THE LIPID DEPENDENCE OF MELITTIN ACTION INVESTIGATED BY DUAL-COLOR FLUORESCENCE BURST ANALYSIS
Geert van den Bogaart 1, Jacek T Mika 1, Victor V Krasnikov 1 and Bert Poolman 1*
1 University of Groningen
* To whom correspondence should be addressed. E-mail: b.poolman{at}rug.nl.
Submitted on February 5, 2007
Revised on February 23, 2007
Accepted on 7 March 2007
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Abstract |
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Dual-color fluorescence-burst analysis (DCFBA) was used to study melittin-induced leakage of macromolecules from liposomes of various lipid compositions. To perform DCFBA, fluorescently-labeled size-marker molecules were encapsulated into liposomes, labeled with a second lipid-attached fluorophore. By correlating the fluorescence bursts, resulting from the liposomes diffusing through the detection volume of a dual-color confocal microscope, the distribution of size-marker molecules over the liposomes was determined. It was found that melittin causes leakage via two different mechanisms: (1) For liposomes composed of neutral bilayer-forming lipids, low melittin concentrations induced pore formation with the pore-size depending on the melittin concentration. (2) For liposomes containing anionic and / or non-bilayer forming lipids, melittin induced fusion or aggregation of liposomes accompanied by a-specific leakage. Experiments with liposomes prepared from Escherichia coli lipid extracts and intact cells of Lactococcus lactis indicate that both mechanisms are physiologically relevant.
Key Words:
fluorescence correlation spectroscopy, lipid dependence, lytic peptide, melittin, membrane fusion, membrane pore
