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

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。

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

ABSTRACT 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. Conclusion: 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.