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.
This paper would give another intuitions and understanding for exploring mechanism of membrane fusion and blocking in vitro.
주름살 제거로 잘 알려진 Botox의 대체 물질에 대한 연구가 진행되는 가운데, 다양한 약물들이 세포막 융합에 중요한 역할을 하는 SNARE 단백질을 저해할 수 있는 가능성을 보여왔다. FRET을 이용하면 이러한 약물들을 생물이나 사람에 투과하는 대신 SNARE단백질이 삽입된 인공 세포막에 투과하여 SNARE단백질과 약물과의 직접적인 반응을 확인할 수 있다. 많은 세포들의 융합을 평균적으로 알아보는 Bulk실험이 이미 진행되었으나 좀 더 구체적으로 개개 세포들의 융합 반응을 확인하고자 single molecule을 이용한 surface FRET실험을 수행하였다. Kaempferol, Cyanidin, Myricetin의 3가지 페놀 화합물의 영향을 연구하였고, 각각의 화합물이 만들어 내는 결과들을 분석하고 정리하였다. 통계적인 데이터 처리를 이용한 FRET data 의 분석과 각 화학물의 결과 특징 비교, 융합에 작용하는 요인들에 대한 고찰을 함께 다루고자 한다.