FM1-43 reports plasma membrane phospholipid scrambling in T-lymphocytes.
We have found using imaging techniques that stimulating Jurkat human leukaemic T-cells with ionomycin in the presence of FM1-43, a dye used to monitor exocytosis and endocytosis, causes large (6--10-fold) increases in FM1-43 fluorescence. These responses are too large to be caused by exocytosis. Instead, three lines of evidence suggest that FM1-43 is responding to phospholipid scrambling. First, ionomycin also stimulates increases in the fluorescence of annexin V, a phosphatidylserine-specific probe, while thapsigargin does not stimulate fluorescence increases of either probe. Secondly, cells that exhibit FM1-43 fluorescence increases after ionomycin stimulation stain with annexin V once FM1-43 is washed out. Thirdly, ionomycin stimulates uptake of 7-nitrobenz-2-oxa-1,3-diazole-labelled phosphatidylcholine, a specific assay for scramblase activity, whereas thapsigargin does not. We find that FM1-43 reports phospholipid scrambling with 'better' kinetics than annexin V, and does require extracellular Ca(2+) to report phospholipid scrambling. We suggest that FM1-43 may be a useful probe to study the dynamics of phospholipid scrambling. The results are the first demonstration that FM1-43 can respond significantly to a biological process other than vesicular trafficking.
Zweifach
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Department of Physiology and Biophysics, University of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262, USA. adam.zweifach@UCHSC.edu
D017304:Annexin A5 / Q000494:pharmacology; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D003952:Diagnostic Imaging / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D019169:Jurkat Cells; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D010743:Phospholipids / Q000378:metabolism*; D011726:Pyridinium Compounds / Q000494:pharmacology*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors
D017304:Annexin A5; D004791:Enzyme Inhibitors; C073804:FM1 43; D005456:Fluorescent Dyes; D007476:Ionophores; D010743:Phospholipids; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D015759:Ionomycin; D019284:Thapsigargin; D016650:Fluorescein-5-isothiocyanate