An electrostatic switch displaces phosphatidylinositol phosphate kinases from the membrane during phagocytosis

Abstract

Plasmalemmal phosphatidylinositol (PI) 4,5-bisphosphate (PI4,5P(2)) synthesized by PI 4-phosphate (PI4P) 5-kinase (PIP5K) is key to the polymerization of actin that drives chemotaxis and phagocytosis. We investigated the means whereby PIP5K is targeted to the membrane and its fate during phagosome formation. Homology modeling revealed that all PIP5K isoforms feature a positively charged face. Together with the substrate-binding loop, this polycationic surface is proposed to constitute a coincidence detector that targets PIP5Ks to the plasmalemma. Accordingly, manipulation of the surface charge displaced PIP5Ks from the plasma membrane. During particle engulfment, PIP5Ks detached from forming phagosomes as the surface charge at these sites decreased. Precluding the change in surface charge caused the PIP5Ks to remain associated with the phagosomal cup. Chemically induced retention of PIP5K-gamma prevented the disappearance of PI4,5P(2) and aborted phagosome formation. We conclude that a bistable electrostatic switch mechanism regulates the association/dissociation of PIP5Ks from the membrane during phagocytosis and likely other processes.

Figure 1.

Expression and localization of PIP5Ks in RAW cells. (a) Immunoblots of RAW cell and brain extracts probed with PIP5K isoform-specific antibodies. (b and c) Colocalization of PIP5K isoforms with their product, PI4,5P₂, at the plasma membrane. RAW cells were transiently cotransfected with GFP or YFP chimeras of the PIP5K isoforms and with either RFP-PH–PLC-δ (b) or PM-RFP used as a plasmalemmal marker (c). The distribution of the fluorescent proteins was analyzed by spinning-disc confocal microscopy, and representative images acquired near the middle of the cell are illustrated. Bars, 3 µm.

Figure 2.

Structure of type II-β PIP4K and models of type I PIP5K-α, -β, and -γ isoforms. (a–d) The reported structure of type II-β PIP4K (a) and the predicted structures of the PIP5K-α, -β, and -γ90 isoforms deduced by homology modeling are shown in an equivalent orientation with their putative membrane-interacting face pointing toward the observer. The surfaces are colored according to the range of electrostatic potential (red, <−10.0 kT/e; blue, >10.0 kT/e [where k = Boltzmann constant, T = absolute temperature, and e = electron]). The electrostatic surface potentials were computed using the continuum solvation model embodied in the Poisson–Boltzmann method, and the adaptive Poisson–Boltzmann solver was implemented in the APBS software. (e–h) The proteins are shown in an orientation corresponding to a 90° rotation from the orientations in a–d such that the presumed membrane-associated face of the proteins points downward. The ATP-binding site and a reported phosphorylation site are highlighted by yellow and green circles, respectively. The dipole moments (yellow arrows) were calculated directly from the atomic models and the atom partial charges using Protein Dipole Moments Server. The magnitude of the arrows is directly proportional to the strength on the dipole (Debye).

Figure 3.

PIP5Ks interact with anionic phospholipids. (a) Purified GST or GST fusion proteins of PIP5K-α and -γ87 were incubated with immobilized phospholipids and detected by immunoblotting using anti-GST antibodies. Chol, cholesterol; SM, sphingomyelin; TAG, triacylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; CL, cardiolipin. Results are representative of three independent experiments. (b) Binding of PIP5K-α to lipid-coated beads. GFP–PIP5K-α, partially purified from HeLa cell extracts, was added to C18 Nucleosil beads coated with PC alone (i), 20% PS + 80% PC (ii), 20% PS + 2% PA + 2% PI4,5P₂ + 76% PC (iii), 0.5% PI4P + 99.5% PC (iv), 5% PI4P + 95% PC (v), or 0.5% PI4P + 20% PS + 2% PA + 2% PI4,5P₂ + 75.5% PC (vi). Binding was assessed measuring the green fluorescence associated with the beads. Data are expressed relative to PC-only beads and are means ± SEM of three separate experiments.

Figure 4.

PIP5Ks interact with anionic phospholipids. (a) Colocalization of PIP5K isoforms with the negative surface charge marker, R-Pre. (b) RAW cells were cotransfected with a kinase-deficient GFP–PIP5K-β and RFP-PH–PLC-δ without or with a construct encoding the 5-phosphatase domain of synaptojanin2 targeted to the membrane by attachment of a C-terminal CaaX box (Sj2-CaaX). (c) Quantification of the membrane association of GFP–PIP5K-β, kinase-deficient GFP–PIP5K-β, and RFP-PH–PLC-δ in the presence or absence of Sj2-CaaX. Data are means ± SEM (n ≥ 25); *, P < 0.001. (d) The amino acid sequence of wild-type (WT) PIP5K-α from residues 410–464. The residues replaced in mutants A and B are highlighted in bold type with asterisks. (e) RAW cells were transiently cotransfected with GFP chimeras of with either wild-type PIP5K-α (left), mutant A (middle), or mutant B and PM-RFP (right). The distribution of the fluorescent proteins was analyzed by spinning-disc confocal microscopy, and representative images acquired near the middle of the cell are illustrated. Bars, 3 µm.

Figure 5.

The localization of PIP5Ks is altered by depressing the surface charge. (a and b) Cells were cotransfected with the specified GFP-PIP5K isoform and RFP-PH–PLC-δ (insets). Images were acquired before and after incubation with either 2-deoxy-glucose plus antimycin for 40 min (a) or with 10 µM ionomycin for 5 min (b). (c) Quantification of the membrane association of GFP-PIP5K isoforms and of PH–PLC-δ before and after the treatments described in a and b. Data are means ± SEM (n ≥ 30). Bars, 3 µm.

Figure 6.

Redistribution of PIP5Ks during phagocytosis. (a) Macrophages were cotransfected with the specified GFP-PIP5K isoform and PM-RFP. Phagocytosis was initiated by exposure to IgG-opsonized beads, and the cells were imaged by spinning-disc microscopy. Arrowheads indicate sealed and internalized phagosomes, and arrows indicate forming phagosomes. The representative images shown were acquired 180 s after initiation of phagocytosis. (b) Quantification of the fluorescence intensity of the phagosomal membrane relative to the plasmalemma for each of the PIP5K isoforms and for PM-RFP. Data are means ± SEM (n ≥ 30). (c) Cells were cotransfected with the indicated constructs and treated with 100 nM wortmannin for 10 min before initiation of phagocytosis. The representative images shown were acquired 180 s after initiation of phagocytosis. Bars, 3 µm.

Figure 7.

Sustained production of PI4,5P₂ inhibits phagocytosis. (a) Schematic of the experimental protocol. The addition of rapamycin induces the heterodimerization of the YFP-FKBP-5K construct with a plasma membrane–targeted form of FRB. (b) Distribution of YFP-FKBP-5K and RFP-PH–PLC-δ before (−Rap) and 5 min after the addition of 10 µM rapamycin (+Rap). Arrowhead identifies sealed and internalized phagosomes, and the arrow highlights forming phagosomes. (c) Internalization and adherence of beads to macrophages was quantified in cells expressing YFP or YFP-FKBP-5K and plasma membrane–targeted FRB with or without the addition of rapamycin. Data are means ± SEM of at least 500 beads per condition.

Figure 8.

Schematic representation of the changes in phospholipid content and surface charge during phagocytosis. Proposed changes in the content of PI4,5P₂ (blue), PI3,4,5P₃ (yellow), and surface charge of the membrane (red) during the various stages of phagocytosis shown chronologically from top to bottom.