Sinard J H, Pollard T D
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore.
Cell Motil Cytoskeleton. 1989;12(1):42-52. doi: 10.1002/cm.970120106.
To study the in vivo role of myosin-II in Acanthamoeba castellanii, motile cells were microinjected with monoclonal antibodies raised against the myosin-II heavy chain. All injected cells underwent a transient shock response. It was found that although injection of buffer alone or of an endogenous Acanthamoeba protein decreased the motility of injected cells from 7 microns/min to approximately 3 microns/min, injection of monoclonal antibodies specific for myosin-II decreased motility further to approximately 0.8 micron/min. This effect was seen whether or not the monoclonal antibody to myosin-II inhibited the actomyosin-II MgATPase activity in vitro. Levels of antibody far in excess of endogenous myosin-II concentrations could not completely block amoeboid movement. The morphology of moving antimyosin-II-injected cells was unusual, suggesting a greater defect in the ability to retract the trailing edge of the cell rather than to extend the leading edge. Endosomes frequently disappeared from injected cells, and although buffer-injected cells rapidly recovered visible endosomes (50% recovery at 5 min), endosomes were not seen in antimyosin-II-injected cells until, on the average, approximately 50 min after injection. Injection of a nonspecific antibody or of a nonspecific exogenous protein (ovalbumin) also decreased the mobility of the injected cells beyond that of buffer-injected cells (to approximately 1 micron/min). These cells tended to recover endosomes more rapidly (approximately 25 min) than cells injected with antimyosin-II monoclonal antibodies. The inability of antibodies to myosin-II to inhibit completely any of the movements studied suggests that although myosin-II probably plays a role in these motilities, the cell either routinely uses or can draw upon another cytoplasmic motor to maintain locomotion, organelle movement, contractile vacuole activity, and endocytosis.
为了研究肌球蛋白-II在卡氏棘阿米巴中的体内作用，将抗肌球蛋白-II重链的单克隆抗体显微注射到运动细胞中。所有注射的细胞都经历了短暂的休克反应。结果发现，虽然单独注射缓冲液或注射内源性棘阿米巴蛋白会使注射细胞的运动能力从7微米/分钟降至约3微米/分钟，但注射针对肌球蛋白-II的单克隆抗体可使运动能力进一步降至约0.8微米/分钟。无论抗肌球蛋白-II单克隆抗体在体外是否抑制肌动球蛋白-II的MgATP酶活性，都能观察到这种效果。远远超过内源性肌球蛋白-II浓度的抗体水平并不能完全阻断阿米巴样运动。注射抗肌球蛋白-II的运动细胞形态异常，这表明细胞在回缩细胞后缘而非延伸前缘的能力方面存在更大缺陷。内体经常从注射的细胞中消失，虽然注射缓冲液的细胞能迅速恢复可见的内体（5分钟时恢复50%），但在注射抗肌球蛋白-II的细胞中，直到注射后平均约50分钟才可见到内体。注射非特异性抗体或非特异性外源蛋白（卵清蛋白）也会使注射细胞的运动能力比注射缓冲液的细胞进一步降低（降至约1微米/分钟）。这些细胞比注射抗肌球蛋白-II单克隆抗体的细胞恢复内体的速度更快（约25分钟）。抗肌球蛋白-II抗体无法完全抑制所研究的任何一种运动，这表明虽然肌球蛋白-II可能在这些运动中起作用，但细胞要么常规使用要么可以利用另一种细胞质马达来维持运动、细胞器移动、收缩泡活动和内吞作用。
