title: Evaluation of fluorescent phosphatidylserine substrates for the aminophospholipid flippase in mammalian cells.
abstract: A series of fluorescent phosphatidylserine and phosphatidylcholine derivatives were prepared and evaluated by cell microscopy for ability to translocate across mammalian plasma membranes via the putative aminophospholipid flippase. Phosphatidylserine derivatives, with either a neutral 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) or a coumarin fluorophore appended to the 2-acyl chain, entered the cytosol of all three cell lines tested and control experiments showed that the translocation was due to flippase activity. In contrast, a phosphatidylserine conjugate containing a charged and polar carboxyfluorescein was not translocated and remained in the cell plasma membrane. The phosphatidylserine-coumarin derivative exhibits bright fluorescence and higher photostability than the NBD analogues, and thus is a promising new fluorescent probe for extended-imaging studies of flippase action in living cells using laser confocal microscopes.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Smith
authors.forename: Edward J
authors.initials: EJ
authors.lastname: O'Neil
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Lampkins
authors.forename: James R
authors.initials: JR
authors.lastname: Johnson
authors.forename: Jung-Jae
authors.initials: JJ
authors.lastname: Lee
authors.forename: Erin L
authors.initials: EL
authors.lastname: Cole
authors.forename: Bradley D
authors.initials: BD
authors.lastname: Smith
mesh_terms: D000818:Animals; D001393:Azoles / Q000378:metabolism; D001692:Biological Transport; D002460:Cell Line; D003374:Coumarins / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D009578:Nitrobenzenes / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins / Q000378:metabolism*
chemical_list: C510797:7-nitrobenz-2-oxa-1,3-diazol-4-yl; D001393:Azoles; D003374:Coumarins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009578:Nitrobenzenes; D010718:Phosphatidylserines; D049294:Phospholipid Transfer Proteins; C024098:6-carboxyfluorescein; C030123:coumarin