Real-time imaging of individual electropores proves their longevity in cells.
Real-time imaging of individual electropores proves their longevity in cells.
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单个电孔的实时成像证明了它们在细胞中的寿命。
DOI：
10.1016/j.bbrc.2023.149408
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发表时间：
2024
影响因子：
3.1
通讯作者：
Pakhomov,AndreiG
中科院分区：
文献类型：
--
作者：
Silkunas,Mantas;Silkuniene,Giedre;Pakhomov,AndreiG
经过 50 多年的电穿孔研究，细胞膜透化的本质仍然难以捉摸。分子模型中电孔的寿命仅限于纳秒或微秒，而电穿孔细胞的透化可以持续几分钟。这项研究旨在解决长期存在的争论，即长期通透是否是由于细胞中长寿命孔的形成所致。我们开发了一种对单个电孔进行动态监测和电导测量的方法。这是通过在装载有 CAL-520 染料并放置在氧化铟锡 (ITO) 表面上的 HEK 细胞中进行延时全内反射荧光 (TIRF) 成像来实现的。在贴片移液管和 ITO 之间施加 1 毫秒、0 至 -400 mV 的脉冲，引起局部 Ca2+ 瞬变，从而识别各个电孔。一些瞬变现象在几毫秒内消失，但另一些则持续了一分钟多。持续瞬变（“Ca2+羽流”）随着时间的推移逐渐减弱到稳定或随机波动的水平，其中可能包括完全静止的时期。单孔电导在电穿孔后 30 秒和 60 秒以 0 至 -50 mV、50 ms 步进测量，范围为 80 至 200 pS。这些实验证明了细胞中电孔的寿命，与分子模拟和脂质双层中的许多发现形成鲜明对比。
With over 50 years of electroporation research, the nature of cell membrane permeabilization remains elusive. The lifetime of electropores in molecular models is limited to nano- or microseconds, whereas the permeabilization of electroporated cells can last minutes. This study aimed at resolving a longstanding debate on whether the prolonged permeabilization is due to the formation of long-lived pores in cells. We developed a method for dynamic monitoring and conductance measurements of individual electropores. This was accomplished by time-lapse total internal reflection fluorescence (TIRF) imaging in HEK cells loaded with CAL-520 dye and placed on an indium tin oxide (ITO) surface. Applying a 1-ms, 0 to −400 mV pulse between the patch pipette and ITO evoked focal Ca2+transients that identified individual electropores. Some transients disappeared in milliseconds but others persisted for over a minute. Persistent transients (“Ca2+plumes”) faded over time to a stable or a randomly fluctuating level that could include periods of full quiescence. Single pore conductance, measured by 0 to −50 mV, 50 ms steps at 30 and 60 s after the electroporation, ranged from 80 to 200 pS. These experiments proved electropore longevity in cells, in stark contrast to molecular simulations and many findings in lipid bilayers.