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An electrostatic switch displaces phosphatidylinositol phosphate kinases from the membrane during phagocytosis.

Fairn GD, Ogata K, Botelho RJ, Stahl PD, Anderson RA, De Camilli P, Meyer T, Wodak S, Grinstein S - J. Cell Biol. (2009)

Bottom Line: During particle engulfment, PIP5Ks detached from forming phagosomes as the surface charge at these sites decreased.Precluding the change in surface charge caused the PIP5Ks to remain associated with the phagosomal cup.We conclude that a bistable electrostatic switch mechanism regulates the association/dissociation of PIP5Ks from the membrane during phagocytosis and likely other processes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Cell Biology and 2 Structural Biology Program, Hospital for Sick Children, Toronto, Ontario, Canada M5G1X8.

ABSTRACT
Plasmalemmal phosphatidylinositol (PI) 4,5-bisphosphate (PI4,5P(2)) synthesized by PI 4-phosphate (PI4P) 5-kinase (PIP5K) is key to the polymerization of actin that drives chemotaxis and phagocytosis. We investigated the means whereby PIP5K is targeted to the membrane and its fate during phagosome formation. Homology modeling revealed that all PIP5K isoforms feature a positively charged face. Together with the substrate-binding loop, this polycationic surface is proposed to constitute a coincidence detector that targets PIP5Ks to the plasmalemma. Accordingly, manipulation of the surface charge displaced PIP5Ks from the plasma membrane. During particle engulfment, PIP5Ks detached from forming phagosomes as the surface charge at these sites decreased. Precluding the change in surface charge caused the PIP5Ks to remain associated with the phagosomal cup. Chemically induced retention of PIP5K-gamma prevented the disappearance of PI4,5P(2) and aborted phagosome formation. We conclude that a bistable electrostatic switch mechanism regulates the association/dissociation of PIP5Ks from the membrane during phagocytosis and likely other processes.

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Sustained production of PI4,5P2 inhibits phagocytosis. (a) Schematic of the experimental protocol. The addition of rapamycin induces the heterodimerization of the YFP-FKBP-5K construct with a plasma membrane–targeted form of FRB. (b) Distribution of YFP-FKBP-5K and RFP-PH–PLC-δ before (−Rap) and 5 min after the addition of 10 µM rapamycin (+Rap). Arrowhead identifies sealed and internalized phagosomes, and the arrow highlights forming phagosomes. (c) Internalization and adherence of beads to macrophages was quantified in cells expressing YFP or YFP-FKBP-5K and plasma membrane–targeted FRB with or without the addition of rapamycin. Data are means ± SEM of at least 500 beads per condition.
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fig7: Sustained production of PI4,5P2 inhibits phagocytosis. (a) Schematic of the experimental protocol. The addition of rapamycin induces the heterodimerization of the YFP-FKBP-5K construct with a plasma membrane–targeted form of FRB. (b) Distribution of YFP-FKBP-5K and RFP-PH–PLC-δ before (−Rap) and 5 min after the addition of 10 µM rapamycin (+Rap). Arrowhead identifies sealed and internalized phagosomes, and the arrow highlights forming phagosomes. (c) Internalization and adherence of beads to macrophages was quantified in cells expressing YFP or YFP-FKBP-5K and plasma membrane–targeted FRB with or without the addition of rapamycin. Data are means ± SEM of at least 500 beads per condition.

Mentions: Previous studies have suggested that disappearance of PI4,5P2 from forming phagosomes is required for termination of actin polymerization, which was in turn proposed to be essential for phagosomal sealing and scission (Scott et al., 2005). Our aforementioned results suggest that an electrostatically induced detachment of PIP5K may contribute to the disappearance of PI4,5P2 by locally terminating its synthesis. To test this notion, we used a form of PIP5K that could be recruited to the membrane in a manner that did not depend on charge interactions. Specifically, we expressed a chimeric construct consisting of the cDNA encoding the full-length 87-kD splice variant of PIP5K-γ fused to the FK506-binding protein (FKBP) and tagged with YFP (YFP-FKBP-5K). Unlike the unmodified PIP5K-γ90 and PIP5K-γ87, this chimeric construct is partly cytosolic (Fig. 7 b). Attachment of the FKBP moiety to the N terminus of PIP5K-γ87 seemingly impairs normal recruitment of the kinase to the membrane, possibly by preventing formation of homodimers or other protein interactions. However, as illustrated diagrammatically in Fig. 7 a, YFP-FKBP-5K can be effectively tethered to the membrane by coexpression of a membrane-targeted form of the rapamycin-binding domain of mTOR (FK506 rapamycin–binding domain [FRB]) followed by addition of rapamycin to induce heterodimerization of the two constructs (Inoue et al., 2005). In the absence of rapamycin, the membrane-associated fraction of YFP-FKBP-5K is displaced from the phagosomal cup in the course of particle ingestion (Fig. 7 b). Under these conditions, the macrophages can eliminate PI4,5P2 from nascent phagosomes and effectively complete particle engulfment (Fig. 7 b). As shown in Fig. 7 c, in the absence of the cross-linker, the phagocytic efficiency of cells expressing YFP-FKBP-5K is only marginally lower than that of cells expressing FRB and YFP only. Upon addition of rapamycin, most of the YFP-FKBP-5K is retained on the plasma membrane by association with FRB and is not released when particles interact with the macrophages (Fig. 7 b). Importantly, the retention of excess PIP5K antagonizes the disappearance of PI4,5P2, which remains at the base of the phagosome, as shown by the persistent association of PH–PLC-δ (Fig. 7 b). As a consequence, actin fails to dissociate from the base of the phagocytic cup, and phagosome closure is impaired. The phagocytic index is markedly reduced when YFP-FKBP-5K is recruited to the plasma membrane by rapamycin (Fig. 7 c). This effect was not caused by rapamycin itself because the cross-linker produced no inhibition when added to cells expressing only YFP and FRB (Fig. 7 c). Together with our previous results, these observations suggest that the timely removal of PI4,5P2 and depolymerization of actin from the base of the cup are required for the completion of phagocytosis.


An electrostatic switch displaces phosphatidylinositol phosphate kinases from the membrane during phagocytosis.

Fairn GD, Ogata K, Botelho RJ, Stahl PD, Anderson RA, De Camilli P, Meyer T, Wodak S, Grinstein S - J. Cell Biol. (2009)

Sustained production of PI4,5P2 inhibits phagocytosis. (a) Schematic of the experimental protocol. The addition of rapamycin induces the heterodimerization of the YFP-FKBP-5K construct with a plasma membrane–targeted form of FRB. (b) Distribution of YFP-FKBP-5K and RFP-PH–PLC-δ before (−Rap) and 5 min after the addition of 10 µM rapamycin (+Rap). Arrowhead identifies sealed and internalized phagosomes, and the arrow highlights forming phagosomes. (c) Internalization and adherence of beads to macrophages was quantified in cells expressing YFP or YFP-FKBP-5K and plasma membrane–targeted FRB with or without the addition of rapamycin. Data are means ± SEM of at least 500 beads per condition.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2806594&req=5

fig7: Sustained production of PI4,5P2 inhibits phagocytosis. (a) Schematic of the experimental protocol. The addition of rapamycin induces the heterodimerization of the YFP-FKBP-5K construct with a plasma membrane–targeted form of FRB. (b) Distribution of YFP-FKBP-5K and RFP-PH–PLC-δ before (−Rap) and 5 min after the addition of 10 µM rapamycin (+Rap). Arrowhead identifies sealed and internalized phagosomes, and the arrow highlights forming phagosomes. (c) Internalization and adherence of beads to macrophages was quantified in cells expressing YFP or YFP-FKBP-5K and plasma membrane–targeted FRB with or without the addition of rapamycin. Data are means ± SEM of at least 500 beads per condition.
Mentions: Previous studies have suggested that disappearance of PI4,5P2 from forming phagosomes is required for termination of actin polymerization, which was in turn proposed to be essential for phagosomal sealing and scission (Scott et al., 2005). Our aforementioned results suggest that an electrostatically induced detachment of PIP5K may contribute to the disappearance of PI4,5P2 by locally terminating its synthesis. To test this notion, we used a form of PIP5K that could be recruited to the membrane in a manner that did not depend on charge interactions. Specifically, we expressed a chimeric construct consisting of the cDNA encoding the full-length 87-kD splice variant of PIP5K-γ fused to the FK506-binding protein (FKBP) and tagged with YFP (YFP-FKBP-5K). Unlike the unmodified PIP5K-γ90 and PIP5K-γ87, this chimeric construct is partly cytosolic (Fig. 7 b). Attachment of the FKBP moiety to the N terminus of PIP5K-γ87 seemingly impairs normal recruitment of the kinase to the membrane, possibly by preventing formation of homodimers or other protein interactions. However, as illustrated diagrammatically in Fig. 7 a, YFP-FKBP-5K can be effectively tethered to the membrane by coexpression of a membrane-targeted form of the rapamycin-binding domain of mTOR (FK506 rapamycin–binding domain [FRB]) followed by addition of rapamycin to induce heterodimerization of the two constructs (Inoue et al., 2005). In the absence of rapamycin, the membrane-associated fraction of YFP-FKBP-5K is displaced from the phagosomal cup in the course of particle ingestion (Fig. 7 b). Under these conditions, the macrophages can eliminate PI4,5P2 from nascent phagosomes and effectively complete particle engulfment (Fig. 7 b). As shown in Fig. 7 c, in the absence of the cross-linker, the phagocytic efficiency of cells expressing YFP-FKBP-5K is only marginally lower than that of cells expressing FRB and YFP only. Upon addition of rapamycin, most of the YFP-FKBP-5K is retained on the plasma membrane by association with FRB and is not released when particles interact with the macrophages (Fig. 7 b). Importantly, the retention of excess PIP5K antagonizes the disappearance of PI4,5P2, which remains at the base of the phagosome, as shown by the persistent association of PH–PLC-δ (Fig. 7 b). As a consequence, actin fails to dissociate from the base of the phagocytic cup, and phagosome closure is impaired. The phagocytic index is markedly reduced when YFP-FKBP-5K is recruited to the plasma membrane by rapamycin (Fig. 7 c). This effect was not caused by rapamycin itself because the cross-linker produced no inhibition when added to cells expressing only YFP and FRB (Fig. 7 c). Together with our previous results, these observations suggest that the timely removal of PI4,5P2 and depolymerization of actin from the base of the cup are required for the completion of phagocytosis.

Bottom Line: During particle engulfment, PIP5Ks detached from forming phagosomes as the surface charge at these sites decreased.Precluding the change in surface charge caused the PIP5Ks to remain associated with the phagosomal cup.We conclude that a bistable electrostatic switch mechanism regulates the association/dissociation of PIP5Ks from the membrane during phagocytosis and likely other processes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Cell Biology and 2 Structural Biology Program, Hospital for Sick Children, Toronto, Ontario, Canada M5G1X8.

ABSTRACT
Plasmalemmal phosphatidylinositol (PI) 4,5-bisphosphate (PI4,5P(2)) synthesized by PI 4-phosphate (PI4P) 5-kinase (PIP5K) is key to the polymerization of actin that drives chemotaxis and phagocytosis. We investigated the means whereby PIP5K is targeted to the membrane and its fate during phagosome formation. Homology modeling revealed that all PIP5K isoforms feature a positively charged face. Together with the substrate-binding loop, this polycationic surface is proposed to constitute a coincidence detector that targets PIP5Ks to the plasmalemma. Accordingly, manipulation of the surface charge displaced PIP5Ks from the plasma membrane. During particle engulfment, PIP5Ks detached from forming phagosomes as the surface charge at these sites decreased. Precluding the change in surface charge caused the PIP5Ks to remain associated with the phagosomal cup. Chemically induced retention of PIP5K-gamma prevented the disappearance of PI4,5P(2) and aborted phagosome formation. We conclude that a bistable electrostatic switch mechanism regulates the association/dissociation of PIP5Ks from the membrane during phagocytosis and likely other processes.

Show MeSH
Related in: MedlinePlus