Mehrle W, Hampp R, Zimmermann U
Institut für Biologie I, Universität Tübingen, F.R.G.
Biochim Biophys Acta. 1989 Jan 30;978(2):267-75. doi: 10.1016/0005-2736(89)90124-7.
Asymmetric breakdown (occurring in only one hemisphere of the cell) was induced in freely suspended and dielectrophoretically aligned vacuole-containing or evacuolated plant protoplasts as well as in isolated vacuoles. In suspended cells breakdown was restricted to the hemisphere facing the anode and in isolated vacuoles to the opposite hemisphere. This difference in the orientation of the asymmetric breakdown can be explained by the opposite direction of the intrinsic membrane potentials of isolated vacuoles and of cells on which the generated potential difference is superimposed. The ensuing permeabilization of the membrane was microscopically monitored by dye uptake and by release of chloroplasts and of cytoplasmic and/or vacuolar solutes. The asymmetric release of intracellular substances (organic acids and/or amino acids) was detected by accumulation of chemotactic bacteria (Pseudomonas aeruginosa) close to the permeabilised membrane area of the cells or vacuoles. Maximum bacteria accumulation required about 5 min and subsequently disappeared after a further 20 min presumably because of the restoration of the original membrane impermeability. With vacuoles retention of the accumulated bacteria was shorter indicating that the resealing process of the tonoplast membrane was faster than that of the plasmalemma. From the kinetics of bacteria accumulation and retention it is therefore possible to deduce information about the life-span and the resealing properties of electropermeabilized membrane areas on the single-cell level. Symmetric breakdown in both hemispheres of the cells could be achieved by electric field-mediated cell rotation of about 180 degrees between two pulses of the same polarity or by application of two pulses of alternating polarity. In dielectrophoretically aligned protoplasts of comparable diameter, breakdown occurred in both hemispheres, even though the breakdown was still asymmetric. It could be demonstrated by the uptake of the vital dye neutral red that the size of the membrane area which was permeabilized was much larger in that hemisphere oriented to the anode than in the other one. The relevance of these observations for further improvement of electroinjection of macromolecules and of electrofusion is discussed. In particular, it is pointed out that positioning of differently sized cells in electric field-mediated hybridisation and the polarity of the breakdown pulse is of great importance with respect to hybrid yield.
在自由悬浮且经介电泳排列的含液泡或无液泡植物原生质体以及分离出的液泡中诱导不对称破裂（仅发生在细胞的一个半球）。在悬浮细胞中，破裂局限于面向阳极的半球，而在分离出的液泡中则局限于相对的半球。不对称破裂方向的这种差异可以用分离出的液泡与叠加了产生的电位差的细胞的固有膜电位的相反方向来解释。随后通过染料摄取以及叶绿体、细胞质和/或液泡溶质的释放，在显微镜下监测膜的通透性。通过趋化细菌（铜绿假单胞菌）在细胞或液泡的通透膜区域附近的聚集来检测细胞内物质（有机酸和/或氨基酸）的不对称释放。最大细菌聚集需要约5分钟，随后在再过20分钟后消失，推测是因为恢复了原来的膜不渗透性。对于液泡，聚集细菌的保留时间较短，表明液泡膜的重新封闭过程比质膜快。因此，从细菌聚集和保留的动力学可以推断出关于单细胞水平上电通透化膜区域的寿命和重新封闭特性的信息。通过在两个相同极性的脉冲之间进行约180度的电场介导细胞旋转或通过施加两个交替极性的脉冲，可以实现细胞两个半球的对称破裂。在介电泳排列的直径相当的原生质体中，两个半球都会发生破裂，尽管破裂仍然是不对称的。通过摄取活体染料中性红可以证明，在面向阳极的半球中通透化的膜面积比另一个半球大得多。讨论了这些观察结果对于进一步改进大分子电注射和电融合的相关性。特别指出，在电场介导的杂交中不同大小细胞的定位以及破裂脉冲的极性对于杂交产量非常重要。
