Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of PIPKI overexpression on phagocytosis. RAW cells transfected with the plasmids indicated below were exposed to IgG-opsonized particles for 8–10 min, then analyzed by laser confocal microscopy either immediately (A and B) or after fixation and staining with Alexa 633-phalloidin (D) or rhodamine-phalloidin (E and F). (A and B) RAW cells transiently transfected with PIPKIβ-YFP and PHPLCδ-CFP. (C and D) Cells transiently transfected with PIPKIβ-YFP. (E and F) Cells transiently transfected with PIPKIγ-GFP. (G) Quantification of the phagocytic efficiency of untransfected cells (control) or cells transiently transfected with PIPKIα-CFP, PIPKIβ-YFP, or PIPKIγ-GFP. Data are the means ± SEM of three experiments, each scoring at least 100 cells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171893&req=5

fig9: Effect of PIPKI overexpression on phagocytosis. RAW cells transfected with the plasmids indicated below were exposed to IgG-opsonized particles for 8–10 min, then analyzed by laser confocal microscopy either immediately (A and B) or after fixation and staining with Alexa 633-phalloidin (D) or rhodamine-phalloidin (E and F). (A and B) RAW cells transiently transfected with PIPKIβ-YFP and PHPLCδ-CFP. (C and D) Cells transiently transfected with PIPKIβ-YFP. (E and F) Cells transiently transfected with PIPKIγ-GFP. (G) Quantification of the phagocytic efficiency of untransfected cells (control) or cells transiently transfected with PIPKIα-CFP, PIPKIβ-YFP, or PIPKIγ-GFP. Data are the means ± SEM of three experiments, each scoring at least 100 cells.

Mentions: The PIPKI isoforms distributed largely to the plasmalemma of RAW cells, in agreement with earlier observations in various cell types (Stephens et al., 1991; Doughman et al., 2003). Opsonized particles bound normally to the overexpressing cells and phagocytic cups formed in these cells. Actin could be seen to accumulate at the cups, as in normal cells (Fig. 9, D and F). Remarkably, closure of the phagosomes was aborted and engulfment was rarely completed. In multiple experiments, where 150 cells were counted, the phagocytic efficiency was inhibited by 95, 72, and 79% by PIPKI α, β, and γ, respectively. Direct assessment of PI(4,5)P2 under these conditions using CFP-tagged PH domain constructs in cells transfected with YFP-PIPKI confirmed that the phosphoinositide remains present and in fact is enriched at the base of the cup (Fig. 9, A and B). Although other explanations can be envisaged, these findings can be most simply explained by the persistence of PI(4,5)P2 at the base of the phagosome due to excessive synthesis.


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 PIPKI overexpression on phagocytosis. RAW cells transfected with the plasmids indicated below were exposed to IgG-opsonized particles for 8–10 min, then analyzed by laser confocal microscopy either immediately (A and B) or after fixation and staining with Alexa 633-phalloidin (D) or rhodamine-phalloidin (E and F). (A and B) RAW cells transiently transfected with PIPKIβ-YFP and PHPLCδ-CFP. (C and D) Cells transiently transfected with PIPKIβ-YFP. (E and F) Cells transiently transfected with PIPKIγ-GFP. (G) Quantification of the phagocytic efficiency of untransfected cells (control) or cells transiently transfected with PIPKIα-CFP, PIPKIβ-YFP, or PIPKIγ-GFP. Data are the means ± SEM of three experiments, each scoring at least 100 cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171893&req=5

fig9: Effect of PIPKI overexpression on phagocytosis. RAW cells transfected with the plasmids indicated below were exposed to IgG-opsonized particles for 8–10 min, then analyzed by laser confocal microscopy either immediately (A and B) or after fixation and staining with Alexa 633-phalloidin (D) or rhodamine-phalloidin (E and F). (A and B) RAW cells transiently transfected with PIPKIβ-YFP and PHPLCδ-CFP. (C and D) Cells transiently transfected with PIPKIβ-YFP. (E and F) Cells transiently transfected with PIPKIγ-GFP. (G) Quantification of the phagocytic efficiency of untransfected cells (control) or cells transiently transfected with PIPKIα-CFP, PIPKIβ-YFP, or PIPKIγ-GFP. Data are the means ± SEM of three experiments, each scoring at least 100 cells.
Mentions: The PIPKI isoforms distributed largely to the plasmalemma of RAW cells, in agreement with earlier observations in various cell types (Stephens et al., 1991; Doughman et al., 2003). Opsonized particles bound normally to the overexpressing cells and phagocytic cups formed in these cells. Actin could be seen to accumulate at the cups, as in normal cells (Fig. 9, D and F). Remarkably, closure of the phagosomes was aborted and engulfment was rarely completed. In multiple experiments, where 150 cells were counted, the phagocytic efficiency was inhibited by 95, 72, and 79% by PIPKI α, β, and γ, respectively. Direct assessment of PI(4,5)P2 under these conditions using CFP-tagged PH domain constructs in cells transfected with YFP-PIPKI confirmed that the phosphoinositide remains present and in fact is enriched at the base of the cup (Fig. 9, A and B). Although other explanations can be envisaged, these findings can be most simply explained by the persistence of PI(4,5)P2 at the base of the phagosome due to excessive synthesis.

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