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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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Comparison of Rac/Cdc42 activity and F-actin in forming phagosomes. RAW cells were transiently transfected with the PBD-YFP chimeric probe (B, C, E, and F, green) or with Cdc42Q61L-GFP (H and I, green) and exposed to IgG-opsonized latex beads for the time indicated. The cells were immediately fixed with 4% PFA, permeabilized and stained with rhodamine-phalloidin to visualize F-actin (A, C, D, F, G, and I, red). (A–C) PBD-YFP expressing cells fixed 2 min after addition of beads. (D–F) PBD-YFP expressing cells fixed 4 min after addition of beads. (G–I) Cdc42Q61L-GFP expressing cells fixed after 4 min. Open arrows point to forming phagosomes and closed arrows point to sealed phagosomes. Inset in G–I shows enlargement of phagosome denoted by the box in main panel. Bars, 5 μm. Images are representative of at least three experiments of each type.
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fig3: Comparison of Rac/Cdc42 activity and F-actin in forming phagosomes. RAW cells were transiently transfected with the PBD-YFP chimeric probe (B, C, E, and F, green) or with Cdc42Q61L-GFP (H and I, green) and exposed to IgG-opsonized latex beads for the time indicated. The cells were immediately fixed with 4% PFA, permeabilized and stained with rhodamine-phalloidin to visualize F-actin (A, C, D, F, G, and I, red). (A–C) PBD-YFP expressing cells fixed 2 min after addition of beads. (D–F) PBD-YFP expressing cells fixed 4 min after addition of beads. (G–I) Cdc42Q61L-GFP expressing cells fixed after 4 min. Open arrows point to forming phagosomes and closed arrows point to sealed phagosomes. Inset in G–I shows enlargement of phagosome denoted by the box in main panel. Bars, 5 μm. Images are representative of at least three experiments of each type.

Mentions: Because of the similar spectral properties of GFP and YFP, actin recruitment and the activation of Rac/Cdc42 were studied separately in the experiments of Figs. 1 and 2. It was conceivable that the differences noted were fortuitous, or that the expression of the PBD-YFP had resulted in an artifactual retardation of phagocytosis. To eliminate these possibilities, we analyzed actin distribution in the same cells transfected with the PBD-YFP probe. This was accomplished by fixation of cells at various stages during phagocytosis, followed by permeabilization and staining of F-actin using labeled phalloidin. As shown in Fig. 3 (A–C), there was an exquisite colocalization of F-actin with PBD-YFP on the forming phagosome at the early (1–2 min) stages of phagocytosis. Note that the less abundant cortical actin found in regions of the membrane not involved in formation of the phagocytic cup was not associated with PBD-YFP, implying that it remains polymerized in a manner that is independent of Rac/Cdc42 activation. The asymmetric accumulation of actin observed after phagosomal closure was also detectable using phalloidin (Fig. 3 D), validating the GFP-actin observations. More importantly, the activation of Rac/Cdc42 clearly persisted in recently closed phagosomes at a time when actin had nearly disappeared from their base (Fig. 3, E and F). These findings confirm the asynchrony between actin disassembly and the deactivation of Rac/Cdc42 and suggest that other factors are involved in the dissociation of actin.


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)

Comparison of Rac/Cdc42 activity and F-actin in forming phagosomes. RAW cells were transiently transfected with the PBD-YFP chimeric probe (B, C, E, and F, green) or with Cdc42Q61L-GFP (H and I, green) and exposed to IgG-opsonized latex beads for the time indicated. The cells were immediately fixed with 4% PFA, permeabilized and stained with rhodamine-phalloidin to visualize F-actin (A, C, D, F, G, and I, red). (A–C) PBD-YFP expressing cells fixed 2 min after addition of beads. (D–F) PBD-YFP expressing cells fixed 4 min after addition of beads. (G–I) Cdc42Q61L-GFP expressing cells fixed after 4 min. Open arrows point to forming phagosomes and closed arrows point to sealed phagosomes. Inset in G–I shows enlargement of phagosome denoted by the box in main panel. Bars, 5 μm. Images are representative of at least three experiments of each type.
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Related In: Results  -  Collection

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fig3: Comparison of Rac/Cdc42 activity and F-actin in forming phagosomes. RAW cells were transiently transfected with the PBD-YFP chimeric probe (B, C, E, and F, green) or with Cdc42Q61L-GFP (H and I, green) and exposed to IgG-opsonized latex beads for the time indicated. The cells were immediately fixed with 4% PFA, permeabilized and stained with rhodamine-phalloidin to visualize F-actin (A, C, D, F, G, and I, red). (A–C) PBD-YFP expressing cells fixed 2 min after addition of beads. (D–F) PBD-YFP expressing cells fixed 4 min after addition of beads. (G–I) Cdc42Q61L-GFP expressing cells fixed after 4 min. Open arrows point to forming phagosomes and closed arrows point to sealed phagosomes. Inset in G–I shows enlargement of phagosome denoted by the box in main panel. Bars, 5 μm. Images are representative of at least three experiments of each type.
Mentions: Because of the similar spectral properties of GFP and YFP, actin recruitment and the activation of Rac/Cdc42 were studied separately in the experiments of Figs. 1 and 2. It was conceivable that the differences noted were fortuitous, or that the expression of the PBD-YFP had resulted in an artifactual retardation of phagocytosis. To eliminate these possibilities, we analyzed actin distribution in the same cells transfected with the PBD-YFP probe. This was accomplished by fixation of cells at various stages during phagocytosis, followed by permeabilization and staining of F-actin using labeled phalloidin. As shown in Fig. 3 (A–C), there was an exquisite colocalization of F-actin with PBD-YFP on the forming phagosome at the early (1–2 min) stages of phagocytosis. Note that the less abundant cortical actin found in regions of the membrane not involved in formation of the phagocytic cup was not associated with PBD-YFP, implying that it remains polymerized in a manner that is independent of Rac/Cdc42 activation. The asymmetric accumulation of actin observed after phagosomal closure was also detectable using phalloidin (Fig. 3 D), validating the GFP-actin observations. More importantly, the activation of Rac/Cdc42 clearly persisted in recently closed phagosomes at a time when actin had nearly disappeared from their base (Fig. 3, E and F). These findings confirm the asynchrony between actin disassembly and the deactivation of Rac/Cdc42 and suggest that other factors are involved in the dissociation of actin.

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