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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 F-actin distribution. IgG-opsonized latex beads were added to RAW cells that were otherwise untreated (A–C) or that had been pretreated with 1 μM U73122 for 10 min (D–F). After 15 min at 37°C, the cells were cooled to 4°C and extracellular beads identified by addition of FITC-conjugated secondary antibody (B and E). The cells were next fixed, permeabilized, and stained with rhodamine-phalloidin (A and D). Corresponding differential interference contrast (DIC) images are shown in C and F. Insets show a magnification of the area indicated by the square in the main panels. Open arrows point to externally accessible beads and closed arrows point to sealed phagosomes. (G) Cortical F-actin thickness, quantified from line scans (white bars) and total cortical actin, calculated by integration (black bars) in control and in U73122-treated cells. Data are the means ± SEM of three experiments, each scoring at least 20 cells. (H) High throughput analysis of F-actin during phagocytosis, determined by averaging the integrated fluorescence of rhodamine-phalloidin in populations of cells. Circles, moderate number of beads. Triangles, high number of beads. Stars, moderate number of beads added after pretreatment with 5 μM U73122 for 10 min. Data show the integrated fluorescence of at least 2,500 cells per time point. Standard error bars were smaller than the size of the symbols.
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fig7: Effect of PLC inhibition on F-actin distribution. IgG-opsonized latex beads were added to RAW cells that were otherwise untreated (A–C) or that had been pretreated with 1 μM U73122 for 10 min (D–F). After 15 min at 37°C, the cells were cooled to 4°C and extracellular beads identified by addition of FITC-conjugated secondary antibody (B and E). The cells were next fixed, permeabilized, and stained with rhodamine-phalloidin (A and D). Corresponding differential interference contrast (DIC) images are shown in C and F. Insets show a magnification of the area indicated by the square in the main panels. Open arrows point to externally accessible beads and closed arrows point to sealed phagosomes. (G) Cortical F-actin thickness, quantified from line scans (white bars) and total cortical actin, calculated by integration (black bars) in control and in U73122-treated cells. Data are the means ± SEM of three experiments, each scoring at least 20 cells. (H) High throughput analysis of F-actin during phagocytosis, determined by averaging the integrated fluorescence of rhodamine-phalloidin in populations of cells. Circles, moderate number of beads. Triangles, high number of beads. Stars, moderate number of beads added after pretreatment with 5 μM U73122 for 10 min. Data show the integrated fluorescence of at least 2,500 cells per time point. Standard error bars were smaller than the size of the symbols.

Mentions: The preceding findings imply that inhibitors of PLC must block phagocytosis at a step preceding the disassembly of phagosomal actin. We therefore analyzed in greater detail the stage at which phagocytosis was arrested in RAW cells treated with U73122. Compared with untreated cells, which displayed distinct actin recruitment to sites of phagocytosis (Fig. 7, A–C), cells pretreated with the PLC inhibitor showed little accumulation of actin at the sites where opsonized particles adhered (Fig. 7, D–F). Instead, the cells appeared rounder, with a thick layer of submembranous F-actin. The increased association of actin with the membrane could be documented both by measurement of the thickness of the actin layer or by integration of the amount of subcellular F-actin in single cells (Fig. 7 G), and by quantitation of total cellular F-actin in populations (Fig. 7 H). These findings suggest that increased deposition of cortical F-actin before exposure to phagocytic particles increased the rigidity of the membrane, impairing the ability of the cells to extend pseudopods and ingest particles.


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 F-actin distribution. IgG-opsonized latex beads were added to RAW cells that were otherwise untreated (A–C) or that had been pretreated with 1 μM U73122 for 10 min (D–F). After 15 min at 37°C, the cells were cooled to 4°C and extracellular beads identified by addition of FITC-conjugated secondary antibody (B and E). The cells were next fixed, permeabilized, and stained with rhodamine-phalloidin (A and D). Corresponding differential interference contrast (DIC) images are shown in C and F. Insets show a magnification of the area indicated by the square in the main panels. Open arrows point to externally accessible beads and closed arrows point to sealed phagosomes. (G) Cortical F-actin thickness, quantified from line scans (white bars) and total cortical actin, calculated by integration (black bars) in control and in U73122-treated cells. Data are the means ± SEM of three experiments, each scoring at least 20 cells. (H) High throughput analysis of F-actin during phagocytosis, determined by averaging the integrated fluorescence of rhodamine-phalloidin in populations of cells. Circles, moderate number of beads. Triangles, high number of beads. Stars, moderate number of beads added after pretreatment with 5 μM U73122 for 10 min. Data show the integrated fluorescence of at least 2,500 cells per time point. Standard error bars were smaller than the size of the symbols.
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Related In: Results  -  Collection

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fig7: Effect of PLC inhibition on F-actin distribution. IgG-opsonized latex beads were added to RAW cells that were otherwise untreated (A–C) or that had been pretreated with 1 μM U73122 for 10 min (D–F). After 15 min at 37°C, the cells were cooled to 4°C and extracellular beads identified by addition of FITC-conjugated secondary antibody (B and E). The cells were next fixed, permeabilized, and stained with rhodamine-phalloidin (A and D). Corresponding differential interference contrast (DIC) images are shown in C and F. Insets show a magnification of the area indicated by the square in the main panels. Open arrows point to externally accessible beads and closed arrows point to sealed phagosomes. (G) Cortical F-actin thickness, quantified from line scans (white bars) and total cortical actin, calculated by integration (black bars) in control and in U73122-treated cells. Data are the means ± SEM of three experiments, each scoring at least 20 cells. (H) High throughput analysis of F-actin during phagocytosis, determined by averaging the integrated fluorescence of rhodamine-phalloidin in populations of cells. Circles, moderate number of beads. Triangles, high number of beads. Stars, moderate number of beads added after pretreatment with 5 μM U73122 for 10 min. Data show the integrated fluorescence of at least 2,500 cells per time point. Standard error bars were smaller than the size of the symbols.
Mentions: The preceding findings imply that inhibitors of PLC must block phagocytosis at a step preceding the disassembly of phagosomal actin. We therefore analyzed in greater detail the stage at which phagocytosis was arrested in RAW cells treated with U73122. Compared with untreated cells, which displayed distinct actin recruitment to sites of phagocytosis (Fig. 7, A–C), cells pretreated with the PLC inhibitor showed little accumulation of actin at the sites where opsonized particles adhered (Fig. 7, D–F). Instead, the cells appeared rounder, with a thick layer of submembranous F-actin. The increased association of actin with the membrane could be documented both by measurement of the thickness of the actin layer or by integration of the amount of subcellular F-actin in single cells (Fig. 7 G), and by quantitation of total cellular F-actin in populations (Fig. 7 H). These findings suggest that increased deposition of cortical F-actin before exposure to phagocytic particles increased the rigidity of the membrane, impairing the ability of the cells to extend pseudopods and ingest particles.

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