Originally published In Press as doi:10.1074/jbc.M313414200 on February 6, 2004
J. Biol. Chem., Vol. 279, Issue 17, 17625-17633, April 23, 2004
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**, and
Donna L. Bratton
From the
Departments of Pediatrics and ¶Medicine, Division of Cell Biology, National Jewish Medical and Research Center, Denver, Colorado 80206, the Departments of Medicine and Pathology and Pediatrics, University of Colorado Health Sciences Center and the ||Department of Medicine, University of Colorado School of Medicine, Denver, Colorado 80262, and the **Department of Hematology, Rigshospitalet-4042, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark
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.
Received for publication, December 8, 2003
, and in revised form, February 4, 2004.
* This work was funded in part by National Institutes of Health Grants HL34303 and AI058228 and by the Danish Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be sent: Dept. of Pediatrics, National Jewish Medical and Research Center, Rm. D506, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1390; Fax: 303-398-1381; E-mail: brattond{at}njc.org.
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Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
