Holohan S-J P, Marston S B
Nation Heart and Lung Institute, Imperial College London, London SW3 6LY, UK.
IEE Proc Nanobiotechnol. 2005 Jun;152(3):113-20. doi: 10.1049/ip-nbt:20045003.
The effect of applying an external load to actin filaments moving in the in vitro motility assay is studied. Bead-tailed actin filaments were made by polymerising actin onto 2.8 microm diameter Dynabeads conjugated with gelsolin-G actin. These were introduced into a motility cell coated with 100 microg/ml rabbit fast skeletal myosin in the presence of ATP and 0.5% methylcellulose. The motility cell was inserted between the pole-pieces of an electromagnet and the fluorescent beads and filaments were observed. The force-current relationship of the electromagnet was determined from the velocity of free beads in viscous solution and Stokes' equation. The magnet produced up to 6 pN force on the Dynabeads at 1 A. Many bead-tailed actin filaments stuck to the surface, but the beads that did move moved at the same speed as unloaded f-actin in the same cell. Bead-tailed filaments slowed down under an increasing magnetic load, eventually stalled and then slid backward under increasing load before detaching from the surface. Single-filament force-velocity curves were constructed and a stalling force of about 0.6 pN/mm of actin filament estimated.
研究了在体外运动分析中对移动的肌动蛋白丝施加外部负载的影响。通过将肌动蛋白聚合到与凝溶胶蛋白 - G肌动蛋白偶联的2.8微米直径的磁珠上制备珠尾肌动蛋白丝。在ATP和0.5%甲基纤维素存在的情况下，将这些丝引入涂有100微克/毫升兔快肌肌球蛋白的运动细胞中。将运动细胞插入电磁铁的极片之间，观察荧光珠和丝。根据粘性溶液中自由珠的速度和斯托克斯方程确定电磁铁的力 - 电流关系。在1 A电流下，磁铁对磁珠产生高达6 pN的力。许多珠尾肌动蛋白丝附着在表面，但移动的珠与同一细胞中未加载的f - 肌动蛋白以相同速度移动。珠尾丝在不断增加的磁负载下减速，最终停止，然后在负载增加时向后滑动，最后从表面脱离。构建了单丝力 - 速度曲线，并估计肌动蛋白丝的失速力约为0.6 pN/mm。
