve؊/؊ Embryos but Normality of PIKfyve؉/؊ Mice under loss of the PtdIns(3,5)P2-synthesizing activity of PIKfyve, achieved through PIKfyve dominant-negative mutation (9), pharmacological inhibition (19), or RNA interference (20, 21), is aberrant endomembrane swelling and vacuolation, which are progressively exacerbated with the dose/time of treatments. To address the requirement of PIKfyve for life in mammals and plausible compensatory production of PtdIns(3,5)P2 and/or PtdIns5P steady-state levels, we generated a murine model with inactivation of the pikfyve gene using the Cre-loxP approach. Our study is the first genetic evidence for the essential and nonredundant function of PIKfyve in cell viability and early embryonic development of mammals
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