O'Brien E T, Salmon E D, Erickson H P
Department of Ophthalmology and Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Cell Motil Cytoskeleton. 1997;36(2):125-35. doi: 10.1002/(SICI)1097-0169(1997)36:2<125::AID-CM3>3.0.CO;2-8.
The effects of calcium (Ca) were assessed using video-enhanced differential interference contrast light microscopy on individual microtubules in vitro. Phosphocellulose-purified (PC) and microtubule associated protein (MAP)-containing preparations of porcine brain tubulin were assembled in a flow chamber onto sperm axoneme fragments and the pattern of growth and shortening of the microtubules was observed. Tubulin plus Ca was then added to the chamber and observation continued. Ca promoted the disassembly of microtubules by specifically promoting the catastrophe reaction in both PC- and MAP-containing microtubules, without an appreciable change in elongation rate. The effect on catastrophe frequency increased very rapidly above 0.5 mM free Ca, implying a possible cooperative effect. The rescue rate remained very high after Ca addition in MAP-containing microtubules, and the shortening rate was unchanged, while in phosphocellulose-purified microtubules, rescue appeared to be decreased by Ca addition and shortening rates increased 4 to 6-fold. These results illustrate that Ca can directly destabilize growing microtubule ends without changing the effective concentration of free tubulin, and that this effect can be seen even against the background of the profound differences in dynamics conferred by the microtubule-associated proteins. Considered within models of the GTP cap, the results imply that high Ca may act to increase the rate of GTP hydrolysis within the cap.
利用视频增强微分干涉相差光学显微镜在体外对单个微管进行观察，评估了钙（Ca）的作用。将磷酸纤维素纯化（PC）的猪脑微管蛋白制剂以及含微管相关蛋白（MAP）的制剂在流动小室中组装到精子轴丝片段上，观察微管的生长和缩短模式。然后向小室中加入微管蛋白加钙，并继续观察。钙通过特异性促进含PC和含MAP微管中的灾难反应，促进了微管的解聚，而伸长率没有明显变化。在游离钙浓度高于0.5 mM时，对灾难频率的影响迅速增加，这意味着可能存在协同效应。在含MAP的微管中加入钙后，挽救率仍然很高，缩短率不变，而在磷酸纤维素纯化的微管中，加入钙后挽救率似乎降低，缩短率增加了4至6倍。这些结果表明，钙可以直接使正在生长的微管末端不稳定，而不改变游离微管蛋白的有效浓度，并且即使在微管相关蛋白赋予的动力学存在巨大差异的背景下，这种效应也能被观察到。在GTP帽模型中考虑，结果表明高钙可能会增加帽内GTP水解的速率。
