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Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis.

Botelho RJ, Teruel M, Dierckman R, Anderson R, Wells A, York JD, Meyer T, Grinstein S - J. Cell Biol. (2000)

Bottom Line: Our results reveal a sequence of exquisitely localized, coordinated steps in phospholipid metabolism: a focal, rapid accumulation of 4,5-PIP(2) accompanied by recruitment of type Ialpha phosphatidylinositol phosphate kinase to the phagosomal cup, followed by disappearance of the phosphoinositide as the phagosome seals.The presence of 4, 5-PIP(2) and active PLCgamma at the phagosome was shown to be essential for effective particle ingestion.The temporal sequence of phosphoinositide metabolism suggests that accumulation of 4,5-PIP(2) is involved in the initial recruitment of actin to the phagocytic cup, while its degradation contributes to the subsequent cytoskeletal remodeling.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Hospital for Sick Children, and Department of Biochemistry, University of Toronto, Ontario M5G 1X8, Canada.

ABSTRACT
Phagocytosis requires localized and transient remodeling of actin filaments. Phosphoinositide signaling is believed to play an important role in cytoskeletal organization, but it is unclear whether lipids, which can diffuse along the membrane, can mediate the focal actin assembly required for phagocytosis. We used imaging of fluorescent chimeras of pleckstrin homology and C1 domains in live macrophages to monitor the distribution of phosphatidylinositol-4,5-bisphosphate (4,5-PIP(2)) and diacylglycerol, respectively, during phagocytosis. Our results reveal a sequence of exquisitely localized, coordinated steps in phospholipid metabolism: a focal, rapid accumulation of 4,5-PIP(2) accompanied by recruitment of type Ialpha phosphatidylinositol phosphate kinase to the phagosomal cup, followed by disappearance of the phosphoinositide as the phagosome seals. Loss of 4,5-PIP(2) correlated with mobilization of phospholipase Cgamma (PLCgamma) and with the localized formation of diacylglycerol. The presence of 4, 5-PIP(2) and active PLCgamma at the phagosome was shown to be essential for effective particle ingestion. The temporal sequence of phosphoinositide metabolism suggests that accumulation of 4,5-PIP(2) is involved in the initial recruitment of actin to the phagocytic cup, while its degradation contributes to the subsequent cytoskeletal remodeling.

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Phagocytosis in macrophages expressing PM-GFP. (A) DIC image and time course of phagocytosis, as described in the legend to Fig. 2 A. (B and C) Enlargements of the areas identified by boxes in A0 and A6. (D) Phagocytosis of 8-μm latex beads. (E) Corresponding DIC image. Bars, 10 μm.
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Figure 3: Phagocytosis in macrophages expressing PM-GFP. (A) DIC image and time course of phagocytosis, as described in the legend to Fig. 2 A. (B and C) Enlargements of the areas identified by boxes in A0 and A6. (D) Phagocytosis of 8-μm latex beads. (E) Corresponding DIC image. Bars, 10 μm.

Mentions: The phagosomal membrane is known to undergo an active maturation process that involves progressive budding and concomitant fusion with endosomes and lysosomes (Beron et al. 1995). It is therefore conceivable that the observed loss of fluorescence from the early phagosomes could result from rapid replacement with endomembranes, which are ostensibly devoid of PLCδ-PH-GFP. This possibility was examined using PM-GFP, an acylated form of GFP that partitions preferentially to the inner monolayer of the plasma membrane (Teruel et al. 1999). Typical results are illustrated in Fig. 3 A. As expected, pseudopods labeled with PM-GFP were observed extending over RBCs (open arrows). However, in contrast to PLCδ-PH-GFP, the association of PM-GFP with the phagosomal membrane was persistent (Fig. 3 A, solid arrows). These observations are clearly illustrated in Fig. 3B and Fig. C, which are magnified images of the enclosed areas in Fig. 3, A0 and A6. In fact, PM-GFP remained bound with phagosomes for at least 20 min after formation (not illustrated). Because RAW cells internalize multiple opsonized particles, this resulted in the progressive intracellular accumulation of brightly labeled phagosomes (Fig. 3 A6).


Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis.

Botelho RJ, Teruel M, Dierckman R, Anderson R, Wells A, York JD, Meyer T, Grinstein S - J. Cell Biol. (2000)

Phagocytosis in macrophages expressing PM-GFP. (A) DIC image and time course of phagocytosis, as described in the legend to Fig. 2 A. (B and C) Enlargements of the areas identified by boxes in A0 and A6. (D) Phagocytosis of 8-μm latex beads. (E) Corresponding DIC image. Bars, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2150667&req=5

Figure 3: Phagocytosis in macrophages expressing PM-GFP. (A) DIC image and time course of phagocytosis, as described in the legend to Fig. 2 A. (B and C) Enlargements of the areas identified by boxes in A0 and A6. (D) Phagocytosis of 8-μm latex beads. (E) Corresponding DIC image. Bars, 10 μm.
Mentions: The phagosomal membrane is known to undergo an active maturation process that involves progressive budding and concomitant fusion with endosomes and lysosomes (Beron et al. 1995). It is therefore conceivable that the observed loss of fluorescence from the early phagosomes could result from rapid replacement with endomembranes, which are ostensibly devoid of PLCδ-PH-GFP. This possibility was examined using PM-GFP, an acylated form of GFP that partitions preferentially to the inner monolayer of the plasma membrane (Teruel et al. 1999). Typical results are illustrated in Fig. 3 A. As expected, pseudopods labeled with PM-GFP were observed extending over RBCs (open arrows). However, in contrast to PLCδ-PH-GFP, the association of PM-GFP with the phagosomal membrane was persistent (Fig. 3 A, solid arrows). These observations are clearly illustrated in Fig. 3B and Fig. C, which are magnified images of the enclosed areas in Fig. 3, A0 and A6. In fact, PM-GFP remained bound with phagosomes for at least 20 min after formation (not illustrated). Because RAW cells internalize multiple opsonized particles, this resulted in the progressive intracellular accumulation of brightly labeled phagosomes (Fig. 3 A6).

Bottom Line: Our results reveal a sequence of exquisitely localized, coordinated steps in phospholipid metabolism: a focal, rapid accumulation of 4,5-PIP(2) accompanied by recruitment of type Ialpha phosphatidylinositol phosphate kinase to the phagosomal cup, followed by disappearance of the phosphoinositide as the phagosome seals.The presence of 4, 5-PIP(2) and active PLCgamma at the phagosome was shown to be essential for effective particle ingestion.The temporal sequence of phosphoinositide metabolism suggests that accumulation of 4,5-PIP(2) is involved in the initial recruitment of actin to the phagocytic cup, while its degradation contributes to the subsequent cytoskeletal remodeling.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Hospital for Sick Children, and Department of Biochemistry, University of Toronto, Ontario M5G 1X8, Canada.

ABSTRACT
Phagocytosis requires localized and transient remodeling of actin filaments. Phosphoinositide signaling is believed to play an important role in cytoskeletal organization, but it is unclear whether lipids, which can diffuse along the membrane, can mediate the focal actin assembly required for phagocytosis. We used imaging of fluorescent chimeras of pleckstrin homology and C1 domains in live macrophages to monitor the distribution of phosphatidylinositol-4,5-bisphosphate (4,5-PIP(2)) and diacylglycerol, respectively, during phagocytosis. Our results reveal a sequence of exquisitely localized, coordinated steps in phospholipid metabolism: a focal, rapid accumulation of 4,5-PIP(2) accompanied by recruitment of type Ialpha phosphatidylinositol phosphate kinase to the phagosomal cup, followed by disappearance of the phosphoinositide as the phagosome seals. Loss of 4,5-PIP(2) correlated with mobilization of phospholipase Cgamma (PLCgamma) and with the localized formation of diacylglycerol. The presence of 4, 5-PIP(2) and active PLCgamma at the phagosome was shown to be essential for effective particle ingestion. The temporal sequence of phosphoinositide metabolism suggests that accumulation of 4,5-PIP(2) is involved in the initial recruitment of actin to the phagocytic cup, while its degradation contributes to the subsequent cytoskeletal remodeling.

Show MeSH
Related in: MedlinePlus