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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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Association of PI(4,5)P2 with forming phagosomes. Phagocytosis was initiated by addition of IgG-opsonized latex beads to RAW cells transiently transfected with PHPLCδ-GFP. Fluorescence was monitored by confocal microscopy. (A–F) Representative time course. The numbers indicate the time in seconds after a bead makes contact with the cell. Bar, 5 μm. (G) The phagosomal accumulation of PHPLCδ-GFP above the cytosolic level was quantified, normalized, and binned into 20-s intervals as in Fig. 1 (solid line). Data are means ± SEM of eight individual determinations. The dashed line is a reproduction of the GFP-actin data of Fig. 1, for comparison. The curves were not significantly different (P > 0.05) at any point.
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fig5: Association of PI(4,5)P2 with forming phagosomes. Phagocytosis was initiated by addition of IgG-opsonized latex beads to RAW cells transiently transfected with PHPLCδ-GFP. Fluorescence was monitored by confocal microscopy. (A–F) Representative time course. The numbers indicate the time in seconds after a bead makes contact with the cell. Bar, 5 μm. (G) The phagosomal accumulation of PHPLCδ-GFP above the cytosolic level was quantified, normalized, and binned into 20-s intervals as in Fig. 1 (solid line). Data are means ± SEM of eight individual determinations. The dashed line is a reproduction of the GFP-actin data of Fig. 1, for comparison. The curves were not significantly different (P > 0.05) at any point.

Mentions: Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays multiple roles in the nucleation, elongation, and bundling of actin filaments (Hilpela et al., 2004). It is therefore conceivable that this phosphoinositide contributes to the remodeling of F-actin during phagocytosis. We therefore compared the distribution and dynamics of PI(4,5)P2 during phagocytosis with that of actin. A chimeric construct consisting of GFP attached to the PH domain of PLCδ (PHPLCδ-GFP; Stauffer et al., 1998; Varnai and Balla, 1998; Botelho et al., 2000) was used to monitor PI(4,5)P2 in live macrophages during the course of particle ingestion. As illustrated in Fig. 5, PI(4,5)P2 is present throughout the plasmalemma before phagocytosis and is clearly visible in the advancing pseudopods during the early stages of particle ingestion (A–C). However, the density of the inositide drops sharply at the base of the phagosome as the pseudopods meet at the top of the particle. PI(4,5)P2 in fact begins to disappear from the base of the phagocytic cup before sealing (Fig. 5, C and D), which is more evident when larger (e.g., 8–μm diam) particles are used (Botelho et al., 2000). The hydrolysis of PI(4,5)P2 during phagocytosis can be validated by conventional chemical methods such as thin layer chromatography (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200412162/DC1), although such global methods are not optimal to detect localized changes. The dynamics of association of PI(4,5)P2 with the phagocytic particle is summarized in Fig. 5 G, where the course of actin assembly is also shown (dashed line) for comparison. Clearly, the asymmetric and rapid disappearance of PI(4,5)P2 from forming phagosomes closely parallels the spatial and temporal pattern of F-actin disassembly. Because PI(4,5)P2 is an important factor in the control of actin polymerization, it is conceivable that hydrolysis of the inositide dictates the disassembly of actin from sealing phagosomes.


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)

Association of PI(4,5)P2 with forming phagosomes. Phagocytosis was initiated by addition of IgG-opsonized latex beads to RAW cells transiently transfected with PHPLCδ-GFP. Fluorescence was monitored by confocal microscopy. (A–F) Representative time course. The numbers indicate the time in seconds after a bead makes contact with the cell. Bar, 5 μm. (G) The phagosomal accumulation of PHPLCδ-GFP above the cytosolic level was quantified, normalized, and binned into 20-s intervals as in Fig. 1 (solid line). Data are means ± SEM of eight individual determinations. The dashed line is a reproduction of the GFP-actin data of Fig. 1, for comparison. The curves were not significantly different (P > 0.05) at any point.
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Related In: Results  -  Collection

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fig5: Association of PI(4,5)P2 with forming phagosomes. Phagocytosis was initiated by addition of IgG-opsonized latex beads to RAW cells transiently transfected with PHPLCδ-GFP. Fluorescence was monitored by confocal microscopy. (A–F) Representative time course. The numbers indicate the time in seconds after a bead makes contact with the cell. Bar, 5 μm. (G) The phagosomal accumulation of PHPLCδ-GFP above the cytosolic level was quantified, normalized, and binned into 20-s intervals as in Fig. 1 (solid line). Data are means ± SEM of eight individual determinations. The dashed line is a reproduction of the GFP-actin data of Fig. 1, for comparison. The curves were not significantly different (P > 0.05) at any point.
Mentions: Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays multiple roles in the nucleation, elongation, and bundling of actin filaments (Hilpela et al., 2004). It is therefore conceivable that this phosphoinositide contributes to the remodeling of F-actin during phagocytosis. We therefore compared the distribution and dynamics of PI(4,5)P2 during phagocytosis with that of actin. A chimeric construct consisting of GFP attached to the PH domain of PLCδ (PHPLCδ-GFP; Stauffer et al., 1998; Varnai and Balla, 1998; Botelho et al., 2000) was used to monitor PI(4,5)P2 in live macrophages during the course of particle ingestion. As illustrated in Fig. 5, PI(4,5)P2 is present throughout the plasmalemma before phagocytosis and is clearly visible in the advancing pseudopods during the early stages of particle ingestion (A–C). However, the density of the inositide drops sharply at the base of the phagosome as the pseudopods meet at the top of the particle. PI(4,5)P2 in fact begins to disappear from the base of the phagocytic cup before sealing (Fig. 5, C and D), which is more evident when larger (e.g., 8–μm diam) particles are used (Botelho et al., 2000). The hydrolysis of PI(4,5)P2 during phagocytosis can be validated by conventional chemical methods such as thin layer chromatography (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200412162/DC1), although such global methods are not optimal to detect localized changes. The dynamics of association of PI(4,5)P2 with the phagocytic particle is summarized in Fig. 5 G, where the course of actin assembly is also shown (dashed line) for comparison. Clearly, the asymmetric and rapid disappearance of PI(4,5)P2 from forming phagosomes closely parallels the spatial and temporal pattern of F-actin disassembly. Because PI(4,5)P2 is an important factor in the control of actin polymerization, it is conceivable that hydrolysis of the inositide dictates the disassembly of actin from sealing phagosomes.

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