Sato M, Grasser W
Department of Bone Biology and Osteoporosis Research, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Cell Motil Cytoskeleton. 1990;17(3):250-63. doi: 10.1002/cm.970170311.
We present microinjection data in support of an indirect approach by which cytoplasmic protein interactions important in the processes of bone resorption can be elucidated. Three polyclonal antibodies (M1, M3, M5) raised against myosin II from perfused rat liver differently affected the actin-activated Mg ATPase of myosin II. These antibodies microinjected into isolated rat osteoclasts affected osteoclast morphology and activity in bone resorption. M1, which completely inhibited myosin ATPase activity at a antibody:myosin ratio of 10:1, initially promoted the extension/retraction motility of lamellipodia but eventually reduced the spread area of osteoclasts along the substrate after 20 hr. M3, which inhibited ATPase activity by 70%, had similar effects; however, M5, which weakly inhibited ATPase activity, neither promoted extension/retraction nor reduced spread area of osteoclasts. Immunofluorescence showed that these antibodies removed myosin II from the majority of actin filaments in injected osteoclasts. Because antibodies that did not bind to a myosin II column had little effect on the extension/retraction of lamellipodia or the osteoclast spread area, these data suggest that myosin II participates in the stabilization of osteoclast lamellipodia along the substrate. M1 injection strongly inhibited injected osteoclasts from excavating resorption lacunae in bone slices, compared to control antibody. M3 and M5 were less effective but also inhibited bone resorption. These data show that myosin II is functionally important in bone resorption and that the osteoclast-differentiated activity of bone resorption is a more sensitive assay for myosin activity than lamellipodia motility or cell morphology.
我们提供了显微注射数据，以支持一种间接方法，通过该方法可以阐明在骨吸收过程中重要的细胞质蛋白相互作用。三种针对灌注大鼠肝脏肌球蛋白II产生的多克隆抗体（M1、M3、M5）对肌球蛋白II的肌动蛋白激活的Mg ATP酶有不同影响。将这些抗体显微注射到分离的大鼠破骨细胞中，会影响破骨细胞的形态和骨吸收活性。M1在抗体与肌球蛋白比例为10:1时完全抑制肌球蛋白ATP酶活性，最初促进了片状伪足的伸展/收缩运动，但最终在20小时后减少了破骨细胞沿底物的铺展面积。M3抑制ATP酶活性70%，有类似效果；然而，M5对ATP酶活性的抑制较弱，既不促进片状伪足的伸展/收缩，也不减少破骨细胞的铺展面积。免疫荧光显示，这些抗体使注射的破骨细胞中大多数肌动蛋白丝上的肌球蛋白II消失。由于未与肌球蛋白II柱结合的抗体对片状伪足的伸展/收缩或破骨细胞铺展面积影响很小，这些数据表明肌球蛋白II参与了破骨细胞片状伪足沿底物的稳定过程。与对照抗体相比，注射M1强烈抑制了注射的破骨细胞在骨切片中挖掘吸收陷窝。M3和M5的效果较差，但也抑制了骨吸收。这些数据表明，肌球蛋白II在骨吸收中具有重要功能，并且破骨细胞分化的骨吸收活性是比片状伪足运动或细胞形态更灵敏的肌球蛋白活性检测方法。
