Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis

Abstract

The Rho GTPases play a critical role in initiating actin polymerization during phagocytosis. In contrast, the factors directing the disassembly of F-actin required for fission of the phagocytic vacuole are ill defined. We used fluorescent chimeric proteins to monitor the dynamics of association of actin and active Cdc42 and Rac1 with the forming phagosome. Although actin was found to disappear from the base of the forming phagosome before sealing was complete, Rac1/Cdc42 activity persisted, suggesting that termination of GTPase activity is not the main determinant of actin disassembly. Furthermore, fully internalized phagosomes engineered to associate constitutively with active Rac1 showed little associated F-actin. The disappearance of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) from the phagosomal membrane closely paralleled the course of actin disassembly. Furthermore, inhibition of PI(4,5)P2 hydrolysis or increased PI(4,5)P2 generation by overexpression of phosphatidylinositol phosphate kinase I prevented the actin disassembly necessary for the completion of phagocytosis. These observations suggest that hydrolysis of PI(4,5)P2 dictates the remodeling of actin necessary for completion of phagocytosis.

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

“…[21][22][23] In agreement with previous observations, 34 we observed that such probe almost exclusively localizes at the plasma membrane where it distributes in discrete microdomains partially colocalizing with lipid rafts (R.G., unpublished data, December 2005). The interaction of NK cells with target cells was followed by a rapid consumption of PIP2 in the area of cytolytic synapse, whereas in contact-free membrane areas or in noncytolytic interactions we did not observe major PIP2 variations, thus ruling out rapid membrane turnover as the process responsible for the localized loss of membrane-associated GFP-PH probe.…”

“…[21][22][23] By means of lentiviral-driven expression, we obtained high transduction efficiency without affecting NK-cell phenotype and functional properties. 25 As illustrated in Figure 1A, in isolated NK cells GFP-PH distributed throughout the plasma membrane where it localized in discrete microdomains; as expected, control GFP distributed homogeneously within the cell ( Figure 1B).…”

“…[12][13][14][15] Increasing evidences indicate that PI5KIs, providing spatially and functionally distinct PIP2 pools, guarantee the regulation of specific cellular events: PI5KI␥, for instance, is required for focal adhesion and synaptic vesicle trafficking, 16,17 PI5KI␤ for constitutive receptor endocytosis, 18 whereas PI5KI␣ acts in the regulation of processes dependent on local changes of actin dynamics, such as membrane ruffling and phagocytosis. 19,20 In this report, we took advantage of the fusion protein consisting of GFP and the pleckstrin homology (PH) domain of PLC␦1, that is known to bind PIP2 with high affinity and specificity, [21][22][23] to monitor the dynamics and function of PIP2 during the cytotoxic event. We present evidences of localized changes in the concentration of PIP2 at the cytolytic synapse area where it is required for granule exocytosis.…”

“…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.…”

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 .…”