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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 and -α depletion by RNAi in CHO-IIA cells. (A) Western blot. The amount of lysate used for PIP5K-γ detection was five times higher than that for other proteins because of its low abundance. (B) TLC analysis (n = 5). (C) Particle attachment and ingestion. (left) Fluorescence/DIC images. Green, PIP5K; blue, external beads; red, phalloidin. (right) Phagocytic indices (n > 40). (D and E) Cells with prebound beads were incubated at 37°C for 5 min. Error bars indicate SEM. (D) Phalloidin staining. Blue, external beads; red, phalloidin. (E) Endogenous PIP5K-α (red) and EGFP-PLCδ1PH (green). Arrowheads highlight phagocytic cups and particles being ingested. Bars, 10 µm.
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fig7: PIP5K-γ and -α depletion by RNAi in CHO-IIA cells. (A) Western blot. The amount of lysate used for PIP5K-γ detection was five times higher than that for other proteins because of its low abundance. (B) TLC analysis (n = 5). (C) Particle attachment and ingestion. (left) Fluorescence/DIC images. Green, PIP5K; blue, external beads; red, phalloidin. (right) Phagocytic indices (n > 40). (D and E) Cells with prebound beads were incubated at 37°C for 5 min. Error bars indicate SEM. (D) Phalloidin staining. Blue, external beads; red, phalloidin. (E) Endogenous PIP5K-α (red) and EGFP-PLCδ1PH (green). Arrowheads highlight phagocytic cups and particles being ingested. Bars, 10 µm.

Mentions: Our results from mice provided definitive evidence for the differential roles of PIP5K-γ and -α in actin remodeling after long-term knockout. We next used RNAi to determine if depletion in the short term in another type of phagocytic cell has similar effects. Because BMM are difficult to transfect, we used the engineered phagocytic CHO-IIA cells, which stably express human FcγRIIA and phagocytize IgG-opsonized particles using a similar (albeit simpler) machinery (Downey et al., 1999). Compared with BMM, CHO-IIA cells had more PIP5K-α than PIP5K-γ (Fig. 1 A). PIP5K knockdown was confirmed by Western blotting (Fig. 7 A). As in knockout BMM, PIP5K-α depletion decreased the PIP2 level in CHO-IIA cells to a larger extent than PIP5K-γ depletion (Fig. 7 B).


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 and -α depletion by RNAi in CHO-IIA cells. (A) Western blot. The amount of lysate used for PIP5K-γ detection was five times higher than that for other proteins because of its low abundance. (B) TLC analysis (n = 5). (C) Particle attachment and ingestion. (left) Fluorescence/DIC images. Green, PIP5K; blue, external beads; red, phalloidin. (right) Phagocytic indices (n > 40). (D and E) Cells with prebound beads were incubated at 37°C for 5 min. Error bars indicate SEM. (D) Phalloidin staining. Blue, external beads; red, phalloidin. (E) Endogenous PIP5K-α (red) and EGFP-PLCδ1PH (green). Arrowheads highlight phagocytic cups and particles being ingested. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig7: PIP5K-γ and -α depletion by RNAi in CHO-IIA cells. (A) Western blot. The amount of lysate used for PIP5K-γ detection was five times higher than that for other proteins because of its low abundance. (B) TLC analysis (n = 5). (C) Particle attachment and ingestion. (left) Fluorescence/DIC images. Green, PIP5K; blue, external beads; red, phalloidin. (right) Phagocytic indices (n > 40). (D and E) Cells with prebound beads were incubated at 37°C for 5 min. Error bars indicate SEM. (D) Phalloidin staining. Blue, external beads; red, phalloidin. (E) Endogenous PIP5K-α (red) and EGFP-PLCδ1PH (green). Arrowheads highlight phagocytic cups and particles being ingested. Bars, 10 µm.
Mentions: Our results from mice provided definitive evidence for the differential roles of PIP5K-γ and -α in actin remodeling after long-term knockout. We next used RNAi to determine if depletion in the short term in another type of phagocytic cell has similar effects. Because BMM are difficult to transfect, we used the engineered phagocytic CHO-IIA cells, which stably express human FcγRIIA and phagocytize IgG-opsonized particles using a similar (albeit simpler) machinery (Downey et al., 1999). Compared with BMM, CHO-IIA cells had more PIP5K-α than PIP5K-γ (Fig. 1 A). PIP5K knockdown was confirmed by Western blotting (Fig. 7 A). As in knockout BMM, PIP5K-α depletion decreased the PIP2 level in CHO-IIA cells to a larger extent than PIP5K-γ depletion (Fig. 7 B).

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