新型荧光探针mMaple3与mEos3.2应用于Fsp27介导脂滴融合的功能研究

摘要

Objective:Fsp27 has been proved to be localized on lipid droplets (LDs) and mediates LD growth.To study the molecular mechanism of Fsp27-mediated LD fusion,here,we investigated the functional effect of two novel fluorescent probes,Fsp27-mMaple3 and Fsp27-mEos3.2,on LD fusion for the super-resolution imaging in physiological studies.Methods:In comparison with arecently used fluorescence fused-protein Fsp27-EGFP,we observed the subcellular localization of thc two novel probes and the sizes of LDs under confocal microscopy;And,fluorescence recovery after photo-bleaching (FRAP) was used to examine the lipid exchange between lipid droplets.Results:LDs expressing Fsp27-mMaple3 and Fsp27-mEos3.2 in 3T3-L1 pre-adipocytes were significantly larger than that without expression of the two probes.The FRAP experiment confirmed the normal exchange between LDs expressing the two probes.Conclusions:The two probes we constructed exhibit their comparable abilities with wild type Fsp27 in LD fusion and growth,and the study provides a functional basis on development of super-resolution imaging.%目的:Fsp27已经被证明定位在脂滴上并且介导脂滴融合与增大.为研究Fsp27介导脂滴融合的动态分子机制,我们构建了Fsp27-mMaple3和Fsp27-mEos3.2两种新型荧光探针的融合蛋白并研究其对脂滴融合的功能影响,进而为研发Fsp27相关生理功能的光学显像技术奠定基础.方法:对照传统绿色荧光的融合蛋白Fsp27-EGFP,在共聚焦显微镜下观察Fsp27-mMaple3和Fsp27-mEos3.2两种新型融合蛋白的亚细胞定位和介导脂滴融合的功能,并利用荧光漂白恢复术(fluorescence recovery afterphoto-bleaching,FRAP)以判断脂滴与脂滴之间是否存在脂的交换.结果:表达Fsp27-mMaple3和Fsp27-mEos3.2两种新型融合蛋白的细胞中脂滴显著增大;同时,融合蛋白皆集中在脂滴与脂滴的接触位点上,且中性脂的交换实验显示脂滴与脂滴之间可以相互连通.结论:我们建构的两种新型荧光探针融合蛋白Fsp27-mMaple3和Fsp27-mEos3.2保持了Fsp27介导脂滴融合的功能,并为我们进一步研发新型的超分辨光学显像技术提供功能基础.