Phospholipid flip-flop and phospholipid scramblase 1 (PLSCR1) co-localize to uropod rafts in formylated Met-Leu-Phe-stimulated neutrophils.
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.
Frasch
S Courtney
SC
Department of Pediatrics, Division of Cell Biology, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.
Henson
Peter M
PM
Nagaosa
Kaz
K
Fessler
Michael B
MB
Borregaard
Niels
N
Bratton
Donna L
DL
D000469:Alkaline Phosphatase / Q000378:metabolism; D017304:Annexin A5 / Q000737:chemistry; D017209:Apoptosis; D015153:Blotting, Western; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin / Q000737:chemistry; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D004705:Endocytosis; D015943:Factor Va / Q000737:chemistry; D005434:Flow Cytometry; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D019169:Jurkat Cells; D017493:Leukocyte Common Antigens / Q000096:biosynthesis; D008055:Lipids / Q000737:chemistry; D008563:Membrane Lipids / Q000737:chemistry; D021962:Membrane Microdomains / Q000737:chemistry*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000737:chemistry*; D009504:Neutrophils / Q000737:chemistry / Q000378:metabolism*; D010718:Phosphatidylserines / Q000737:chemistry; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D010767:Phosphorylcholine / Q000737:chemistry; D010873:Pinocytosis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D017304:Annexin A5; D002352:Carrier Proteins; D003902:Detergents; D007546:Isoquinolines; D008055:Lipids; D008563:Membrane Lipids; D008565:Membrane Proteins; D010718:Phosphatidylserines; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D010767:Phosphorylcholine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015943:Factor Va; D004247:DNA; D002772:Cholera Toxin; C017475:lucifer yellow; D000469:Alkaline Phosphatase; D017493:Leukocyte Common Antigens