The Phosphoinositide Kinase PIKfyve Is Vital in Early Embryonic Development

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.Reversible phosphorylation by kinases and phosphatases at positions 3, 4, and/or 5 of the inositol head group in phosphatidylinositol (PtdIns) 2 generates a family of seven phosphoinositide species (1-6). They all are now found to function as versatile membrane-anchored signals that control diverse and essential cellular processes. Consequently, mutations in the genes encoding the phosphoinositide-metabolizing enzymes are associated with an increasing number of human diseases (1-6). The mammalian enzyme that makes PtdIns(3,5)P 2 from PtdIns3P and PtdIns5P from PtdIns is PIKfyve (7,8). It is an evolutionarily conserved large protein of ϳ230 kDa, a product of a single gene in the animal kingdom, whose function is required for proper performance of the endosomal system and certain signaling pathways (9 -11). PIKfyve harbors a PtdIns3P-binding module, i.e. the FYVE finger domain that associates with the PtdIns3P-enriched endosomal membranes, assuring a rapid PIKfyve recruitment to this low abundance substrate of its catalytic activity (12). PIKfyve interacts physically or functionally with multiple partner proteins; the ones involved in PtdIns(3,5)P 2 homeostasis are the best studied (9). Thus, PIKfyve physically associates with the antagonistic enzyme, i.e. the PtdIns(3,5)P 2 -specific phosphatase Sac3, which ...

Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence

“…Disruption of PIKfyve activity causes several major physiological defects including neurological decay, lethal embryonic development, and inflammatory disease, among others (Ikonomov et al, 2011;Min et al, 2014;Chow et al, 2007;McCartney et al, 2014). It is unclear how these defects arise, but they likely relate to disruption of endo/lysosome homeostasis, including impaired hydrolytic function, reduced rates of autophagic flux, and endo/lysosome swelling (Martin et al, 2013;de Lartigue et al, 2009;Min et al, 2014;Kim et al, 2016).…”

“…Inactivation of PIKfyve or of its regulators, Vac14/ArPIKfyve and Fig4/Sac3, causes a variety of physiological problems including embryonic lethality, neurodegeneration and immune malfunction (Cai et al, 2013;Chow et al, 2007;Jin et al, 2008;Ho et al, 2012;Mccartney et al, 2014;Sbrissa et al, 2007;Ferguson et al, 2010;Min et al, 2014;Lenk et al, 2016;Ikonomov et al, 2011). At the cellular level, these defects relate to impaired autophagic flux, altered delivery to lysosomes, dysregulation of lysosomal Ca 2+ transport, and massive enlargement of lysosomes (Ferguson et al, 2009;Dong et al, 2010;de Lartigue et al, 2009;Ferguson et al, 2010;Kim et al, 2014;Ho et al, 2012;Mccartney et al, 2014;Kim et al, 2016).…”

Class III PI 3-kinase is the main source of PtdIns3P substrate and membrane recruitment signal for PIKfyve constitutive function in podocyte endomembrane homeostasis

“…Pikfyve and Vps34 ( fl/fl or wt/wt ) MEFs were isolated from mouse E12.5 embryos derived from the transgenic models (14,33) following previously described procedures (14). Glomerular cells were isolated from kidneys of Vps34 fl/fl mice (33).…”

“…PIKfyve controls both constitutive and regulated endocytic trafficking as evidenced by the presence of its lipid products in quiescent cells and their elevation upon activation of endosome dynamics by cues such as insulin, EGF or viral infection (2–8). A hallmark of PIKfyve cellular inactivation, achieved by means of PIKfyve dominant-negative kinase-deficient mutants (9,10), pharmacological inhibition (5,11,12), siRNA-mediated silencing (13) or Cre-mediated gene disruption (14,15), is progressive dilation of endosomal membranes, culminating in the appearance of multiple translucent cytoplasmic vacuoles readily seen by light microscopy in cells grown in complete media. Because the vacuoles are selectively rescued by cytoplasmic microinjections of PtdIns(3,5)P 2 but not PtdIns5P (10), the aberrant phenotype has been widely used as a sensitive intracellular indicator for selective PIKfyve-dependent PtdIns(3,5)P 2 deficiency [reviewed in (1,2)].…”

PIKfyve Sensitivity of hERG Channels

“…Lack of PIKfyve is apparently not compatible with survival, an observation highlighting the functional significance of PIKfyve-dependent regulation [58]. PIKfyve is obviously required for early embryonic development [58].…”