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Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis.

Scott CC, Dobson W, Botelho RJ, Coady-Osberg N, Chavrier P, Knecht DA, Heath C, Stahl P, Grinstein S - J. Cell Biol. (2005)

Bottom Line: Although actin was found to disappear from the base of the forming phagosome before sealing was complete, Rac1/Cdc42 activity persisted, suggesting that termination of GTPase activity is not the main determinant of actin disassembly.Furthermore, fully internalized phagosomes engineered to associate constitutively with active Rac1 showed little associated F-actin.These observations suggest that hydrolysis of PI(4,5)P(2) dictates the remodeling of actin necessary for completion of phagocytosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.

ABSTRACT
The Rho GTPases play a critical role in initiating actin polymerization during phagocytosis. In contrast, the factors directing the disassembly of F-actin required for fission of the phagocytic vacuole are ill defined. We used fluorescent chimeric proteins to monitor the dynamics of association of actin and active Cdc42 and Rac1 with the forming phagosome. Although actin was found to disappear from the base of the forming phagosome before sealing was complete, Rac1/Cdc42 activity persisted, suggesting that termination of GTPase activity is not the main determinant of actin disassembly. Furthermore, fully internalized phagosomes engineered to associate constitutively with active Rac1 showed little associated F-actin. The disappearance of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) from the phagosomal membrane closely paralleled the course of actin disassembly. Furthermore, inhibition of PI(4,5)P(2) hydrolysis or increased PI(4,5)P(2) generation by overexpression of phosphatidylinositol phosphate kinase I prevented the actin disassembly necessary for the completion of phagocytosis. These observations suggest that hydrolysis of PI(4,5)P(2) dictates the remodeling of actin necessary for completion of phagocytosis.

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Effect of PLC inhibition on the completion of phagocytosis. RAW cells transiently transfected with PHPLCδ-GFP were either left untreated (A and B), treated with 100 μM LY294002 for 30 min (C and D), treated with 1 μM U73122 for 10 min (not depicted), or treated with 100 μM LY294002 for 30 min, with 1 μM U73122 present for the last 10 min of the incubation, followed by removal of LY294002 while maintaining the U73122 for an additional 10 min (E and F). The cells were then exposed to IgG-opsonized particles for 10 min and finally fixed, permeabilized, and stained with rhodamine-phalloidin (B, D, and F). PHPLCδ-GFP fluorescence is shown in A, C, and E. Bars, 5 μm. (G) The phagocytic index of cells treated as above was determined as described in Materials and methods. Data are the means ± SEM of three experiments, each scoring at least 50 cells.
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fig8: Effect of PLC inhibition on the completion of phagocytosis. RAW cells transiently transfected with PHPLCδ-GFP were either left untreated (A and B), treated with 100 μM LY294002 for 30 min (C and D), treated with 1 μM U73122 for 10 min (not depicted), or treated with 100 μM LY294002 for 30 min, with 1 μM U73122 present for the last 10 min of the incubation, followed by removal of LY294002 while maintaining the U73122 for an additional 10 min (E and F). The cells were then exposed to IgG-opsonized particles for 10 min and finally fixed, permeabilized, and stained with rhodamine-phalloidin (B, D, and F). PHPLCδ-GFP fluorescence is shown in A, C, and E. Bars, 5 μm. (G) The phagocytic index of cells treated as above was determined as described in Materials and methods. Data are the means ± SEM of three experiments, each scoring at least 50 cells.

Mentions: When added before exposure of the cells to phagocytic targets, U73122 prevented cup formation and failed to provide evidence of the role of PI(4,5)P2 hydrolysis in cytoskeletal disassembly during the course of particle internalization. To circumvent the effects of PLC inhibitors on the early stages of actin remodeling, we attempted to use the inhibitors only after phagocytosis had been initiated. However, their slow permeation rate requires extended incubation periods, precluding this experimental paradigm. Instead, we sought to design experiments where phagocytosis could be arrested after formation of the phagocytic cup, but before completion of phagocytosis, affording us the opportunity to load the cells with PLC antagonists and assess their effect on actin disassembly. This was made possible by the use of LY294002, a reversible PI3-K inhibitor (Vlahos et al., 1994). Treatment with PI3-K inhibitors arrested phagocytosis at an intermediate stage, after actin assembly and partial extension of pseudopodia (Fig. 8, C and D; Araki et al., 1996; Cox et al., 1999; Vieira et al., 2001). The profound inhibition of phagocytosis exerted by LY294002 is illustrated in Fig. 8 G. Unlike wortmannin, which covalently inactivates PI3-K, LY294002 is reversible and most of the phagocytic activity was restored shortly after the inhibitor was removed (Fig. 8 G, second column). The actin accumulated at the aborted phagocytic cup disappeared almost entirely from the periphery of the formed phagosomes when the inhibitor was removed (not depicted). This enabled us to abort phagocytosis after actin cups formed, and subsequently incubate the cells for 10 min with U73122 to allow its entry to the cells. LY294002 was then removed while maintaining U73122 in the incubation medium, and the effects of inhibition of PLC on phagocytosis and on the disassembly of the actin cup were monitored. Fig. 8 G shows that U73122 was as potent a blocker of phagocytosis when added after cup formation, as it was when added before phagocytosis (compare third column with fourth column). More importantly, the PLC antagonist prevented the disassembly of F-actin from the cups, which remained fully formed yet unable to seal (Fig. 8, E and F). These observations support the concept that PI(4,5)P2 hydrolysis is required for the disassembly of actin that is associated with phagosomal closure.


Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis.

Scott CC, Dobson W, Botelho RJ, Coady-Osberg N, Chavrier P, Knecht DA, Heath C, Stahl P, Grinstein S - J. Cell Biol. (2005)

Effect of PLC inhibition on the completion of phagocytosis. RAW cells transiently transfected with PHPLCδ-GFP were either left untreated (A and B), treated with 100 μM LY294002 for 30 min (C and D), treated with 1 μM U73122 for 10 min (not depicted), or treated with 100 μM LY294002 for 30 min, with 1 μM U73122 present for the last 10 min of the incubation, followed by removal of LY294002 while maintaining the U73122 for an additional 10 min (E and F). The cells were then exposed to IgG-opsonized particles for 10 min and finally fixed, permeabilized, and stained with rhodamine-phalloidin (B, D, and F). PHPLCδ-GFP fluorescence is shown in A, C, and E. Bars, 5 μm. (G) The phagocytic index of cells treated as above was determined as described in Materials and methods. Data are the means ± SEM of three experiments, each scoring at least 50 cells.
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fig8: Effect of PLC inhibition on the completion of phagocytosis. RAW cells transiently transfected with PHPLCδ-GFP were either left untreated (A and B), treated with 100 μM LY294002 for 30 min (C and D), treated with 1 μM U73122 for 10 min (not depicted), or treated with 100 μM LY294002 for 30 min, with 1 μM U73122 present for the last 10 min of the incubation, followed by removal of LY294002 while maintaining the U73122 for an additional 10 min (E and F). The cells were then exposed to IgG-opsonized particles for 10 min and finally fixed, permeabilized, and stained with rhodamine-phalloidin (B, D, and F). PHPLCδ-GFP fluorescence is shown in A, C, and E. Bars, 5 μm. (G) The phagocytic index of cells treated as above was determined as described in Materials and methods. Data are the means ± SEM of three experiments, each scoring at least 50 cells.
Mentions: When added before exposure of the cells to phagocytic targets, U73122 prevented cup formation and failed to provide evidence of the role of PI(4,5)P2 hydrolysis in cytoskeletal disassembly during the course of particle internalization. To circumvent the effects of PLC inhibitors on the early stages of actin remodeling, we attempted to use the inhibitors only after phagocytosis had been initiated. However, their slow permeation rate requires extended incubation periods, precluding this experimental paradigm. Instead, we sought to design experiments where phagocytosis could be arrested after formation of the phagocytic cup, but before completion of phagocytosis, affording us the opportunity to load the cells with PLC antagonists and assess their effect on actin disassembly. This was made possible by the use of LY294002, a reversible PI3-K inhibitor (Vlahos et al., 1994). Treatment with PI3-K inhibitors arrested phagocytosis at an intermediate stage, after actin assembly and partial extension of pseudopodia (Fig. 8, C and D; Araki et al., 1996; Cox et al., 1999; Vieira et al., 2001). The profound inhibition of phagocytosis exerted by LY294002 is illustrated in Fig. 8 G. Unlike wortmannin, which covalently inactivates PI3-K, LY294002 is reversible and most of the phagocytic activity was restored shortly after the inhibitor was removed (Fig. 8 G, second column). The actin accumulated at the aborted phagocytic cup disappeared almost entirely from the periphery of the formed phagosomes when the inhibitor was removed (not depicted). This enabled us to abort phagocytosis after actin cups formed, and subsequently incubate the cells for 10 min with U73122 to allow its entry to the cells. LY294002 was then removed while maintaining U73122 in the incubation medium, and the effects of inhibition of PLC on phagocytosis and on the disassembly of the actin cup were monitored. Fig. 8 G shows that U73122 was as potent a blocker of phagocytosis when added after cup formation, as it was when added before phagocytosis (compare third column with fourth column). More importantly, the PLC antagonist prevented the disassembly of F-actin from the cups, which remained fully formed yet unable to seal (Fig. 8, E and F). These observations support the concept that PI(4,5)P2 hydrolysis is required for the disassembly of actin that is associated with phagosomal closure.

Bottom Line: Although actin was found to disappear from the base of the forming phagosome before sealing was complete, Rac1/Cdc42 activity persisted, suggesting that termination of GTPase activity is not the main determinant of actin disassembly.Furthermore, fully internalized phagosomes engineered to associate constitutively with active Rac1 showed little associated F-actin.These observations suggest that hydrolysis of PI(4,5)P(2) dictates the remodeling of actin necessary for completion of phagocytosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.

ABSTRACT
The Rho GTPases play a critical role in initiating actin polymerization during phagocytosis. In contrast, the factors directing the disassembly of F-actin required for fission of the phagocytic vacuole are ill defined. We used fluorescent chimeric proteins to monitor the dynamics of association of actin and active Cdc42 and Rac1 with the forming phagosome. Although actin was found to disappear from the base of the forming phagosome before sealing was complete, Rac1/Cdc42 activity persisted, suggesting that termination of GTPase activity is not the main determinant of actin disassembly. Furthermore, fully internalized phagosomes engineered to associate constitutively with active Rac1 showed little associated F-actin. The disappearance of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) from the phagosomal membrane closely paralleled the course of actin disassembly. Furthermore, inhibition of PI(4,5)P(2) hydrolysis or increased PI(4,5)P(2) generation by overexpression of phosphatidylinositol phosphate kinase I prevented the actin disassembly necessary for the completion of phagocytosis. These observations suggest that hydrolysis of PI(4,5)P(2) dictates the remodeling of actin necessary for completion of phagocytosis.

Show MeSH
Related in: MedlinePlus