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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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Assessment of Rac/Cdc42 activity in forming phagosomes. Phagocytosis was initiated by addition of IgG-opsonized latex beads to RAW cells transiently transfected with PBD-YFP. 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 PBD-YFP above the cytosolic level was quantified, normalized and binned into 20-s intervals as in Fig. 1 (solid line). Abscissa: time in seconds after the bead made contact with cell. Ordinate: relative fluorescence, normalized as in Fig. 1 G. Data are means ± SEM of six individual determinations. The dashed line is a reproduction of the GFP-actin data of Fig. 1, for comparison. Asterisks indicate instances where the difference between the curves is significant (P < 0.05).
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fig2: Assessment of Rac/Cdc42 activity in forming phagosomes. Phagocytosis was initiated by addition of IgG-opsonized latex beads to RAW cells transiently transfected with PBD-YFP. 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 PBD-YFP above the cytosolic level was quantified, normalized and binned into 20-s intervals as in Fig. 1 (solid line). Abscissa: time in seconds after the bead made contact with cell. Ordinate: relative fluorescence, normalized as in Fig. 1 G. Data are means ± SEM of six individual determinations. The dashed line is a reproduction of the GFP-actin data of Fig. 1, for comparison. Asterisks indicate instances where the difference between the curves is significant (P < 0.05).

Mentions: The preceding observations indicate that actin depolymerization commences shortly after phagosomal closure and that it occurs asymmetrically. Because activation of Rac1 and Cdc42 is thought to underlie the assembly of actin at the phagosome (Cox et al., 1997; Caron and Hall, 1998; Massol et al., 1998) we considered the possibility that deactivation of the GTPases was responsible for the pattern of actin disassembly. To this end, we measured the kinetics of activation of the GTPases using a fusion protein consisting of the p21-binding domain of PAK fused to YFP (PBD-YFP; Srinivasan et al., 2003). In quiescent RAW cells the PBD-YFP probe was distributed almost exclusively in the cytosol, indicating minimal activation of Rac1 and Cdc42 (Fig. 2 A). Upon addition of opsonized beads, PBD-YFP accumulated in the budding pseudopods (Fig. 2 B) and was ultimately visible all around the phagosome, closely resembling the early stages of actin accumulation (Fig. 2, D and E). Unlike actin, however, the deactivation of Rac and/or Cdc42 occurred homogeneously throughout the phagosomal circumference (Fig. 2, E and F). Moreover, the association of PBD-YFP with the phagosome persisted after actin disassembly was obvious. The differential behavior of actin and of the active GTPases became more apparent when the kinetics of PBD-YFP association with the phagosome was quantified as described above. The results of six experiments, summarized in Fig. 2 G, indicate that Rac/Cdc42 activation is maximal only after ≈200 s and is still detectable after 300 s, clearly lagging behind the kinetics of actin assembly.


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)

Assessment of Rac/Cdc42 activity in forming phagosomes. Phagocytosis was initiated by addition of IgG-opsonized latex beads to RAW cells transiently transfected with PBD-YFP. 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 PBD-YFP above the cytosolic level was quantified, normalized and binned into 20-s intervals as in Fig. 1 (solid line). Abscissa: time in seconds after the bead made contact with cell. Ordinate: relative fluorescence, normalized as in Fig. 1 G. Data are means ± SEM of six individual determinations. The dashed line is a reproduction of the GFP-actin data of Fig. 1, for comparison. Asterisks indicate instances where the difference between the curves is significant (P < 0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171893&req=5

fig2: Assessment of Rac/Cdc42 activity in forming phagosomes. Phagocytosis was initiated by addition of IgG-opsonized latex beads to RAW cells transiently transfected with PBD-YFP. 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 PBD-YFP above the cytosolic level was quantified, normalized and binned into 20-s intervals as in Fig. 1 (solid line). Abscissa: time in seconds after the bead made contact with cell. Ordinate: relative fluorescence, normalized as in Fig. 1 G. Data are means ± SEM of six individual determinations. The dashed line is a reproduction of the GFP-actin data of Fig. 1, for comparison. Asterisks indicate instances where the difference between the curves is significant (P < 0.05).
Mentions: The preceding observations indicate that actin depolymerization commences shortly after phagosomal closure and that it occurs asymmetrically. Because activation of Rac1 and Cdc42 is thought to underlie the assembly of actin at the phagosome (Cox et al., 1997; Caron and Hall, 1998; Massol et al., 1998) we considered the possibility that deactivation of the GTPases was responsible for the pattern of actin disassembly. To this end, we measured the kinetics of activation of the GTPases using a fusion protein consisting of the p21-binding domain of PAK fused to YFP (PBD-YFP; Srinivasan et al., 2003). In quiescent RAW cells the PBD-YFP probe was distributed almost exclusively in the cytosol, indicating minimal activation of Rac1 and Cdc42 (Fig. 2 A). Upon addition of opsonized beads, PBD-YFP accumulated in the budding pseudopods (Fig. 2 B) and was ultimately visible all around the phagosome, closely resembling the early stages of actin accumulation (Fig. 2, D and E). Unlike actin, however, the deactivation of Rac and/or Cdc42 occurred homogeneously throughout the phagosomal circumference (Fig. 2, E and F). Moreover, the association of PBD-YFP with the phagosome persisted after actin disassembly was obvious. The differential behavior of actin and of the active GTPases became more apparent when the kinetics of PBD-YFP association with the phagosome was quantified as described above. The results of six experiments, summarized in Fig. 2 G, indicate that Rac/Cdc42 activation is maximal only after ≈200 s and is still detectable after 300 s, clearly lagging behind the kinetics of actin assembly.

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