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

Novel Fluorescent Probes mMaple3 and mEos3.2 for The Functional Study of Fsp27-mediated Lipid Droplet Fusion

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 after photo-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。

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 the 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.