Excited Fluorophores Enhance the Opening of Vesicles at Electrode Surfaces in Vesicle Electrochemical Cytometry.
Electrochemical cytometry is a method developed recently to determine the content of an individual cell vesicle. The mechanism of vesicle rupture at the electrode surface involves the formation of a pore at the interface between a vesicle and the electrode through electroporation, which leads to the release and oxidation of the vesicle's chemical cargo. We have manipulated the membrane properties using excited fluorophores conjugated to lipids, which appears to make the membrane more susceptible to electroporation. We propose that by having excited fluorophores in close contact with the membrane, membrane lipids (and perhaps proteins) are oxidized upon production of reactive oxygen species, which then leads to changes in membrane properties and the formation of water defects. This is supported by experiments in which the fluorophores were placed on the lipid tail instead of the headgroup, which leads to a more rapid onset of vesicle opening. Additionally, application of DMSO to the vesicles, which increases the membrane area per lipid, and decreasing the membrane thickness result in the same enhancement in vesicle opening, which confirms the mechanism of vesicle opening with excited fluorophores in the membrane. Light-induced manipulation of membrane vesicle pore opening might be an attractive means of controlling cell activity and exocytosis. Additionally, our data confirm that in experiments in which cells or vesicle membranes are labeled for fluorescence monitoring, the properties of the excited membrane change substantially.
Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Kemivägen 10, 41296, Gothenburg, Sweden.
Neda
N
Najafinobar
Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Kemivägen 10, 41296, Gothenburg, Sweden.
Jelena
J
Lovrić
Department of Chemistry and Molecular Biology, University of Gothenburg, Kemivägen 10, 41296, Gothenburg, Sweden.
Soodabeh
S
Majdi
Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Kemivägen 10, 41296, Gothenburg, Sweden.
Johan
J
Dunevall
Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Kemivägen 10, 41296, Gothenburg, Sweden.
Ann-Sofie
AS
Cans
Department of Chemistry and Molecular Biology, University of Gothenburg, Kemivägen 10, 41296, Gothenburg, Sweden.|Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Kemivägen 10, 41296, Gothenburg, Sweden.
Andrew
0000-0002-2084-0133
A
Ewing
D000818:Animals; D019439:Chromaffin Cells / Q000166:cytology* / Q000378:metabolism; D055664:Electrochemical Techniques; D004566:Electrodes; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D017382:Reactive Oxygen Species / Q000378:metabolism; D013499:Surface Properties
D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species
chemical cytometry; electroporation; fluorophores; fusion pores; vesicles