Localized Biphasic Changes in Phosphatidylinositol-4,5-Bisphosphate at Sites of Phagocytosis

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-PIP2) 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-PIP2 accompanied by recruitment of type Iα phosphatidylinositol phosphate kinase to the phagosomal cup, followed by disappearance of the phosphoinositide as the phagosome seals. Loss of 4,5-PIP2 correlated with mobilization of phospholipase Cγ (PLCγ) and with the localized formation of diacylglycerol. The presence of 4,5-PIP2 and active PLCγ at the phagosome was shown to be essential for effective particle ingestion. The temporal sequence of phosphoinositide metabolism suggests that accumulation of 4,5-PIP2 is involved in the initial recruitment of actin to the phagocytic cup, while its degradation contributes to the subsequent cytoskeletal remodeling.

Lateral Sequestration of Phosphatidylinositol 4,5-Bisphosphate by the Basic Effector Domain of Myristoylated Alanine-rich C Kinase Substrate Is Due to Nonspecific Electrostatic Interactions

“…In fact, such demarcations (i.e. steep gradients) for polyphosphoinositides are indeed observed at the borders of membrane ruffles 3 and nascent phagosomes (109,110). The interpretation of these experiments, however, is not straightforward because the probe used to detect the lateral organization of PIP 2 in the plasma membrane, the PH domain of PLC-␦ 1 , is itself negatively charged when bound to PIP 2 , and a green fluorescent protein construct of this PH domain, like PIP 2 , is also electrostatically sequestered by the basic residues in the effector domain of MARCKS.…”

“…Furthermore, MARCKS is not uniformly distributed in the plasma membrane of some cell types (103); it is concentrated in the membrane ruffles of fibroblasts (104) and, along with MacMARCKS, in the nascent phagosomes of macrophages (105,106). If MARCKS sequesters PIP 2 , this lipid should colocalize with MARCKS in these regions, and it does (107)(108)(109). However, elucidating the mechanism of colocalization is complicated by the fact that PIP 2 -producing kinases are also concentrated in ruffles and nascent phagosomes.…”

Annexin A2 Regulates Phagocytosis of Photoreceptor Outer Segments in the Mouse Retina

“…Actin polymerization drives phagosome formation, and in other phagocytic cell types it has been reported that F-actin begins to depolymerize at or before the point at which phagosome closure is complete (May and Machesky, 2001). It has been proposed that phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P 2 ) at the site of formation of the phagocytic cup and its subsequent hydrolysis as the cup closes are the key signaling events regulating the cycle of actin polymerization and depolymerization (Botelho et al, 2000;Scott et al, 2005). Because anx A2 is a potent regulator of actin dynamics (Merrifield et al, 2001;Hayes et al, 2006) and binds both cholesterol-rich membranes, Ca 2ϩ , and PtdIns4,5P 2 Rescher et al, 2004;Gerke et al, 2005), it is well placed to function as a sensor of these second messengers and signaling intermediates, linking .…”

PI5KI-dependent signals are critical regulators of the cytolytic secretory pathway

“…on April 7, 2019. by guest www.bloodjournal.org From Interestingly, the reduction of PIP2 levels in the synapse area is reminiscent of PIP2 disappearance observed in forming phagosomes. 23,30 The contribution of individual metabolic pathways to PIP2 use is likely attributable to its enzymatic conversion into critical signaling intermediates, such as IP3 and diacylglycerol or PIP3, by PLC␥, and PI3K, respectively. However, PIP2 interaction with ligands with higher affinity may also contribute to the displacement of the probe during the cytolytic event.…”

“…29,30 Primary cultured NK cells were sorted by FACS to isolate cells expressing GFP-PH at high levels. Cell viability was not affected by PIP2 masking (F.M., unpublished data, January 15, 2006).…”