Hosu Basarab G, Sun Mingzhai, Marga Françoise, Grandbois Michel, Forgacs Gabor
Department of Biological Sciences, University of Missouri, Columbia, MO 65211, USA.
Phys Biol. 2007 Apr 19;4(2):67-78. doi: 10.1088/1478-3975/4/2/001.
Membrane nanotubes, under physiological conditions, typically form en masse. We employed magnetic tweezers (MTW) to extract tethers from human brain tumor cells and compared their biophysical properties with tethers extracted after disruption of the cytoskeleton and from a strongly differing cell type, Chinese hamster ovary cells. In this method, the constant force produced with the MTW is transduced to cells through super-paramagnetic beads attached to the cell membrane. Multiple sudden jumps in bead velocity were manifest in the recorded bead displacement-time profiles. These discrete events were interpreted as successive ruptures of individual tethers. Observation with scanning electron microscopy supported the simultaneous existence of multiple tethers. The physical characteristics, in particular, the number and viscoelastic properties of the extracted tethers were determined from the analytic fit to bead trajectories, provided by a standard model of viscoelasticity. Comparison of tethers formed with MTW and atomic force microscopy (AFM), a technique where the cantilever-force transducer is moved at constant velocity, revealed significant differences in the two methods of tether formation. Our findings imply that extreme care must be used to interpret the outcome of tether pulling experiments performed with single molecular techniques (MTW, AFM, optical tweezers, etc). First, the different methods may be testing distinct membrane structures with distinct properties. Second, as soon as a true cell membrane (as opposed to that of a vesicle) can attach to a substrate, upon pulling on it, multiple nonspecific membrane tethers may be generated. Therefore, under physiological conditions, distinguishing between tethers formed through specific and nonspecific interactions is highly nontrivial if at all possible.
在生理条件下，膜纳米管通常会大量形成。我们使用磁性镊子（MTW）从人脑肿瘤细胞中提取系链，并将其生物物理特性与细胞骨架破坏后以及与一种差异很大的细胞类型——中国仓鼠卵巢细胞中提取的系链进行比较。在这种方法中，MTW产生的恒定力通过附着在细胞膜上的超顺磁性珠子传递到细胞上。在记录的珠子位移-时间曲线上出现了珠子速度的多次突然跳跃。这些离散事件被解释为单个系链的连续断裂。扫描电子显微镜观察支持了多个系链的同时存在。提取的系链的物理特性，特别是数量和粘弹性特性，是根据由粘弹性标准模型提供的对珠子轨迹的解析拟合来确定的。对用MTW和原子力显微镜（AFM）形成的系链进行比较，AFM是一种悬臂力传感器以恒定速度移动的技术，结果显示两种系链形成方法存在显著差异。我们的研究结果表明，在解释用单分子技术（MTW、AFM、光镊等）进行的系链拉伸实验结果时必须格外小心。首先，不同的方法可能在测试具有不同特性的不同膜结构。其次，一旦真正的细胞膜（与囊泡的膜相对）能够附着在底物上，在对其进行拉伸时，可能会产生多个非特异性膜系链。因此，在生理条件下，区分通过特异性和非特异性相互作用形成的系链即使有可能也是非常困难的。
