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Guillaume Drin - One of the best experts on this subject based on the ideXlab platform.

  • In Vitro Strategy to Measure Sterol/Phosphatidylinositol-4-Phosphate Exchange Between Membranes
    Intracellular Lipid Transport, 2019
    Co-Authors: Nicolas-frédéric Lipp, Guillaume Drin
    Abstract:

    Recent findings unveiled that Oxysterol-binding protein-related proteins (ORP)/Oxysterol-binding homology (Osh) proteins, which constitute a major family of lipid transfer proteins (LTPs), conserved among eukaryotes, are not all mere sterol transporters or sensors. Indeed, some of them are able to exchange sterol for Phosphatidylinositol-4-Phosphate (PI4P) or phosphatidylserine (PS) for PI4P between membranes and thereby to use PI4P metabolism to generate sterol or PS gradients in the cell, respectively. Here, we describe a full strategy to measure in vitro a sterol/PI4P exchange process between artificial membranes using Förster resonance energy transfer (FRET)-based assays and a standard spectrofluorometer. Such an approach can serve to better characterize the activity of known sterol/PI4P exchangers, but also to reveal whether ill-defined ORP/Osh proteins or LTPs belonging to other families have such an exchange activity. Besides, this protocol is amenable to test whether molecules can act as Orphilins, which have been found to inhibit the sterol/PI4P exchange activity of certain ORPs. Last, our strategy to measure in real-time PI4P transport using a known lipid-binding domain can serve as a basis for the design of novel in vitro protocols aiming to detect other lipid species.

  • in vitro strategy to measure sterol Phosphatidylinositol 4 Phosphate exchange between membranes
    Methods of Molecular Biology, 2019
    Co-Authors: Nicolas-frédéric Lipp, Guillaume Drin
    Abstract:

    Recent findings unveiled that Oxysterol-binding protein-related proteins (ORP)/Oxysterol-binding homology (Osh) proteins, which constitute a major family of lipid transfer proteins (LTPs), conserved among eukaryotes, are not all mere sterol transporters or sensors. Indeed, some of them are able to exchange sterol for Phosphatidylinositol-4-Phosphate (PI4P) or phosphatidylserine (PS) for PI4P between membranes and thereby to use PI4P metabolism to generate sterol or PS gradients in the cell, respectively. Here, we describe a full strategy to measure in vitro a sterol/PI4P exchange process between artificial membranes using Forster resonance energy transfer (FRET)-based assays and a standard spectrofluorometer. Such an approach can serve to better characterize the activity of known sterol/PI4P exchangers, but also to reveal whether ill-defined ORP/Osh proteins or LTPs belonging to other families have such an exchange activity. Besides, this protocol is amenable to test whether molecules can act as Orphilins, which have been found to inhibit the sterol/PI4P exchange activity of certain ORPs. Last, our strategy to measure in real-time PI4P transport using a known lipid-binding domain can serve as a basis for the design of novel in vitro protocols aiming to detect other lipid species.

  • phosphatidylserine transport by orp osh proteins is driven by Phosphatidylinositol 4 Phosphate
    Science, 2015
    Co-Authors: Joachim Moser Von Filseck, V. Delfosse, Stefano Vanni, Catherine L. Jackson, William Bourguet, Alenka Copic, Guillaume Drin
    Abstract:

    In eukaryotic cells, phosphatidylserine (PS) is synthesized in the endoplasmic reticulum (ER) but is highly enriched in the plasma membrane (PM), where it contributes negative charge and to specific recruitment of signaling proteins. This distribution relies on transport mechanisms whose nature remains elusive. Here, we found that the PS transporter Osh6p extracted Phosphatidylinositol 4-Phosphate (PI4P) and exchanged PS for PI4P between two membranes. We solved the crystal structure of Osh6p:PI4P complex and demonstrated that the transport of PS by Osh6p depends on PI4P recognition in vivo. Finally, we showed that the PI4P-phosphatase Sac1p, by maintaining a PI4P gradient at the ER/PM interface, drove PS transport. Thus, PS transport by oxysterol-binding protein–related protein (ORP)/oxysterol-binding homology (Osh) proteins is fueled by PI4P metabolism through PS/PI4P exchange cycles.

  • Phosphatidylserine transport by ORP/Osh proteins is driven by Phosphatidylinositol 4-Phosphate
    Science, 2015
    Co-Authors: Joachim Moser Von Filseck, Alenka Čopič, V. Delfosse, Stefano Vanni, Catherine L. Jackson, William Bourguet, Guillaume Drin
    Abstract:

    In eukaryotic cells, phosphatidylserine (PS) is synthesized in the endoplasmic reticulum (ER) but is highly enriched in the plasma membrane (PM), where it contributes negative charge and to specific recruitment of signaling proteins. This distribution relies on transport mechanisms whose nature remains elusive. Here, we found that the PS transporter Osh6p extracted Phosphatidylinositol 4-Phosphate (PI4P) and exchanged PS for PI4P between two membranes. We solved the crystal structure of Osh6p:PI4P complex and demonstrated that the transport of PS by Osh6p depends on PI4P recognition in vivo. Finally, we showed that the PI4P-phosphatase Sac1p, by maintaining a PI4P gradient at the ER/PM interface, drove PS transport. Thus, PS transport by oxysterol-binding protein–related protein (ORP)/oxysterol-binding homology (Osh) proteins is fueled by PI4P metabolism through PS/PI4P exchange cycles.

  • A Phosphatidylinositol-4-Phosphate powered exchange mechanism to create a lipid gradient between membranes
    Nature communications, 2015
    Co-Authors: Joachim Moser Von Filseck, Stefano Vanni, Bruno Antonny, Bruno Mesmin, Guillaume Drin
    Abstract:

    Lipids are unevenly distributed within eukaryotic cells, thus defining organelle identity. How non-vesicular transport mechanisms generate these lipid gradients between membranes remains a central question. Here using quantitative, real-time lipid transport assays, we demonstrate that Osh4p, a sterol/Phosphatidylinositol-4-Phosphate (PI(4)P) exchanger of the ORP/Osh family, transports sterol against its gradient between two membranes by dissipating the energy of a PI(4)P gradient. Sterol transport is sustained through the maintenance of this PI(4)P gradient by the PI(4)P-phosphatase Sac1p. Differences in lipid packing between membranes can stabilize sterol gradients generated by Osh4p and modulate its lipid exchange capacity. The ability of Osh4p to recognize sterol and PI(4)P via distinct modalities and the dynamics of its N-terminal lid govern its activity. We thus demonstrate that an intracellular lipid transfer protein actively functions to create a lipid gradient between membranes.

Gregory D. Fairn - One of the best experts on this subject based on the ideXlab platform.

  • Enforced expression of Phosphatidylinositol 4-Phosphate 5-kinase homolog alters PtdIns(4,5)P2 distribution and the localization of small G-proteins.
    Scientific reports, 2019
    Co-Authors: Yanbo Yang, Miriam Park, Masashi Maekawa, Gregory D. Fairn
    Abstract:

    The generation of Phosphatidylinositol 4,5-bisPhosphate (PtdIns(4,5)P2) by Phosphatidylinositol 4-Phosphate 5-kinases (PIP5Ks) is essential for many functions including control of the cytoskeleton, signal transduction, and endocytosis. Due to its presence in the plasma membrane and anionic charge, PtdIns(4,5)P2, together with phosphatidylserine, provide the inner leaflet of the plasma membrane with a negative surface charge. This negative charge helps to define the identity of the plasma membrane, as it serves to recruit or regulate a multitude of peripheral and membrane proteins that contain polybasic domains or patches. Here, we determine that the Phosphatidylinositol 4-Phosphate 5-kinase homolog (PIPKH) alters the subcellular distribution of PtdIns(4,5)P2 by re-localizing the three PIP5Ks to endomembranes. We find a redistribution of the PIP5K family members to endomembrane structures upon PIPKH overexpression that is accompanied by accumulation of PtdIns(4,5)P2 and Phosphatidylinositol 3,4,5-trisPhosphate (PtdIns(3,4,5)P3). PIP5Ks are targeted to membranes in part due to electrostatic interactions; however, the interaction between PIPKH and PIP5K is maintained following hydrolysis of PtdIns(4,5)P2. Expression of PIPKH did not impair bulk endocytosis as monitored by FM4-64 uptake but did result in clustering of FM4-64 positive endosomes. Finally, we demonstrate that accumulation of polyphosphoinositides increases the negative surface charge of endosomes and in turn, leads to relocalization of surface charge probes as well as the polycationic proteins K-Ras and Rac1.

  • Enforced expression of Phosphatidylinositol 4-Phosphate 5-kinase homolog (PIPKH) alters Phosphatidylinositol 4,5-bisPhosphate distribution and the localization of small G-proteins
    bioRxiv, 2018
    Co-Authors: Yamin Yang, Minyoung Park, Gregory D. Fairn
    Abstract:

    The generation of Phosphatidylinositol 4,5-bisPhosphate (PtdIns(4,5)P2) by Phosphatidylinositol 4-Phosphate 5-kinases (PIP5Ks) is essential for many of the functions including the control of cytoskeleton, signal transduction and endocytosis. Additionally, due to its presence in the plasma membrane and its anionic charge PtdIns(4,5)P2, together with phosphatidylserine, imbue the inner leaflet of the plasma membrane with a negative surface charge. This negative charge helps to define the identity of the plasma membrane as serves to recruit or regulate a multitude of proteins that contain polybasic domains or patches. Here we determine that the Phosphatidylinositol 4-Phosphate 5-kinase homolog (PIPKH) alters the subcellular distribution of PtdIns(4,5)P2 by re-localizing the PIP5Ks to endomembranes. Consistently, we find a redistribution of the PIP5K family members to endomembrane structures upon PIPKH overexpression that is accompanied by an accumulation of PtdIns(4,5)P2 and Phosphatidylinositol 3,4,5-trisPhosphate (PtdIns(3,4,5)P3), which further influences the distribution of endosomes and lysosomes. Additionally, we demonstrate that the accumulation of polyphosphoinositides increases their negative surface charge that in turn leads to the relocalization of surface charge probes as well as the polycationic proteins K-Ras and Rac1.

  • Multiphasic dynamics of Phosphatidylinositol 4-Phosphate during phagocytosis
    Molecular Biology of the Cell, 2016
    Co-Authors: Roni Levin, Tamas Balla, Gerald R.v. Hammond, Gregory D. Fairn, Pietro De Camilli, Sergio Grinstein
    Abstract:

    : We analyzed the distribution, fate, and functional role of Phosphatidylinositol 4-Phosphate (PtdIns4P) during phagosome formation and maturation. To this end, we used genetically encoded probes consisting of the PtdIns4P-binding domain of the bacterial effector SidM. PtdIns4P was found to undergo complex, multiphasic changes during phagocytosis. The phosphoinositide, which is present in the plasmalemma before engagement of the target particle, is transiently enriched in the phagosomal cup. Soon after the phagosome seals, PtdIns4P levels drop precipitously due to the hydrolytic activity of Sac2 and phospholipase C, becoming undetectable for ∼10 min. PtdIns4P disappearance coincides with the emergence of phagosomal PtdIns3P. Conversely, the disappearance of PtdIns3P that signals the transition from early to late phagosomes is accompanied by resurgence of PtdIns4P, which is associated with the recruitment of Phosphatidylinositol 4-kinase 2A. The reacquisition of PtdIns4P can be prevented by silencing expression of the kinase and can be counteracted by recruitment of a 4-phosphatase with a heterodimerization system. Using these approaches, we found that the secondary accumulation of PtdIns4P is required for proper phagosomal acidification. Defective acidification may be caused by impaired recruitment of Rab7 effectors, including RILP, which were shown earlier to displace phagosomes toward perinuclear lysosomes. Our results show multimodal dynamics of PtdIns4P during phagocytosis and suggest that the phosphoinositide plays important roles during the maturation of the phagosome.

  • Multiphasic dynamics of Phosphatidylinositol 4-Phosphate during phagocytosis
    Molecular biology of the cell, 2016
    Co-Authors: Roni Levin, Tamas Balla, Gerald R.v. Hammond, Gregory D. Fairn, Pietro De Camilli, Sergio Grinstein
    Abstract:

    Phosphatidylinositol 4-Phosphate undergoes striking multiphasic changes during phagosome formation and maturation. The molecular mechanisms underlying these changes and the role of phosphatidylinos...

  • The oxysterol binding protein Kes1p regulates Golgi apparatus Phosphatidylinositol-4-Phosphate function
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: Gregory D. Fairn, Amy J. Curwin, Christopher J. Stefan, Christopher R. Mcmaster
    Abstract:

    The Saccharomyces cerevisiae phosphatidylcholine/Phosphatidylinositol transfer protein Sec14p is required for Golgi apparatus-derived vesicular transport through coordinate regulation of phospholipid metabolism. Sec14p is normally essential. The essential requirement for SEC14 can be bypassed by inactivation of (i) the CDP–choline pathway for phosphatidylcholine synthesis or (ii) KES1, which encodes an oxysterol binding protein. A unique screen was used to determine genome-wide genetic interactions for the essential gene SEC14 and to assess whether the two modes of “sec14 bypass” were similar or distinct. The results indicate that inactivation of the CDP–choline pathway allows cells with inactivated SEC14 to live through a mechanism distinct from that of inactivation of KES1. We go on to demonstrate an important biological function of Kes1p. Kes1p regulates Golgi apparatus-derived vesicular transport by inhibiting the function of Pik1p-generated Golgi apparatus Phosphatidylinositol-4-Phosphate (PI-4P). Kes1p affects both the availability and level of Golgi apparatus PI-4P. A set of potential PI-4P-responsive proteins that include the Rab GTPase Ypt31p and its GTP exchange factor are described.

Ronald F. Coburn - One of the best experts on this subject based on the ideXlab platform.

  • Effects of polyamines and calcium and sodium ions on smooth muscle cytoskeleton-associated Phosphatidylinositol (4)-Phosphate 5-kinase.
    Journal of Cellular Physiology, 1998
    Co-Authors: H Chen, Carl B. Baron, T. Griffiths, P. Greeley, Ronald F. Coburn
    Abstract:

    : In many different cell types, including smooth muscle cells (Baron et al., 1989, Am. J. Physiol., 256: C375-383; Baron et al., J. Pharmacol. Exp. Ther. 266: 8-15), Phosphatidylinositol (4)-Phosphate 5-kinase plays a critical role in the regulation of membrane concentrations of Phosphatidylinositol (4,5)-bisPhosphate and formation of inositol (1,4,5)-trisPhosphate. In unstimulated porcine trachealis smooth muscle, 70% of total cellular Phosphatidylinositol (4)-Phosphate 5-kinase activity was associated with cytoskeletal proteins and only trace activity was detectable in isolated sarcolemma. Using two different preparations, we studied cytoskeleton-associated phosphatidyl inositol (4)-Phosphate 5-kinase under conditions that attempted to mimic the ionic and thermal cytoplasmic environment of living cells. The cytoskeleton-associated enzyme, studied using Phosphatidylinositol (4)-Phosphate substrate concentrations that produced Phosphatidylinositol 4,5-bisPhosphate at about 10% of the maximal rate, was sensitive to free [Mg2+], had an absolute requirement for phosphatidylserine, phosphatidic acid, or Phosphatidylinositol, and included type I isoforms. At 0.5 mM free [Mg2+], physiological spermine concentrations, 0.2-0.4 mM, increased Phosphatidylinositol (4)-Phosphate 5-kinase activity two to four times compared to controls run without spermine. The EC50 for spermine-evoked increases in activity was 0.17 +/- 0.02 mM. Spermine-evoked enzyme activity was a function of both free [Mg2+] and substrate concentration. Cytoskeleton-associated Phosphatidylinositol (4)-Phosphate 5-kinase was inhibited by free [Ca2+] over a physiological range for cytoplasm--10(-8) to 10(-5) M, an effect independent of the presence of calmodulin. Na+ over the range 20 to 50 mM also inhibited this enzyme activated by 5 mM Mg2+ but had no effect on spermine-activated enzyme. Na+, Ca2+, and spermine appear to be physiological modulators of smooth muscle cytoskeleton-bound Phosphatidylinositol (4)-Phosphate 5-kinase.

  • Effects of polyamines and calcium and sodium ions on smooth muscle cytoskeleton-associated Phosphatidylinositol (4)-Phosphate 5-kinase.
    Journal of Cellular Physiology, 1998
    Co-Authors: H Chen, Carl B. Baron, T. Griffiths, P. Greeley, Ronald F. Coburn
    Abstract:

    : In many different cell types, including smooth muscle cells (Baron et al., 1989, Am. J. Physiol., 256: C375-383; Baron et al., J. Pharmacol. Exp. Ther. 266: 8-15), Phosphatidylinositol (4)-Phosphate 5-kinase plays a critical role in the regulation of membrane concentrations of Phosphatidylinositol (4,5)-bisPhosphate and formation of inositol (1,4,5)-trisPhosphate. In unstimulated porcine trachealis smooth muscle, 70% of total cellular Phosphatidylinositol (4)-Phosphate 5-kinase activity was associated with cytoskeletal proteins and only trace activity was detectable in isolated sarcolemma. Using two different preparations, we studied cytoskeleton-associated phosphatidyl inositol (4)-Phosphate 5-kinase under conditions that attempted to mimic the ionic and thermal cytoplasmic environment of living cells. The cytoskeleton-associated enzyme, studied using Phosphatidylinositol (4)-Phosphate substrate concentrations that produced Phosphatidylinositol 4,5-bisPhosphate at about 10% of the maximal rate, was sensitive to free [Mg2+], had an absolute requirement for phosphatidylserine, phosphatidic acid, or Phosphatidylinositol, and included type I isoforms. At 0.5 mM free [Mg2+], physiological spermine concentrations, 0.2-0.4 mM, increased Phosphatidylinositol (4)-Phosphate 5-kinase activity two to four times compared to controls run without spermine. The EC50 for spermine-evoked increases in activity was 0.17 +/- 0.02 mM. Spermine-evoked enzyme activity was a function of both free [Mg2+] and substrate concentration. Cytoskeleton-associated Phosphatidylinositol (4)-Phosphate 5-kinase was inhibited by free [Ca2+] over a physiological range for cytoplasm--10(-8) to 10(-5) M, an effect independent of the presence of calmodulin. Na+ over the range 20 to 50 mM also inhibited this enzyme activated by 5 mM Mg2+ but had no effect on spermine-activated enzyme. Na+, Ca2+, and spermine appear to be physiological modulators of smooth muscle cytoskeleton-bound Phosphatidylinositol (4)-Phosphate 5-kinase.

H Chen - One of the best experts on this subject based on the ideXlab platform.

  • Effects of polyamines and calcium and sodium ions on smooth muscle cytoskeleton-associated Phosphatidylinositol (4)-Phosphate 5-kinase.
    Journal of Cellular Physiology, 1998
    Co-Authors: H Chen, Carl B. Baron, T. Griffiths, P. Greeley, Ronald F. Coburn
    Abstract:

    : In many different cell types, including smooth muscle cells (Baron et al., 1989, Am. J. Physiol., 256: C375-383; Baron et al., J. Pharmacol. Exp. Ther. 266: 8-15), Phosphatidylinositol (4)-Phosphate 5-kinase plays a critical role in the regulation of membrane concentrations of Phosphatidylinositol (4,5)-bisPhosphate and formation of inositol (1,4,5)-trisPhosphate. In unstimulated porcine trachealis smooth muscle, 70% of total cellular Phosphatidylinositol (4)-Phosphate 5-kinase activity was associated with cytoskeletal proteins and only trace activity was detectable in isolated sarcolemma. Using two different preparations, we studied cytoskeleton-associated phosphatidyl inositol (4)-Phosphate 5-kinase under conditions that attempted to mimic the ionic and thermal cytoplasmic environment of living cells. The cytoskeleton-associated enzyme, studied using Phosphatidylinositol (4)-Phosphate substrate concentrations that produced Phosphatidylinositol 4,5-bisPhosphate at about 10% of the maximal rate, was sensitive to free [Mg2+], had an absolute requirement for phosphatidylserine, phosphatidic acid, or Phosphatidylinositol, and included type I isoforms. At 0.5 mM free [Mg2+], physiological spermine concentrations, 0.2-0.4 mM, increased Phosphatidylinositol (4)-Phosphate 5-kinase activity two to four times compared to controls run without spermine. The EC50 for spermine-evoked increases in activity was 0.17 +/- 0.02 mM. Spermine-evoked enzyme activity was a function of both free [Mg2+] and substrate concentration. Cytoskeleton-associated Phosphatidylinositol (4)-Phosphate 5-kinase was inhibited by free [Ca2+] over a physiological range for cytoplasm--10(-8) to 10(-5) M, an effect independent of the presence of calmodulin. Na+ over the range 20 to 50 mM also inhibited this enzyme activated by 5 mM Mg2+ but had no effect on spermine-activated enzyme. Na+, Ca2+, and spermine appear to be physiological modulators of smooth muscle cytoskeleton-bound Phosphatidylinositol (4)-Phosphate 5-kinase.

  • Effects of polyamines and calcium and sodium ions on smooth muscle cytoskeleton-associated Phosphatidylinositol (4)-Phosphate 5-kinase.
    Journal of Cellular Physiology, 1998
    Co-Authors: H Chen, Carl B. Baron, T. Griffiths, P. Greeley, Ronald F. Coburn
    Abstract:

    : In many different cell types, including smooth muscle cells (Baron et al., 1989, Am. J. Physiol., 256: C375-383; Baron et al., J. Pharmacol. Exp. Ther. 266: 8-15), Phosphatidylinositol (4)-Phosphate 5-kinase plays a critical role in the regulation of membrane concentrations of Phosphatidylinositol (4,5)-bisPhosphate and formation of inositol (1,4,5)-trisPhosphate. In unstimulated porcine trachealis smooth muscle, 70% of total cellular Phosphatidylinositol (4)-Phosphate 5-kinase activity was associated with cytoskeletal proteins and only trace activity was detectable in isolated sarcolemma. Using two different preparations, we studied cytoskeleton-associated phosphatidyl inositol (4)-Phosphate 5-kinase under conditions that attempted to mimic the ionic and thermal cytoplasmic environment of living cells. The cytoskeleton-associated enzyme, studied using Phosphatidylinositol (4)-Phosphate substrate concentrations that produced Phosphatidylinositol 4,5-bisPhosphate at about 10% of the maximal rate, was sensitive to free [Mg2+], had an absolute requirement for phosphatidylserine, phosphatidic acid, or Phosphatidylinositol, and included type I isoforms. At 0.5 mM free [Mg2+], physiological spermine concentrations, 0.2-0.4 mM, increased Phosphatidylinositol (4)-Phosphate 5-kinase activity two to four times compared to controls run without spermine. The EC50 for spermine-evoked increases in activity was 0.17 +/- 0.02 mM. Spermine-evoked enzyme activity was a function of both free [Mg2+] and substrate concentration. Cytoskeleton-associated Phosphatidylinositol (4)-Phosphate 5-kinase was inhibited by free [Ca2+] over a physiological range for cytoplasm--10(-8) to 10(-5) M, an effect independent of the presence of calmodulin. Na+ over the range 20 to 50 mM also inhibited this enzyme activated by 5 mM Mg2+ but had no effect on spermine-activated enzyme. Na+, Ca2+, and spermine appear to be physiological modulators of smooth muscle cytoskeleton-bound Phosphatidylinositol (4)-Phosphate 5-kinase.

Yasunori Kanaho - One of the best experts on this subject based on the ideXlab platform.

  • Phosphatidylinositol 4-Phosphate 5-Kinase Is Indispensable for Mouse Spermatogenesis
    Biology of Reproduction, 2012
    Co-Authors: Hiroshi Hasegawa, Misuzu Yamashita, Risa Okada, Rika Sugimoto, Momoko Furuya, Takamitsu Unoki, Yuji Funakoshi, Junko Noguchi, Tadashi Baba, Yasunori Kanaho
    Abstract:

    ABSTRACT The lipid kinase Phosphatidylinositol 4-Phosphate 5-kinase (PIP5K) produces a versatile signaling phospholipid, Phosphatidylinositol 4,5-bisPhosphate. Three PIP5K isozymes, PIP5K1A, PIP5K1B, and PIP5K1C, have been identified in mammals so far. Although the functions of these three PIP5K isozymes have been extensively studied in vitro, the in vivo physiological roles of these PIP5K isozymes remain largely unknown. In this study, we examined the functions of PIP5K1A and PIP5K1B in spermatogenesis, using Pip5k1a-knockout (KO), Pip5k1b-KO, and Pip5k1a/Pip5k1b double (D)-KO mice. Pip5k1a-KO and D-KO males were subfertile and completely sterile, respectively. F-actin in the seminiferous epithelium was disorganized in the D-KO mice, although F-actin bundles at the apical ectoplasmic specialization was not affected. D-KO seminiferous tubules contained a greatly decreased number of elongated spermatids. Flagella of sperm from Pip5k1a-KO and D-KO mice remarkably underwent morphological change, whereas Pip5...

  • Phosphatidylinositol 4-Phosphate 5-Kinase Is Indispensable for Mouse Spermatogenesis
    Biology of reproduction, 2012
    Co-Authors: Hiroshi Hasegawa, Misuzu Yamashita, Risa Okada, Rika Sugimoto, Momoko Furuya, Takamitsu Unoki, Yuji Funakoshi, Junko Noguchi, Tadashi Baba, Yasunori Kanaho
    Abstract:

    The lipid kinase Phosphatidylinositol 4-Phosphate 5-kinase (PIP5K) produces a versatile signaling phospholipid, Phosphatidylinositol 4,5-bisPhosphate. Three PIP5K isozymes, PIP5K1A, PIP5K1B, and PIP5K1C, have been identified in mammals so far. Although the functions of these three PIP5K isozymes have been extensively studied in vitro, the in vivo physiological roles of these PIP5K isozymes remain largely unknown. In this study, we examined the functions of PIP5K1A and PIP5K1B in spermatogenesis, using Pip5k1a-knockout (KO), Pip5k1b-KO, and Pip5k1a/Pip5k1b double (D)-KO mice. Pip5k1a-KO and D-KO males were subfertile and completely sterile, respectively. F-actin in the seminiferous epithelium was disorganized in the D-KO mice, although F-actin bundles at the apical ectoplasmic specialization was not affected. D-KO seminiferous tubules contained a greatly decreased number of elongated spermatids. Flagella of sperm from Pip5k1a-KO and D-KO mice remarkably underwent morphological change, whereas Pip5k1b-KO sperm were morphologically normal. Notably, the flagellar shape of D-KO sperm was more severely impaired than that of Pip5k1a-KO sperm. These results suggest that PIP5K1A and PIP5K1B may coordinately and/or redundantly function in the maintenance of sperm number and morphology during spermatogenesis.

  • Regulation of Phosphatidylinositol 4-Phosphate 5-kinase activity by partner proteins.
    Methods in Enzymology, 2007
    Co-Authors: Yasunori Kanaho, Michael A Frohman, Kazuhisa Nakayama, Takeaki Yokozeki
    Abstract:

    Abstract The remarkably versatile phospholipid, Phosphatidylinositol 4,5-bisPhosphate [PI(4,5)P 2 ], plays crucial roles in signal transduction, actin cytoskeleton reorganization, clathrin-dependent endocytosis, and regulation of membrane morphology. In mammalian cells, PI(4,5)P 2 is synthesized predominantly by Phosphatidylinositol 4-Phosphate [PI(4)P] 5-kinase (PIP5K) through phosphorylation of PI(4)P at the D-5 position of the inositol ring. PIP5K is composed of three isoforms, PIP5Kα, β, and γ, and three splicing variants of the γ isozyme. Although the PIP5Kγ splicing variant PIP5Kγ661 appears to be very specifically activated by talin, which plays a crucial role in focal adhesion formation, and the adaptor complex AP-2, the regulation of activities of other PIP5K isozymes is not fully understood at present. To understand the activation mechanism and the physiological function specific to each PIP5K isozyme, it is required to identify a specific activator of each PIP5K isozyme. This chapter describes common assays used to measure interaction and activation of PIP5K isozymes by activators thus far identified. In addition, procedures for preparation of PIP5K isozymes and activators are described.

  • Phosphatidylinositol 4 Phosphate 5 kinase is essential for rock mediated neurite remodeling
    Journal of Biological Chemistry, 2002
    Co-Authors: Masakazu Yamazaki, Hideyuki Miyazaki, Tomohiko Maehama, Michael A Frohman, Takehiko Sasaki, Hiroshi Watanabe, Yasunori Kanaho
    Abstract:

    Abstract Phosphatidylinositol 4-Phosphate 5-kinase (PIP-5kin) regulates actin cytoskeletal reorganization through its product Phosphatidylinositol 4,5-bisPhosphate. In the present study we demonstrate that PIP-5kin is essential for neurite remodeling, which is regulated by actin cytoskeletal reorganization in neuroblastoma N1E-115 cells. Overexpression of wild-type mouse PIP-5kin-α inhibits the neurite formation that is normally stimulated by serum depletion, whereas a lipid kinase-defective mutant of PIP-5kin-α, D266A, triggers neurite extension even in the presence of serum and blocks lysophosphatidic acid-induced neurite retraction. These results phenocopy those previously reported for the small GTPase RhoA and its effector p160 Rho-associated coiled coil-forming protein kinase (ROCK). However, the ROCK-specific inhibitor Y-27632 failed to block the inhibition by PIP-5kin-α of neurite extension, whereas D266A did block the neurite retraction induced by overexpression of ROCK. These results, taken together, suggest that PIP-5kin-α functions as a downstream effector for RhoA/ROCK to couple lysophosphatidic acid signaling to neurite retraction presumably through its product Phosphatidylinositol 4,5-bisPhosphate.

  • Phosphatidylinositol-4-Phosphate 5-Kinase Localized on the Plasma Membrane Is Essential for Yeast Cell Morphogenesis
    Journal of Biological Chemistry, 1998
    Co-Authors: Keiichi Homma, Masayo Minemura, Hiroshi Qadota, Yasuhiro Anraku, Yasunori Kanaho, Sachiko Terui, Yoshikazu Ohya
    Abstract:

    Abstract Phosphatidylinositol 4,5-biPhosphate (PtdIns(4,5)P2), an important element in eukaryotic signal transduction, is synthesized either by Phosphatidylinositol-4-Phosphate 5-kinase (PtdIns(4)P 5K) from Phosphatidylinositol 4-Phosphate (PtdIns(4)P) or by Phosphatidylinositol-5-Phosphate 4-kinase (PtdIns(5)P 4K) from Phosphatidylinositol 5-Phosphate (PtdIns(5)P). Two Saccharomyces cerevisiae genes, MSS4 and FAB1, are homologous to mammalian PtdIns(4)P 5Ks and PtdIns(5)P 4Ks. We show here that MSS4 is a functional homolog of mammalian PtdIns(4)P 5K but not of PtdIns(5)P 4K in vivo. We constructed a hemagglutinin epitope-tagged form of Mss4p and found that Mss4p has PtdIns(4)P 5K activity. Immunofluorescent and fractionation studies of the epitope-tagged Mss4p suggest that Mss4p is localized on the plasma membrane, whereas Fab1p is reportedly localized on the vacuolar membrane. A temperature-sensitive mss4-1 mutant was isolated, and its phenotypes at restrictive temperatures were found to include increased cell size, round shape, random distribution of actin patches, and delocalized staining of cell wall chitin. Thus, biochemical and genetic analyses on Mss4p indicated that yeast PtdIns(4)P 5K localized on the plasma membrane is required for actin organization.