Single vesicle FRET verifies phenol chemical inhibition effects on neuronal SNARE driven membrane fusion in vitro

Abstract

Effect of some phenolic chemicals on SNARE driven membrane fusion is analyzed using single vesicle FRET. Kaempferol, Myricetin, Cyanidin, which are previously known as blocking chemicals were chosen. Lipidic vesicles of DII(donor dye) and DID(acceptor dye) probe the vesicle fusion. Full fusion blocking effect of three chemicals were re-confirmed. And it is shown that each chemical has different degree of full fusion blocking. After careful examination of interaction between chemical, lipid, surface, and protein, dual function of chemicals were found. Chemicals interacts both with surface and vesicle. In the absense of SNARE protein, chemical mediated membrane fusion occurs. However, when protein is incorporated, chemical effect on membrane is reduced and vesicle fusion is mainly driven by SNARE protein. Chemicals block forming of SNARE complex, therefore chemicals could work as a fusion blocker. SNARE protein incorporation seems to block direct vesicle-vesicle interaction driven by chemicals,making distance between vesicles. SNARE-free DII labeled vesicles were used as control. Intensity and FRET distribution of vesicles were chosen as criteria. As chemical-vesicle interaction is not thoroughly elimiated, vesicle fusion blocking are affected by protein and lipid. However, SNARE protein plays a main role in membrane fusion and blocking of fusion is mainly incurred by chemical-SNARE protein interaction. FRET value of each vesicles were calculated and histrogram of FRET population were shown. Charged coupled device(CCD) camera obtains the images from the sample in multiple local spots. For each sample, 19-20 images were gathered and analyzed. For full fusion population, the ratio of fully fused vesicle to total vesicle number were adopted as normalized method. Additionally, the number of fully fused vesicles in each spot was counted(absolute number method) based on assumption that fully fused vesicles attachment on the surface is not affected by chemicals...