Winkelmann D A, Bourdieu L, Ott A, Kinose F, Libchaber A
Department of Pathology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Biophys J. 1995 Jun;68(6):2444-53. doi: 10.1016/S0006-3495(95)80426-1.
We have analyzed the dependence of actin filament sliding movement on the mode of myosin attachment to surfaces. Monoclonal antibodies (mAbs) that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain (LC2) located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. This method of attachment provides a means of controlling the flexibility and density of myosin on the surface. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these mAbs, and the sliding movement of fluorescently labeled actin filaments was analyzed by video microscopy. Each of these antibodies produced stable myosin-coated surfaces that supported uniform motion of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM mAbs yielded significantly higher velocities (10 microns/s at 30 degrees C) than attachment through anti-LC2 (4-5 microns/s at 30 degrees C). For each antibody, we observed a characteristic value of the myosin density for the onset of F-actin motion and a second critical density for velocity saturation. The specific mode of attachment influences the velocity of actin filaments and the characteristic surface density needed to support movement.
我们分析了肌动蛋白丝滑动运动对肌球蛋白附着于表面方式的依赖性。使用与三个不同位点结合的单克隆抗体（mAb）将肌球蛋白 tether 到硝酸纤维素包被的玻璃上。一种抗体与位于头部 - 杆连接处的调节轻链（LC2）上的表位反应。另外两种与杆结构域中的位点反应，一个在靠近 S2 - LMM 铰链的 S2 区域，另一个在肌球蛋白杆的 C 末端。这种附着方法提供了一种控制表面上肌球蛋白柔韧性和密度的手段。快速骨骼肌肌球蛋白单体通过与这些 mAb 的特异性相互作用结合到表面，并且通过视频显微镜分析荧光标记的肌动蛋白丝的滑动运动。这些抗体中的每一种都产生了稳定的肌球蛋白包被表面，在数小时内支持肌动蛋白的均匀运动。通过抗 S2 和抗 LMM mAb 附着肌球蛋白产生的速度（30℃时为 10 微米/秒）明显高于通过抗 LC2 附着（30℃时为 4 - 5 微米/秒）。对于每种抗体，我们观察到 F - 肌动蛋白运动开始时肌球蛋白密度的特征值以及速度饱和的第二个临界密度。特定的附着方式影响肌动蛋白丝的速度以及支持运动所需的特征表面密度。
