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Abstract Voltage-sensing phosphatase (VSP) exhibits voltage-dependent phosphatase activity toward phosphoinositides. VSP generates a specialized phosphoinositide environment in mammalian sperm flagellum. However, the voltage-sensing mechanism of VSP in spermatozoa is not yet characterized. Here, we found that VSP is activated during sperm maturation, indicating that electric signals in immature spermatozoa are essential. Using a heterologous expression system, we show the voltage-sensing property of mouse VSP (mVSP). The voltage-sensing threshold of mVSP is approximately −30 mV, which is sensitive enough to activate mVSP in immature spermatozoa. We also report several knock-in mice in which we manipulate the voltage-sensitivity or electrochemical coupling of mVSP. Notably, the V312R mutant, with a minor voltage-sensitivity change, exhibits abnormal sperm motility after, but not before, capacitation. Additionally, the V312R mutant shows a significant change in the acyl-chain profile of phosphoinositide. Our findings suggest that electrical signals during sperm maturation are crucial for establishing the optimal phosphoinositide environment in spermatozoa.

Different phosphoinositide isomers are involved in a variety of physiological and pathological processes. Here, the authors combine chiral column chromatography and mass spectrometry to measure phosphoinositide regioisomers, revealing their dynamic changes in intra- and extracellular cancer cell milieus.

Abstract Purpose Penile research is expected to reveal new targets for treatment and prevention of the complex mechanisms of its disorder including erectile dysfunction (ED). Thus, analyses of the molecular processes of penile ED and continuous erection as priapism are essential issues of reproductive medicine. Methods By performing mouse N‐ethyl‐N‐nitrosourea mutagenesis and exome sequencing, we established a novel mouse line displaying protruded genitalia phenotype (PGP; priapism‐like phenotype) and identified a novel Pitpna gene mutation for PGP. Extensive histological analyses on the Pitpna mutant and intracavernous pressure measurement (ICP) and liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI/MS)/MS analyses were performed. Results We evaluated the role of phospholipids during erection for the first time and showed the mutants of inducible phenotypes of priapism. Moreover, quantitative analysis using LC–ESI/MS/MS revealed that the level of phosphatidylinositol (PI) was significantly lower in the mutant penile samples. These results imply that PI may contribute to penile erection by PITPα. Conclusions Our findings suggest that the current mutant is a mouse model for priapism and abnormalities in PI signaling pathways through PITPα may lead to priapism providing an attractive novel therapeutic target in its treatment.

Phagocytosis is a highly conserved process whereby phagocytic cells engulf pathogens and apoptotic bodies. The present study focused on the role of inositol polyphosphate-4-phosphatase type I (Inpp4a) in phagocytosis. Raw264.7 cells that express shRNA against Inpp4a (shInpp4a cells) showed significantly increased phagocytic activity. The introduction of shRNA-resistant human Inpp4a abolished this increase. Macrophages from Inpp4a knockout mice showed similar increases in the phagocytic activity. Inpp4a was recruited to the phagosome membrane by a mechanism other than the direct interaction with Rab5. PtdIns(3,4)P2 increased on the phagosome of shInpp4a cells, while PtdIns(3)P significantly decreased. The results indicate that Inpp4a negatively regulates the phagocytic activity of macrophages as a member of the sequential dephosphorylation system that metabolizes phagosomal PtdIns(3,4,5)P3 to PtdIns(3)P.

The voltage-sensing phosphatase (VSP) is a unique protein that shows voltage-dependent phosphoinositide phosphatase activity. Here we report that VSP is activated in mice sperm flagellum and generates a unique subcellular distribution pattern of PtdIns(4,5)P2. Sperm from VSP-/- mice show more Ca2+ influx upon capacitation than VSP+/- mice and abnormal circular motion. VSP-deficient sperm showed enhanced activity of Slo3, a PtdIns(4,5)P2-sensitive K+ channel, which selectively localizes to the principal piece of the flagellum and indirectly enhances Ca2+ influx. Most interestingly, freeze-fracture electron microscopy analysis indicates that normal sperm have much less PtdIns(4,5)P2 in the principal piece than in the midpiece of the flagellum, and this polarized PtdIns(4,5)P2 distribution disappeared in VSP-deficient sperm. Thus, VSP appears to optimize PtdIns(4,5)P2 distribution of the principal piece. These results imply that flagellar PtdIns(4,5)P2 distribution plays important roles in ion channel regulation as well as sperm motility. [ABSTRACT FROM AUTHOR]

TMEM55a is an enzyme that dephosphorylates phosphatidylinositol (PtdIns) (4,5)P2 to form PtdIns(5)P in vitro. However, the in vivo conversion of the polyphosphoinositide to PtdIns(5)P by the phosphatase has not yet been demonstrated, and its role remains poorly understood. Mouse macrophages (Raw264.7) deficient in TMEM55a showed an increased engulfment of large particles without affecting the phagocytosis of Escherichia coli. Transfection of a bacterial phosphatase with similar substrate specificity to TMEM55a, IpgD to Raw264.7 cells, inhibited the engulfment of IgG-erythrocytes in a manner dependent on its phosphatase activity. In contrast, cells transfected with PIP4K2a, which catalyzes PtdIns(4,5)P2 production from PtdIns(5)P, increased phagocytosis. Fluorescent TMEM55a transfected into Raw264.7 cells substantially localized to the phagosome. The accumulation of PtdIns(4,5)P2, PtdIns(3,4,5)P3 and F-actin on the phagocytic cup is increased in TMEM55a deficient cells, as monitored by live-cell imaging. Phagosomal PtdIns(5)P was decreased in the knock down cells, but the augmentation of phagocytosis in these cells was unaffected by the exogenous addition of PtdIns(5)P. Together, these results suggest that TMEM55a negatively regulates the phagocytosis of large particles by reducing phagosomal PtdIns(4,5)P2 accumulation during cup formation. [ABSTRACT FROM AUTHOR]

Voltage-sensing phosphatase (VSP) exhibits voltage-dependent phosphatase activity toward phosphoinositides. VSP generates a specialized phosphoinositide environment in mammalian sperm flagellum. However, the voltage-sensing mechanism of VSP in spermatozoa is not yet characterized. Here, we found that VSP is activated during sperm maturation, indicating that electric signals in immature spermatozoa are essential. Using a heterologous expression system, we show the voltage-sensing property of mouse VSP (mVSP). The voltage-sensing threshold of mVSP is approximately −30 mV, which is sensitive enough to activate mVSP in immature spermatozoa. We also report several knock-in mice in which we manipulate the voltage-sensitivity or electrochemical coupling of mVSP. Notably, the V312R mutant, with a minor voltage-sensitivity change, exhibits abnormal sperm motility after, but not before, capacitation. Additionally, the V312R mutant shows a significant change in the acyl-chain profile of phosphoinositide. Our findings suggest that electrical signals during sperm maturation are crucial for establishing the optimal phosphoinositide environment in spermatozoa. Membrane potentials in living cells can function as electrical signals. Here, the authors report that spermatozoa utilize their electrical signals during the maturing stage to create appropriate membrane lipids environment that is important for sperm motility. [ABSTRACT FROM AUTHOR]

Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes. Due to the existence of multiple phosphoinositide regioisomers and their low intracellular concentrations, profiling these lipids and linking a specific acyl variant to a change in biological state have been difficult. To enable the comprehensive analysis of phosphoinositide phosphorylation status and acyl chain identity, we develop PRMC-MS (Phosphoinositide Regioisomer Measurement by Chiral column chromatography and Mass Spectrometry). Using this method, we reveal a severe skewing in acyl chains in phosphoinositides in Pten-deficient prostate cancer tissues, extracellular mobilization of phosphoinositides upon expression of oncogenic PIK3CA, and a unique profile for exosomal phosphoinositides. Thus, our approach allows characterizing the dynamics of phosphoinositide acyl variants in intracellular and extracellular milieus. Different phosphoinositide isomers are involved in a variety of physiological and pathological processes. Here, the authors combine chiral column chromatography and mass spectrometry to measure phosphoinositide regioisomers, revealing their dynamic changes in intra- and extracellular cancer cell milieus. [ABSTRACT FROM AUTHOR]

Purpose: Penile research is expected to reveal new targets for treatment and prevention of the complex mechanisms of its disorder including erectile dysfunction (ED). Thus, analyses of the molecular processes of penile ED and continuous erection as priapism are essential issues of reproductive medicine. Methods: By performing mouse N‐ethyl‐N‐nitrosourea mutagenesis and exome sequencing, we established a novel mouse line displaying protruded genitalia phenotype (PGP; priapism‐like phenotype) and identified a novel Pitpna gene mutation for PGP. Extensive histological analyses on the Pitpna mutant and intracavernous pressure measurement (ICP) and liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI/MS)/MS analyses were performed. Results: We evaluated the role of phospholipids during erection for the first time and showed the mutants of inducible phenotypes of priapism. Moreover, quantitative analysis using LC–ESI/MS/MS revealed that the level of phosphatidylinositol (PI) was significantly lower in the mutant penile samples. These results imply that PI may contribute to penile erection by PITPα. Conclusions: Our findings suggest that the current mutant is a mouse model for priapism and abnormalities in PI signaling pathways through PITPα may lead to priapism providing an attractive novel therapeutic target in its treatment. [ABSTRACT FROM AUTHOR]

Lysophosphatidylinositol‐acyltransferase‐1 (LPIAT1) specifically catalyzes the transfer of arachidonoyl‐CoA to lysophosphoinositides. LPIAT−/− mice have been shown to have severe defects in the brain and liver; however, the exact molecular mechanisms behind these conditions are not well understood. As immune cells have been implicated in liver inflammation based on disfunction of LPIAT1, we generated Raw264.7 macrophages deficient in LPIAT1, using shRNA and CRISPR/Cas9. The amount of C38:4 species in phosphoinositides, especially in PtdInsP2, was remarkably decreased in these cells. Unlike in wild‐type cells, LPIAT1‐deficient cells showed prolonged oscillations of intracellular Ca2+ upon UDP stimulation, which is known to activate phospholipase Cβ through the Gq‐coupled P2Y6 receptor, even in the absence of extracellular Ca2+. It is speculated that the prolonged Ca2+ response may be relevant to the increased risk of liver inflammation induced by LPIAT1 disfunction. [ABSTRACT FROM AUTHOR]

The relative abundance of phosphoinositide (PI) species on the phagosome membrane fluctuates over the course of phagocytosis. PtdIns(3,4,5)P³ and PtdIns(3,4)P² rapidly increase in the forming of the phagocytic cup, following which they disappear after sealing of the cup. In the present study, we monitored the clearance of these PI species using the enhanced green fluorescent protein-fused pleckstrin homology domain of Akt, a fluorescence probe that binds both PtdIns(3,4,5)P³ and PtdIns(3,4)P² in Raw 264.7 macrophages. The clearance of PIs was much faster when the phagocytosed particles were coated with IgG. The effect of IgG was not observed in the macrophages deficient in FcγRIIb, an inhibitory IgG receptor. To identify the lipid phosphatases responsible for the FcγRIIb-accelerated PI clearance, we prepared a panel of lipid phosphatase-deficient cells. The lack of a PI 5-phosphatase Src homology 2 domain-containing inositol-5-phosphatase (SHIP)1 or SHIP² impaired the FcγRIIb-accelerated clearance of PIs. The lack of a PI 4-phosphatase Inpp4a also impaired the accelerated PIs clearance. In the FcγRIIb- and Inpp4a-deficient cells, acidification of the formed phagosome was slowed. These results suggested that FcγRIIb drives the sequential dephosphorylation system comprising SHIPs and Inpp4a, and accelerates phagosome acidification. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 3-kinase (PI3K)/Akt signaling has been implicated in the anti-inflammatory response in a mouse model of endotoxemia and sepsis. The present study focused on the role of inositol polyphosphate-4-phosphatase type I (Inpp4a), which dephosphorylates PtdIns(3,4)P2 to PtdIns(3)P, in bacterial infections. We prepared myeloid cell-specific Inpp4a-conditional knockout mice. Macrophages from these mice showed increased Akt phosphorylation and reduced production of inflammatory cytokines in response to LPS or Escherichia coli in vitro. The Inpp4a knockout mice survived for a shorter time than wild type mice after i.p. infection with E. coli, with less production of inflammatory cytokines. Additionally, E. coli clearance from blood and lung was significantly impaired in the knockout mice. A likely mechanism is that the Inpp4a-catalyzed dephosphorylation of PtdIns(3,4)P2 down-regulates Akt pathways, which, in turn, increases the production of inflammatory mediators. This mechanism at least fits the decreased E. coli clearance and short survival in the Inpp4a knockout mice. [ABSTRACT FROM AUTHOR]

Phosphoinositide 5'-phosphatases have been implicated in the regulation of phagocytosis. However, their precise roles in the phagocytic process are poorly understood. We prepared RAW264.7 macrophages deficient in Inpp5e (shInpp5e) to clarify the role of this lipid phosphatase. In the shInpp5e cells, the uptake of solid particles was increased and the rate of phagosome acidification was accelerated. As expected, levels of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 were increased and decreased respectively, on the forming phagocytic cups of these cells. Unexpectedly, the most prominent consequence of the Inpp5e deficiency was the decreased accumulation of PtdIns3P and Rab5 on the phagosome. The expression of a constitutively active form of Rab5b in the shInpp5e cells rescued the PtdIns3P accumulation. Rab20 has been reported to regulate the activity of Rabex5, a guanine nucleotide exchange factor for Rab5. The association of Rab20 with the phagosome was remarkably abrogated in the shInpp5e cells. Over-expression of Rab20 increased phagosomal PtdIns3P accumulation and delayed its elimination. These results suggest that Inpp5e, through functional interactions with Rab20 on the phagosome, activates Rab5, which, in turn, increases PtdIns3P and delays phagosome acidification. [ABSTRACT FROM AUTHOR]