How to unveil self-quenched fluorophores and subsequently map the subcellular distribution of exogenous peptides.
How to unveil self-quenched fluorophores and subsequently map the subcellular distribution of exogenous peptides.
复制标题
如何揭示自淬灭的荧光团，然后绘制外源肽的亚细胞分布。
DOI：
10.1038/srep20237
复制
发表时间：
2016-02-03
影响因子：
4.6
通讯作者：
Lavielle S
中科院分区：
文献类型：
--
作者：
Swiecicki JM;Thiebaut F;Di Pisa M;Gourdin-Bertin S;Tailhades J;Mansuy C;Burlina F;Chwetzoff S;Trugnan G;Chassaing G;Lavielle S
共聚焦激光扫描显微镜（CLSM）是用于绘制荧光分子的亚细胞分布的最流行的技术，并且被广泛用于研究外源大分子（例如细胞穿透肽（CPP））在细胞内的穿透特性。尽管所有这些CPP的膜缔合倾向，荧光标记的CPP的信号不与质膜共定位。我们研究了这种荧光消光的起源和CLSM图片的细胞内定位的解释的总体后果。我们证明，这种差异源于荧光自猝灭。通过“稀释”方案，即通过改变荧光/非荧光CPP的比率来揭示荧光。这种策略使我们能够对几种CPP的亚细胞分布进行置信度排名，有助于阐明渗透机制。更一般地说，这项研究提出了一个广泛适用的和可靠的方法来研究任何荧光标记分子的亚细胞分布。
Confocal laser scanning microscopy (CLSM) is the most popular technique for mapping the subcellular distribution of a fluorescent molecule and is widely used to investigate the penetration properties of exogenous macromolecules, such as cell-penetrating peptides (CPPs), within cells. Despite the membrane-association propensity of all these CPPs, the signal of the fluorescently labeled CPPs did not colocalize with the plasma membrane. We studied the origin of this fluorescence extinction and the overall consequence on the interpretation of intracellular localizations from CLSM pictures. We demonstrated that this discrepancy originated from fluorescence self-quenching. The fluorescence was unveiled by a “dilution” protocol, i.e. by varying the ratio fluorescent/non-fluorescent CPP. This strategy allowed us to rank with confidence the subcellular distribution of several CPPs, contributing to the elucidation of the penetration mechanism. More generally, this study proposes a broadly applicable and reliable method to study the subcellular distribution of any fluorescently labeled molecules.