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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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Disappearance of PI(4,5)P2 from Rac1-V1–induced phagosomes. RBL-2H3 cells engineered to express stably the two constructs described in Fig. 4 were transiently transfected with PHPLCδ-GFP (A). Beads coated with anti-CD25 antibodies were then added and the cells were incubated for 60 min at 37°C, then rapidly cooled to 4°C and treated with Cy5-conjugated anti–mouse antibodies to identify beads that were accessible from the medium, i.e., not completely internalized (C). A small amount of light refracted by latex is visible in both internal and external beads. This refraction is apparent only at the center of the beads and is clearly distinguishable from the more peripheral antibody labeling. The cells were next fixed, permeabilized and immunostained with anti-myc antibodies to reveal the location of myc-Rac1-V12 (B). Open arrows point to externally accessible beads and closed arrows point to sealed phagosomes. Bar, 5 μm. Insets show a magnification of the area indicated by the square in the main panels. (D) Phagocytosis was induced in the transfected RBL-2H3 cells as described in Fig. 4. The cells were otherwise untreated, or were pretreated with the PLC inhibitors U73122 (1 μM) or ET-18-OCH3 (25 μM) for 10 min before phagocytosis. Extracellular beads were labeled and the number of beads ingested after 20 min was quantified under a fluorescence microscope. The phagocytic index was normalized to allow comparison of multiple experiments. The data are the means ± SEM of three experiments, each scoring at least 50 cells.
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fig6: Disappearance of PI(4,5)P2 from Rac1-V1–induced phagosomes. RBL-2H3 cells engineered to express stably the two constructs described in Fig. 4 were transiently transfected with PHPLCδ-GFP (A). Beads coated with anti-CD25 antibodies were then added and the cells were incubated for 60 min at 37°C, then rapidly cooled to 4°C and treated with Cy5-conjugated anti–mouse antibodies to identify beads that were accessible from the medium, i.e., not completely internalized (C). A small amount of light refracted by latex is visible in both internal and external beads. This refraction is apparent only at the center of the beads and is clearly distinguishable from the more peripheral antibody labeling. The cells were next fixed, permeabilized and immunostained with anti-myc antibodies to reveal the location of myc-Rac1-V12 (B). Open arrows point to externally accessible beads and closed arrows point to sealed phagosomes. Bar, 5 μm. Insets show a magnification of the area indicated by the square in the main panels. (D) Phagocytosis was induced in the transfected RBL-2H3 cells as described in Fig. 4. The cells were otherwise untreated, or were pretreated with the PLC inhibitors U73122 (1 μM) or ET-18-OCH3 (25 μM) for 10 min before phagocytosis. Extracellular beads were labeled and the number of beads ingested after 20 min was quantified under a fluorescence microscope. The phagocytic index was normalized to allow comparison of multiple experiments. The data are the means ± SEM of three experiments, each scoring at least 50 cells.

Mentions: As described in connection with Fig. 4, actin dissociates from phagosomes despite the sustained presence of active Rac1. We therefore investigated whether this dissociation is similarly accompanied by the hydrolysis of PI(4,5)P2 from myc-Rac1-V12–induced phagosomes. To this end, we transiently transfected the engineered RBL phagocytes with the PH domain-GFP chimera to monitor the fate of PI(4,5)P2. After internalization of the particles the amount of PI(4,5)P2 associated with the phagocytic vacuoles is greatly reduced compared with the originating plasma membrane (Fig. 6 A). It is noteworthy that this PI(4,5)P2 depletion occurred in spite of the continued presence of active Rac1 that can directly associate with and activate phosphatidylinositol phosphate kinase type I (PIPKI), the enzyme responsible for PI(4,5)P2 generation (Tolias and Carpenter, 2000; Weernink et al., 2004). Therefore, stimulation of PI(4,5)P2 catabolism probably accounts for the observed drop in its density during phagocytosis, whereas inhibition of synthesis is less likely.


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)

Disappearance of PI(4,5)P2 from Rac1-V1–induced phagosomes. RBL-2H3 cells engineered to express stably the two constructs described in Fig. 4 were transiently transfected with PHPLCδ-GFP (A). Beads coated with anti-CD25 antibodies were then added and the cells were incubated for 60 min at 37°C, then rapidly cooled to 4°C and treated with Cy5-conjugated anti–mouse antibodies to identify beads that were accessible from the medium, i.e., not completely internalized (C). A small amount of light refracted by latex is visible in both internal and external beads. This refraction is apparent only at the center of the beads and is clearly distinguishable from the more peripheral antibody labeling. The cells were next fixed, permeabilized and immunostained with anti-myc antibodies to reveal the location of myc-Rac1-V12 (B). Open arrows point to externally accessible beads and closed arrows point to sealed phagosomes. Bar, 5 μm. Insets show a magnification of the area indicated by the square in the main panels. (D) Phagocytosis was induced in the transfected RBL-2H3 cells as described in Fig. 4. The cells were otherwise untreated, or were pretreated with the PLC inhibitors U73122 (1 μM) or ET-18-OCH3 (25 μM) for 10 min before phagocytosis. Extracellular beads were labeled and the number of beads ingested after 20 min was quantified under a fluorescence microscope. The phagocytic index was normalized to allow comparison of multiple experiments. The data are the means ± SEM of three experiments, each scoring at least 50 cells.
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fig6: Disappearance of PI(4,5)P2 from Rac1-V1–induced phagosomes. RBL-2H3 cells engineered to express stably the two constructs described in Fig. 4 were transiently transfected with PHPLCδ-GFP (A). Beads coated with anti-CD25 antibodies were then added and the cells were incubated for 60 min at 37°C, then rapidly cooled to 4°C and treated with Cy5-conjugated anti–mouse antibodies to identify beads that were accessible from the medium, i.e., not completely internalized (C). A small amount of light refracted by latex is visible in both internal and external beads. This refraction is apparent only at the center of the beads and is clearly distinguishable from the more peripheral antibody labeling. The cells were next fixed, permeabilized and immunostained with anti-myc antibodies to reveal the location of myc-Rac1-V12 (B). Open arrows point to externally accessible beads and closed arrows point to sealed phagosomes. Bar, 5 μm. Insets show a magnification of the area indicated by the square in the main panels. (D) Phagocytosis was induced in the transfected RBL-2H3 cells as described in Fig. 4. The cells were otherwise untreated, or were pretreated with the PLC inhibitors U73122 (1 μM) or ET-18-OCH3 (25 μM) for 10 min before phagocytosis. Extracellular beads were labeled and the number of beads ingested after 20 min was quantified under a fluorescence microscope. The phagocytic index was normalized to allow comparison of multiple experiments. The data are the means ± SEM of three experiments, each scoring at least 50 cells.
Mentions: As described in connection with Fig. 4, actin dissociates from phagosomes despite the sustained presence of active Rac1. We therefore investigated whether this dissociation is similarly accompanied by the hydrolysis of PI(4,5)P2 from myc-Rac1-V12–induced phagosomes. To this end, we transiently transfected the engineered RBL phagocytes with the PH domain-GFP chimera to monitor the fate of PI(4,5)P2. After internalization of the particles the amount of PI(4,5)P2 associated with the phagocytic vacuoles is greatly reduced compared with the originating plasma membrane (Fig. 6 A). It is noteworthy that this PI(4,5)P2 depletion occurred in spite of the continued presence of active Rac1 that can directly associate with and activate phosphatidylinositol phosphate kinase type I (PIPKI), the enzyme responsible for PI(4,5)P2 generation (Tolias and Carpenter, 2000; Weernink et al., 2004). Therefore, stimulation of PI(4,5)P2 catabolism probably accounts for the observed drop in its density during phagocytosis, whereas inhibition of synthesis is less likely.

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