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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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Actin association with myc-Rac1-V12–induced phagosomes. (A) Strategy used to generate phagosomes by recruitment of active Rac1 to the membrane. RBL-2H3 cells were stably transfected with two separate constructs: a soluble, myc-tagged Rac1-V12/FKBP2 chimera (myc-Rac1-V12) and a transmembrane CD25/FRB chimera (A). Association between the two chimeras is induced by addition of rapamycin (A′). Beads coated with anti-CD25 antibodies are then added to cross-link the complexes at defined sites (A′′). (B–D) After binding the beads, the cells were incubated for 10 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. The cells were next fixed, permeabilized, and stained for F-actin and immunostained with anti-myc antibodies to reveal the location of myc-Rac1-V12. (E–G) After binding the beads, the cells were incubated for 60 min at 37°C and treated as in B–D. Bars, 5 μm. Open arrows point to forming phagosomes and closed arrows point to sealed phagosomes. Insets show a magnification of the area indicated by the square in the main panels. Images are representative of three experiments.
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fig4: Actin association with myc-Rac1-V12–induced phagosomes. (A) Strategy used to generate phagosomes by recruitment of active Rac1 to the membrane. RBL-2H3 cells were stably transfected with two separate constructs: a soluble, myc-tagged Rac1-V12/FKBP2 chimera (myc-Rac1-V12) and a transmembrane CD25/FRB chimera (A). Association between the two chimeras is induced by addition of rapamycin (A′). Beads coated with anti-CD25 antibodies are then added to cross-link the complexes at defined sites (A′′). (B–D) After binding the beads, the cells were incubated for 10 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. The cells were next fixed, permeabilized, and stained for F-actin and immunostained with anti-myc antibodies to reveal the location of myc-Rac1-V12. (E–G) After binding the beads, the cells were incubated for 60 min at 37°C and treated as in B–D. Bars, 5 μm. Open arrows point to forming phagosomes and closed arrows point to sealed phagosomes. Insets show a magnification of the area indicated by the square in the main panels. Images are representative of three experiments.

Mentions: To more conclusively dissociate the disassembly of actin from the deactivation of Rac1 we sought to prolong the association of Rac1-GTP with the phagosomal membrane. This was accomplished using the system illustrated in Fig. 4 A, originally described by Castellano et al. (2000), designed to recruit constitutively active Rac1 (myc-Rac1-V12) to the cytoplasmic face of the plasma membrane at the site of bead attachment. The system consists of RBL cells transfected with two separate vectors: one encodes for a soluble form of myc-tagged Rac1-V12 and the other for a transmembrane membrane protein with an extracellular epitope (CD25). Each one of the constructs is in addition fused to different rapamycin-binding moieties. Upon addition of rapamycin, which is membrane permeant, the active Rac1 is recruited to the membrane, where it associates with the transmembrane construct (Fig. 4 A′). The latter can then be clustered on the surface by binding to beads bearing anti-CD25 antibodies (Fig. 4 A′′). The result of these joint maneuvers is the recruitment of constitutively active Rac1 to the cytosolic face of the membrane lining the bead. This was shown earlier to suffice for particle engulfment, recapitulating phagocytosis (Castellano et al., 2000). As shown in Fig. 4 C, the recruitment of Rac1 to the sites of bead attachment can be verified by immunostaining for the myc epitope linked to the GTPase. When the beads have not been fully internalized, as shown by their accessibility to externally added antibodies (Fig. 4 D), polymerization of F-actin can be readily demonstrated at sites where phagosomes are being formed, by staining with phalloidin derivatives (Fig. 4 B). At later times, the beads become fully internalized, because they can no longer be stained by externally added antibodies (Fig. 4 G), and are displaced from the cell periphery toward the center of the cell (Fig. 4 F). Remarkably, F-actin is no longer associated with these fully internalized beads, even though they remain lined by active Rac1, which was confirmed by immunostaining the myc epitope (Fig. 4, E and F). These findings clearly indicate that F-actin can dissociate from the phagosomal membrane despite the continued presence of active Rac1. Together with the preceding findings, these observations imply that factors other than deactivation of the Rho family GTPases are responsible for the rapid and asymmetric disassembly of phagosomal 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)

Actin association with myc-Rac1-V12–induced phagosomes. (A) Strategy used to generate phagosomes by recruitment of active Rac1 to the membrane. RBL-2H3 cells were stably transfected with two separate constructs: a soluble, myc-tagged Rac1-V12/FKBP2 chimera (myc-Rac1-V12) and a transmembrane CD25/FRB chimera (A). Association between the two chimeras is induced by addition of rapamycin (A′). Beads coated with anti-CD25 antibodies are then added to cross-link the complexes at defined sites (A′′). (B–D) After binding the beads, the cells were incubated for 10 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. The cells were next fixed, permeabilized, and stained for F-actin and immunostained with anti-myc antibodies to reveal the location of myc-Rac1-V12. (E–G) After binding the beads, the cells were incubated for 60 min at 37°C and treated as in B–D. Bars, 5 μm. Open arrows point to forming phagosomes and closed arrows point to sealed phagosomes. Insets show a magnification of the area indicated by the square in the main panels. Images are representative of three experiments.
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Related In: Results  -  Collection

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fig4: Actin association with myc-Rac1-V12–induced phagosomes. (A) Strategy used to generate phagosomes by recruitment of active Rac1 to the membrane. RBL-2H3 cells were stably transfected with two separate constructs: a soluble, myc-tagged Rac1-V12/FKBP2 chimera (myc-Rac1-V12) and a transmembrane CD25/FRB chimera (A). Association between the two chimeras is induced by addition of rapamycin (A′). Beads coated with anti-CD25 antibodies are then added to cross-link the complexes at defined sites (A′′). (B–D) After binding the beads, the cells were incubated for 10 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. The cells were next fixed, permeabilized, and stained for F-actin and immunostained with anti-myc antibodies to reveal the location of myc-Rac1-V12. (E–G) After binding the beads, the cells were incubated for 60 min at 37°C and treated as in B–D. Bars, 5 μm. Open arrows point to forming phagosomes and closed arrows point to sealed phagosomes. Insets show a magnification of the area indicated by the square in the main panels. Images are representative of three experiments.
Mentions: To more conclusively dissociate the disassembly of actin from the deactivation of Rac1 we sought to prolong the association of Rac1-GTP with the phagosomal membrane. This was accomplished using the system illustrated in Fig. 4 A, originally described by Castellano et al. (2000), designed to recruit constitutively active Rac1 (myc-Rac1-V12) to the cytoplasmic face of the plasma membrane at the site of bead attachment. The system consists of RBL cells transfected with two separate vectors: one encodes for a soluble form of myc-tagged Rac1-V12 and the other for a transmembrane membrane protein with an extracellular epitope (CD25). Each one of the constructs is in addition fused to different rapamycin-binding moieties. Upon addition of rapamycin, which is membrane permeant, the active Rac1 is recruited to the membrane, where it associates with the transmembrane construct (Fig. 4 A′). The latter can then be clustered on the surface by binding to beads bearing anti-CD25 antibodies (Fig. 4 A′′). The result of these joint maneuvers is the recruitment of constitutively active Rac1 to the cytosolic face of the membrane lining the bead. This was shown earlier to suffice for particle engulfment, recapitulating phagocytosis (Castellano et al., 2000). As shown in Fig. 4 C, the recruitment of Rac1 to the sites of bead attachment can be verified by immunostaining for the myc epitope linked to the GTPase. When the beads have not been fully internalized, as shown by their accessibility to externally added antibodies (Fig. 4 D), polymerization of F-actin can be readily demonstrated at sites where phagosomes are being formed, by staining with phalloidin derivatives (Fig. 4 B). At later times, the beads become fully internalized, because they can no longer be stained by externally added antibodies (Fig. 4 G), and are displaced from the cell periphery toward the center of the cell (Fig. 4 F). Remarkably, F-actin is no longer associated with these fully internalized beads, even though they remain lined by active Rac1, which was confirmed by immunostaining the myc epitope (Fig. 4, E and F). These findings clearly indicate that F-actin can dissociate from the phagosomal membrane despite the continued presence of active Rac1. Together with the preceding findings, these observations imply that factors other than deactivation of the Rho family GTPases are responsible for the rapid and asymmetric disassembly of phagosomal 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