| 黄 超,郁 苗,汪文敏,廖榕玉,徐 俐,周林康.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[J].,2018,(4):601-605 |
| 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 |
| 投稿时间：2017-06-12 修订日期：2017-06-30 |
| DOI：10.13241/j.cnki.pmb.2018.04.001 |
| 中文关键词: 脂滴 荧光探针 亚细胞定位 Fsp27 |
| 英文关键词: Lipid droplet Fluorescent probes Subcellular localization Fsp27 |
| 基金项目:中国博士后科学基金项目(2013T60103，2012M520249)；周林康博士受中国科协2016-2018年度青年人才托举计划的支持 |
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| 中文摘要: |
| 摘要 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。 |
| 英文摘要: |
| 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. |
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