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Essential and unique roles of PIP5K-gamma and -alpha in Fcgamma receptor-mediated phagocytosis.

Mao YS, Yamaga M, Zhu X, Wei Y, Sun HQ, Wang J, Yun M, Wang Y, Di Paolo G, Bennett M, Mellman I, Abrams CS, De Camilli P, Lu CY, Yin HL - J. Cell Biol. (2009)

Bottom Line: The actin cytoskeleton is dynamically remodeled during Fcgamma receptor (FcgammaR)-mediated phagocytosis in a phosphatidylinositol (4,5)-bisphosphate (PIP(2))-dependent manner.Delivery of exogenous PIP(2) rescued these defects.In addition, PIP5K-gamma but not PIP5K-alpha is transiently activated by spleen tyrosine kinase-mediated phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

ABSTRACT
The actin cytoskeleton is dynamically remodeled during Fcgamma receptor (FcgammaR)-mediated phagocytosis in a phosphatidylinositol (4,5)-bisphosphate (PIP(2))-dependent manner. We investigated the role of type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) gamma and alpha isoforms, which synthesize PIP(2), during phagocytosis. PIP5K-gamma-/- bone marrow-derived macrophages (BMM) have a highly polymerized actin cytoskeleton and are defective in attachment to IgG-opsonized particles and FcgammaR clustering. Delivery of exogenous PIP(2) rescued these defects. PIP5K-gamma knockout BMM also have more RhoA and less Rac1 activation, and pharmacological manipulations establish that they contribute to the abnormal phenotype. Likewise, depletion of PIP5K-gamma by RNA interference inhibits particle attachment. In contrast, PIP5K-alpha knockout or silencing has no effect on attachment but inhibits ingestion by decreasing Wiskott-Aldrich syndrome protein activation, and hence actin polymerization, in the nascent phagocytic cup. In addition, PIP5K-gamma but not PIP5K-alpha is transiently activated by spleen tyrosine kinase-mediated phosphorylation. We propose that PIP5K-gamma acts upstream of Rac/Rho and that the differential regulation of PIP5K-gamma and -alpha allows them to work in tandem to modulate the actin cytoskeleton during the attachment and ingestion phases of phagocytosis.

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PIP5K-α−/− BMM are defective in actin polymerization during ingestion. BMM incubated with IgG-opsonized beads at 4°C for 10 min were warmed to 37°C for 1 min. (A) Quantitation of polymerized actin in situ. (left) Fluorescence images. Green, phalloidin; red, external beads. (middle) Line scans of phalloidin fluorescence intensity spanning the bottom of a nascent phagocytic cup (x) and its contralateral PM (y). Data shown were taken from representative cells. (right) Ratio of phalloidin intensity (x/y; n = 30 cups). Error bars indicate SEM. (B) Red, active WASP; green, phalloidin; blue, external beads. (inset) Total WASP (red) in another cell. (C) p34-Arc (red). Bars, 10 µm.
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fig6: PIP5K-α−/− BMM are defective in actin polymerization during ingestion. BMM incubated with IgG-opsonized beads at 4°C for 10 min were warmed to 37°C for 1 min. (A) Quantitation of polymerized actin in situ. (left) Fluorescence images. Green, phalloidin; red, external beads. (middle) Line scans of phalloidin fluorescence intensity spanning the bottom of a nascent phagocytic cup (x) and its contralateral PM (y). Data shown were taken from representative cells. (right) Ratio of phalloidin intensity (x/y; n = 30 cups). Error bars indicate SEM. (B) Red, active WASP; green, phalloidin; blue, external beads. (inset) Total WASP (red) in another cell. (C) p34-Arc (red). Bars, 10 µm.

Mentions: We performed additional studies to determine if WASP-mediated activation of the Arp2/3 complex was compromised. WASP is required for efficient phagocytosis (Lorenzi et al., 2000; May et al., 2000), and it is activated coordinately by PIP2 and Cdc42 (Rohatgi et al., 1999). Overexpression of PIP5K promotes WASP and Arp2/3-dependent actin polymerization (Rozelle et al., 2000). Using a WASP antibody that recognizes the active open conformation (Labno et al., 2003), we found that there was no detectible staining in the cytoplasm of WT BMM but strong staining in the cups (Fig. 6 B). However, the antibody that detects total WASP stained both the cups and cytoplasm (Fig. 6 B, inset). Consistent with WASP activation in the cups, p34-Arc, a component of the Arp2/3 complex that binds active WASP, also accumulated there (Fig. 6 C).


Essential and unique roles of PIP5K-gamma and -alpha in Fcgamma receptor-mediated phagocytosis.

Mao YS, Yamaga M, Zhu X, Wei Y, Sun HQ, Wang J, Yun M, Wang Y, Di Paolo G, Bennett M, Mellman I, Abrams CS, De Camilli P, Lu CY, Yin HL - J. Cell Biol. (2009)

PIP5K-α−/− BMM are defective in actin polymerization during ingestion. BMM incubated with IgG-opsonized beads at 4°C for 10 min were warmed to 37°C for 1 min. (A) Quantitation of polymerized actin in situ. (left) Fluorescence images. Green, phalloidin; red, external beads. (middle) Line scans of phalloidin fluorescence intensity spanning the bottom of a nascent phagocytic cup (x) and its contralateral PM (y). Data shown were taken from representative cells. (right) Ratio of phalloidin intensity (x/y; n = 30 cups). Error bars indicate SEM. (B) Red, active WASP; green, phalloidin; blue, external beads. (inset) Total WASP (red) in another cell. (C) p34-Arc (red). Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654300&req=5

fig6: PIP5K-α−/− BMM are defective in actin polymerization during ingestion. BMM incubated with IgG-opsonized beads at 4°C for 10 min were warmed to 37°C for 1 min. (A) Quantitation of polymerized actin in situ. (left) Fluorescence images. Green, phalloidin; red, external beads. (middle) Line scans of phalloidin fluorescence intensity spanning the bottom of a nascent phagocytic cup (x) and its contralateral PM (y). Data shown were taken from representative cells. (right) Ratio of phalloidin intensity (x/y; n = 30 cups). Error bars indicate SEM. (B) Red, active WASP; green, phalloidin; blue, external beads. (inset) Total WASP (red) in another cell. (C) p34-Arc (red). Bars, 10 µm.
Mentions: We performed additional studies to determine if WASP-mediated activation of the Arp2/3 complex was compromised. WASP is required for efficient phagocytosis (Lorenzi et al., 2000; May et al., 2000), and it is activated coordinately by PIP2 and Cdc42 (Rohatgi et al., 1999). Overexpression of PIP5K promotes WASP and Arp2/3-dependent actin polymerization (Rozelle et al., 2000). Using a WASP antibody that recognizes the active open conformation (Labno et al., 2003), we found that there was no detectible staining in the cytoplasm of WT BMM but strong staining in the cups (Fig. 6 B). However, the antibody that detects total WASP stained both the cups and cytoplasm (Fig. 6 B, inset). Consistent with WASP activation in the cups, p34-Arc, a component of the Arp2/3 complex that binds active WASP, also accumulated there (Fig. 6 C).

Bottom Line: The actin cytoskeleton is dynamically remodeled during Fcgamma receptor (FcgammaR)-mediated phagocytosis in a phosphatidylinositol (4,5)-bisphosphate (PIP(2))-dependent manner.Delivery of exogenous PIP(2) rescued these defects.In addition, PIP5K-gamma but not PIP5K-alpha is transiently activated by spleen tyrosine kinase-mediated phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

ABSTRACT
The actin cytoskeleton is dynamically remodeled during Fcgamma receptor (FcgammaR)-mediated phagocytosis in a phosphatidylinositol (4,5)-bisphosphate (PIP(2))-dependent manner. We investigated the role of type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) gamma and alpha isoforms, which synthesize PIP(2), during phagocytosis. PIP5K-gamma-/- bone marrow-derived macrophages (BMM) have a highly polymerized actin cytoskeleton and are defective in attachment to IgG-opsonized particles and FcgammaR clustering. Delivery of exogenous PIP(2) rescued these defects. PIP5K-gamma knockout BMM also have more RhoA and less Rac1 activation, and pharmacological manipulations establish that they contribute to the abnormal phenotype. Likewise, depletion of PIP5K-gamma by RNA interference inhibits particle attachment. In contrast, PIP5K-alpha knockout or silencing has no effect on attachment but inhibits ingestion by decreasing Wiskott-Aldrich syndrome protein activation, and hence actin polymerization, in the nascent phagocytic cup. In addition, PIP5K-gamma but not PIP5K-alpha is transiently activated by spleen tyrosine kinase-mediated phosphorylation. We propose that PIP5K-gamma acts upstream of Rac/Rho and that the differential regulation of PIP5K-gamma and -alpha allows them to work in tandem to modulate the actin cytoskeleton during the attachment and ingestion phases of phagocytosis.

Show MeSH
Related in: MedlinePlus