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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 GFP-actin with forming phagosomes. Phagocytosis was initiated by addition of IgG-opsonized latex beads (3.1-μm diam) to RAW cells stably transfected with GFP-actin. Fluorescence was monitored by confocal microscopy. (A–F) Representative time course. The numbers indicate the time in seconds after a bead makes contact at the site indicated by the open arrow in A. Note that another bead has just been engulfed near the top of the cell and still shows remnant actin at the site of sealing. Insets show enlargements of the phagosome noted by arrow. Open arrows point to forming phagosomes and closed arrows point to formed, sealed phagosomes. Bar, 5 μm. (G) The phagosomal accumulation of GFP-actin above the cytosolic level was quantified and binned into 20-s intervals as detailed in Materials and methods. Abscissa: time in seconds after the bead made contact with cell. Ordinate: relative fluorescence. To allow comparison between experiments, phagosomal fluorescence was normalized to the maximum recorded for each individual phagosome. Data are means ± SEM of seven individual determinations.
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fig1: Association of GFP-actin with forming phagosomes. Phagocytosis was initiated by addition of IgG-opsonized latex beads (3.1-μm diam) to RAW cells stably transfected with GFP-actin. Fluorescence was monitored by confocal microscopy. (A–F) Representative time course. The numbers indicate the time in seconds after a bead makes contact at the site indicated by the open arrow in A. Note that another bead has just been engulfed near the top of the cell and still shows remnant actin at the site of sealing. Insets show enlargements of the phagosome noted by arrow. Open arrows point to forming phagosomes and closed arrows point to formed, sealed phagosomes. Bar, 5 μm. (G) The phagosomal accumulation of GFP-actin above the cytosolic level was quantified and binned into 20-s intervals as detailed in Materials and methods. Abscissa: time in seconds after the bead made contact with cell. Ordinate: relative fluorescence. To allow comparison between experiments, phagosomal fluorescence was normalized to the maximum recorded for each individual phagosome. Data are means ± SEM of seven individual determinations.

Mentions: To study actin dynamics during phagocytosis, RAW 264.7 macrophages (referred to hereafter as RAW cells) were stably transfected with GFP-actin. Phagocytosis was induced by exposure of the cells to latex beads opsonized with IgG and the distribution of actin was monitored in live cells by laser confocal microscopy. As illustrated in Fig. 1 and reported previously (Allison et al., 1971; Henry et al., 2004), there is a marked accumulation of actin in the region of the forming phagosome. At the earliest stages, extension of actin-rich pseudopods around the latex bead was consistently observed (Fig. 1, B and C). Upon phagosome closure, when the advancing pseudopods meet and fuse, actin transiently surrounds the entire phagosome, appearing as a nearly homogeneous, continuous ring (Fig. 1 D). Importantly, actin disassembly occurs asymmetrically after phagosomal sealing, with loss of fluorescence occurring initially at the base of the phagosome (Fig. 1 E, the innermost half), whereas a “cap” of actin persists for at least 240 s in the space between the phagosome and the plasmalemma. Eventually, this outermost cap also disassembles and actin accumulation is no longer detectable around the internalized phagosome (Fig. 1 F). For beads of 3.1-μm diam, such as those used in Fig. 1, the entire process is completed in 4–5 min at 37°C. Predictably, this time course is slightly more protracted than that reported by Defacque and colleagues (Defacque et al., 2000), who monitored actin polymerization using smaller (1 μm) beads. Slower kinetics of actin association was reported by Henry et al. (2004) who used somewhat larger particles, namely sheep RBCs.


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 GFP-actin with forming phagosomes. Phagocytosis was initiated by addition of IgG-opsonized latex beads (3.1-μm diam) to RAW cells stably transfected with GFP-actin. Fluorescence was monitored by confocal microscopy. (A–F) Representative time course. The numbers indicate the time in seconds after a bead makes contact at the site indicated by the open arrow in A. Note that another bead has just been engulfed near the top of the cell and still shows remnant actin at the site of sealing. Insets show enlargements of the phagosome noted by arrow. Open arrows point to forming phagosomes and closed arrows point to formed, sealed phagosomes. Bar, 5 μm. (G) The phagosomal accumulation of GFP-actin above the cytosolic level was quantified and binned into 20-s intervals as detailed in Materials and methods. Abscissa: time in seconds after the bead made contact with cell. Ordinate: relative fluorescence. To allow comparison between experiments, phagosomal fluorescence was normalized to the maximum recorded for each individual phagosome. Data are means ± SEM of seven individual determinations.
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Related In: Results  -  Collection

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fig1: Association of GFP-actin with forming phagosomes. Phagocytosis was initiated by addition of IgG-opsonized latex beads (3.1-μm diam) to RAW cells stably transfected with GFP-actin. Fluorescence was monitored by confocal microscopy. (A–F) Representative time course. The numbers indicate the time in seconds after a bead makes contact at the site indicated by the open arrow in A. Note that another bead has just been engulfed near the top of the cell and still shows remnant actin at the site of sealing. Insets show enlargements of the phagosome noted by arrow. Open arrows point to forming phagosomes and closed arrows point to formed, sealed phagosomes. Bar, 5 μm. (G) The phagosomal accumulation of GFP-actin above the cytosolic level was quantified and binned into 20-s intervals as detailed in Materials and methods. Abscissa: time in seconds after the bead made contact with cell. Ordinate: relative fluorescence. To allow comparison between experiments, phagosomal fluorescence was normalized to the maximum recorded for each individual phagosome. Data are means ± SEM of seven individual determinations.
Mentions: To study actin dynamics during phagocytosis, RAW 264.7 macrophages (referred to hereafter as RAW cells) were stably transfected with GFP-actin. Phagocytosis was induced by exposure of the cells to latex beads opsonized with IgG and the distribution of actin was monitored in live cells by laser confocal microscopy. As illustrated in Fig. 1 and reported previously (Allison et al., 1971; Henry et al., 2004), there is a marked accumulation of actin in the region of the forming phagosome. At the earliest stages, extension of actin-rich pseudopods around the latex bead was consistently observed (Fig. 1, B and C). Upon phagosome closure, when the advancing pseudopods meet and fuse, actin transiently surrounds the entire phagosome, appearing as a nearly homogeneous, continuous ring (Fig. 1 D). Importantly, actin disassembly occurs asymmetrically after phagosomal sealing, with loss of fluorescence occurring initially at the base of the phagosome (Fig. 1 E, the innermost half), whereas a “cap” of actin persists for at least 240 s in the space between the phagosome and the plasmalemma. Eventually, this outermost cap also disassembles and actin accumulation is no longer detectable around the internalized phagosome (Fig. 1 F). For beads of 3.1-μm diam, such as those used in Fig. 1, the entire process is completed in 4–5 min at 37°C. Predictably, this time course is slightly more protracted than that reported by Defacque and colleagues (Defacque et al., 2000), who monitored actin polymerization using smaller (1 μm) beads. Slower kinetics of actin association was reported by Henry et al. (2004) who used somewhat larger particles, namely sheep RBCs.

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