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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-γ−/− BMM have abnormal RhoA and Rac1 activation. (A) GTP-Rac1 or -RhoA pull down by GST-PBD or -RBD, respectively. Samples were blotted with anti-Rac1 or -RhoA. (left) Western blot. Three times more WT BMM lysate and GST-RBD pull-down sample were loaded than PIP5K-γ−/− BMM. (right) Ratios of GTP-bound to total GTPase, expressed relative to the levels of WT BMM (n = 4 for Rac1 and 2 for RhoA). (B) Rescue of particle binding defect by C3T. BMM were incubated with C3T at 37°C for 4 h before the exposure of IgG-opsonized particles at 4°C. (left) Fluorescence staining. Green, external beads; red, phalloidin. (right) Binding indices (n > 70). (C) Effects of manipulating Rac activation. BMM were transduced with 600 nM Tat-Rac1L61 or N17 at 37°C for 30 min. (left) Fluorescence images. Green, external beads; red, phalloidin. Arrowheads indicate dorsal ruffles. (top right) Particle binding indices (n > 50). (bottom right) Fluorometric phalloidin quantitation (n = 3). Data are expressed as the percentage of WT BMM without transduced Rac1. Error bars indicate SEM. Bars, 10 µm.
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fig4: PIP5K-γ−/− BMM have abnormal RhoA and Rac1 activation. (A) GTP-Rac1 or -RhoA pull down by GST-PBD or -RBD, respectively. Samples were blotted with anti-Rac1 or -RhoA. (left) Western blot. Three times more WT BMM lysate and GST-RBD pull-down sample were loaded than PIP5K-γ−/− BMM. (right) Ratios of GTP-bound to total GTPase, expressed relative to the levels of WT BMM (n = 4 for Rac1 and 2 for RhoA). (B) Rescue of particle binding defect by C3T. BMM were incubated with C3T at 37°C for 4 h before the exposure of IgG-opsonized particles at 4°C. (left) Fluorescence staining. Green, external beads; red, phalloidin. (right) Binding indices (n > 70). (C) Effects of manipulating Rac activation. BMM were transduced with 600 nM Tat-Rac1L61 or N17 at 37°C for 30 min. (left) Fluorescence images. Green, external beads; red, phalloidin. Arrowheads indicate dorsal ruffles. (top right) Particle binding indices (n > 50). (bottom right) Fluorometric phalloidin quantitation (n = 3). Data are expressed as the percentage of WT BMM without transduced Rac1. Error bars indicate SEM. Bars, 10 µm.

Mentions: Actin remodeling is regulated by a balance between the activation states of Rac and Rho GTPases (Burridge and Wennerberg, 2004). The PIP5K-γ−/− actin phenotype could be explained by an alteration in GTPase activation or their downstream effector functions. To distinguish between these possibilities, we determined if PIP5K-γ−/− knockdown disrupts Rac/Rho activation. GTPase effector pull-down assays showed that PIP5K-γ−/− BMM had a sixfold increase in GTP-RhoA and a 70% decrease in GTP-Rac1 (Fig. 4 A). Thus, PIP5K-γ knockout altered RhoA and Rac1 activation in opposite directions to tip the balance toward RhoA domination.


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-γ−/− BMM have abnormal RhoA and Rac1 activation. (A) GTP-Rac1 or -RhoA pull down by GST-PBD or -RBD, respectively. Samples were blotted with anti-Rac1 or -RhoA. (left) Western blot. Three times more WT BMM lysate and GST-RBD pull-down sample were loaded than PIP5K-γ−/− BMM. (right) Ratios of GTP-bound to total GTPase, expressed relative to the levels of WT BMM (n = 4 for Rac1 and 2 for RhoA). (B) Rescue of particle binding defect by C3T. BMM were incubated with C3T at 37°C for 4 h before the exposure of IgG-opsonized particles at 4°C. (left) Fluorescence staining. Green, external beads; red, phalloidin. (right) Binding indices (n > 70). (C) Effects of manipulating Rac activation. BMM were transduced with 600 nM Tat-Rac1L61 or N17 at 37°C for 30 min. (left) Fluorescence images. Green, external beads; red, phalloidin. Arrowheads indicate dorsal ruffles. (top right) Particle binding indices (n > 50). (bottom right) Fluorometric phalloidin quantitation (n = 3). Data are expressed as the percentage of WT BMM without transduced Rac1. Error bars indicate SEM. Bars, 10 µm.
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Related In: Results  -  Collection

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fig4: PIP5K-γ−/− BMM have abnormal RhoA and Rac1 activation. (A) GTP-Rac1 or -RhoA pull down by GST-PBD or -RBD, respectively. Samples were blotted with anti-Rac1 or -RhoA. (left) Western blot. Three times more WT BMM lysate and GST-RBD pull-down sample were loaded than PIP5K-γ−/− BMM. (right) Ratios of GTP-bound to total GTPase, expressed relative to the levels of WT BMM (n = 4 for Rac1 and 2 for RhoA). (B) Rescue of particle binding defect by C3T. BMM were incubated with C3T at 37°C for 4 h before the exposure of IgG-opsonized particles at 4°C. (left) Fluorescence staining. Green, external beads; red, phalloidin. (right) Binding indices (n > 70). (C) Effects of manipulating Rac activation. BMM were transduced with 600 nM Tat-Rac1L61 or N17 at 37°C for 30 min. (left) Fluorescence images. Green, external beads; red, phalloidin. Arrowheads indicate dorsal ruffles. (top right) Particle binding indices (n > 50). (bottom right) Fluorometric phalloidin quantitation (n = 3). Data are expressed as the percentage of WT BMM without transduced Rac1. Error bars indicate SEM. Bars, 10 µm.
Mentions: Actin remodeling is regulated by a balance between the activation states of Rac and Rho GTPases (Burridge and Wennerberg, 2004). The PIP5K-γ−/− actin phenotype could be explained by an alteration in GTPase activation or their downstream effector functions. To distinguish between these possibilities, we determined if PIP5K-γ−/− knockdown disrupts Rac/Rho activation. GTPase effector pull-down assays showed that PIP5K-γ−/− BMM had a sixfold increase in GTP-RhoA and a 70% decrease in GTP-Rac1 (Fig. 4 A). Thus, PIP5K-γ knockout altered RhoA and Rac1 activation in opposite directions to tip the balance toward RhoA domination.

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