Detection of the entry of nonlabeled transportan 10 into single vesicles
Detection of the entry of nonlabeled transportan 10 into single vesicles
复制标题
检测非标记转运蛋白 10 进入单个囊泡
DOI：
10.1021/acs.biochem.0c00102
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发表时间：
2020
期刊：
影响因子：
2.9
通讯作者：
M. Yamazaki
中科院分区：
文献类型：
--
作者：
M. L. Shuma;M. M. R. Moghal;M. Yamazaki
细胞穿透肽（CPP）进入活细胞和脂质囊泡已经使用探针（例如，荧光染料）标记的CPP。然而，探针标记可以改变CPP与膜的相互作用。我们已经开发了一种新的方法来检测进入单一的巨大的单层囊泡（GUV）没有孔形成的GUV膜的内腔的未标记的CPPs。GUV含有大的单层囊泡（LUV），其内腔含有高（自猝灭）浓度的荧光染料钙黄绿素。如果CPP进入GUV腔并与这些LUV相互作用以诱导钙黄绿素渗漏，则由于GUV腔中的钙黄绿素而引起的荧光强度（FI）增加。LUV和GUV的脂质组成允许从LUV而不是从GUV泄漏。我们应用该方法检测转运蛋白10（TP10）进入包含二油酰磷脂酰甘油和二油酰磷脂酰胆碱的单个GUV，并检查低浓度的未标记的TP10与单个GUV（其内腔含有Alexa Fluor 647酰肼（AF 647）和上述LUV）的相互作用。由于钙黄绿素，GUV腔的FI随时间连续增加而没有AF 647的泄漏，表明TP10进入GUV而没有在GUV膜中形成孔。钙黄绿素引起的腔强度随着TP 10浓度的增加而增加，表明TP 10进入GUV腔的速率增加。我们估计的最低TP10浓度在GUV管腔检测到这种方法。我们讨论了非标记TP 10的输入和这种方法的特点。
The entry of cell-penetrating peptides (CPPs) into live cells and lipid vesicles has been monitored using probe (e.g., fluorescent dye)-labeled CPPs. However, probe labeling may alter the interaction of CPPs with membranes. We have developed a new method to detect the entry of nonlabeled CPPs into the lumens of single giant unilamellar vesicles (GUVs) without pore formation in the GUV membrane. The GUVs contain large unilamellar vesicles (LUVs) whose lumens contain a high (self-quenching) concentration of the fluorescent dye calcein. If the CPPs enter the GUV lumen and interact with these LUVs to induce calcein leakage, the fluorescence intensity (FI) due to calcein in the GUV lumen increases. The lipid compositions of the LUVs and GUVs allow leakage from LUVs but not from the GUVs. We applied this method to detect the entry of transportan 10 (TP10) into single GUVs comprising dioleoylphosphatidylglycerol and dioleoylphosphatidylcholine and examined the interaction of low concentrations of nonlabeled TP10 with single GUVs whose lumens contain Alexa Fluor 647 hydrazide (AF647) and the LUVs mentioned above. The FI of the GUV lumen due to calcein increased continuously with time without leakage of AF647, indicating that TP10 entered the GUV without pore formation in the GUV membrane. The lumen intensity due to calcein increased with TP10 concentration, indicating that the rate of entry of TP10 into the GUV lumen increased. We estimated the minimum TP10 concentration in a GUV lumen detected by this method. We discuss the entry of nonlabeled TP10 and the characteristics of this method.