Phospholipid Flip-Flop and Phospholipid Scramblase 1 (PLSCR1) Co-localize to Uropod Rafts in Formylated Met-Leu-Phe-stimulated Neutrophils

S Courtney Frasch,Peter M Henson,Kaz Nagaosa,Michael B Fessler,Niels Borregaard,Donna L Bratton

doi:10.1074/jbc.m313414200

Abstract

Movement of phosphatidylserine (PS) to the plasma membrane outer leaflet is a nearly universal marker of apoptosis and occurs during activation of many cells. Neutrophils stimulated with the chemotactic peptide formylated Met-Leu-Phe (fMLP) demonstrated transient PS exposure. Stimulated outward movement of PS was accompanied by enhanced inward movement of several phosphorylcholine lipid probes and was associated with enhanced FM 1-43 staining indicative of phospholipid packing changes. Unlike apoptosis, inward movement of exogenously added fluorescent PS did not decline, and DNA was not cleaved during fMLP stimulation. Movement of phospholipids occurred within minutes following stimulation, was independent of endocytosis/pinocytosis, and was consistent with bidirectional, transbilayer phospholipid flip-flop. While the role of phospholipid scramblase 1 (PLSCR1) is controversial in flip-flop, we sought evidence for its role in enhanced phospholipid movements during fMLP stimulation. Using antibodies to the carboxyl-terminal domain of PLSCR1, its presence in the plasma membranes of non-permeabilized neutrophils was confirmed by flow cytometry. Additionally subcellular fractionation demonstrated that PLSCR1 was also located in secretory vesicles and tertiary and secondary granules. Activation of neutrophils with fMLP, however, did not significantly alter surface labeling suggesting that stimulated phospholipid flip-flop does not require additional mobilization of PLSCR1 to the plasma membrane. As expected for palmitoylated proteins, PLSCR1 was enriched in detergent-insoluble membranes and co-localized with raft markers at the neutrophil uropod after stimulation. Of note, PS exposure, phospholipid uptake, and FM 1-43 staining also localized to the uropod following stimulation demonstrating that both PLSCR1 and phospholipid flip-flop characterize this specialized domain of polarized neutrophils.

Highlights

Movement of phosphatidylserine (PS) to the plasma membrane outer leaflet is a nearly universal marker of apoptosis and occurs during activation of many cells
PS exposure during formylated Met-Leu-Phe (fMLP) stimulation was not accompanied by loss of aminophospholipid translocase activity as was seen during UV-induced apoptosis (Fig. 1B); PS uptake during fMLP stimulation appeared to be enhanced
Phospholipid Flip-Flop Occurs in Neutrophil Rafts—We have shown that neutrophils exhibit enhanced nonspecific phospholipid flip-flop in response to fMLP and localized phospholipid scramblase 1 (PLSCR1), a candidate protein proposed to play a role in mediating phospholipid flip-flop, to raft domains at the uropod

Summary

EXPERIMENTAL PROCEDURES

Reagents—Cholera toxin B (CT-B)-Alexa 555, FM 1-43, annexin VAlexa 488, fluorescein isothiocyanate-phalloidin, 6-((N-(7-nitrobenz-2oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosyl phosphocholine (NBDsphingomyelin), and Lucifer Yellow were from Molecular Probes (Eugene, OR). 1-Palmitoyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoyl[-sn-glycero-3-phosphoserine] (NBD-PS) and 1-palmitoyl-2[12-[(7-nitro-2–1,3-benzoxadiazol-4-yl)amino]hexanoyl[-sn-glycero-3phosphocholine] (NBD-PC) were from Avanti Polar Lipids (Alabaster, AL). For annexin V staining, 1 ϫ 106 cells in 100 ␮l of NBD buffer with 2.5 mM CaCl2, annexin V-Alexa 488 (1:50), and 5 ␮g/ml propidium iodide were incubated for 15 min at room temperature, diluted with 400 ␮l of ice-cold NBD buffer with 2.5 mM CaCl2, and analyzed by flow cytometry. Cells were washed once and incubated with 100 ␮l of anti-mouse IgG F(abЈ) (1:100 in NBD buffer plus 1 mM CaCl2 and 0.1% BSA) for 30 min on ice, washed once, and analyzed by flow cytometry. Neutrophils (in KRPD buffer with 0.25% BSA) were stimulated with fMLP for 10 min at 37 °C, fixed in 3% paraformaldehyde, 3% sucrose in PBS at room temperature for 10 min, washed twice with PBS, and incubated with Cy3 goat anti-Rabbit F(abЈ) diluted 1:100 for 30 min on ice. Cells were washed twice more with PBS and analyzed by flow cytometry. Plasma membrane and secretory vesicles from control neutrophils were isolated by free flow electrophoresis as described previously [33]

RESULTS

PS Exposure Was Accompanied by Enhanced Uptake of Other

DISCUSSION