Kowalski R J, Williams R C
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235.
Cell Motil Cytoskeleton. 1993;26(4):282-90. doi: 10.1002/cm.970260403.
To understand the mechanism of dynamic instability of microtubule growth and shortening, one needs a means of reliably determining the polarity of the microtubules under investigation. Sea urchin sperm-tail axonemal fragments nucleate the growth of both plus-ended and minus-ended microtubules, but their polarity is not apparent by video-enhanced DIC microscopy. The polarity of a microtubule is usually assessed by observing differences between the rates and lengths of growth and shortening excursions of the two ends. In practice, though, a significant fraction of the population of microtubules displays characteristics intermediate between the average characteristics of either end, thereby escaping classification. Excluding these "intermediate" microtubules from the measured populations introduces bias into the understanding of microtubule dynamic instability. We circumvent this problem by making use of the plus-end directed movement of the microtubule-dependent molecular motor kinesin to determine the polarity of any given microtubule unambiguously. Carboxylated-microspheres coated with kinesin, which are clearly visible by DIC microscopy, were used to determine the polarity of a microtubule. The dynamics were then observed. Kinesin was found to have no marked effect on dynamic instability. By this technique, we show that the distributions of properties that describe microtubule dynamic instability (rates and lengths of growth and shortening as well as frequencies of interconversion between these phases) of plus-ends overlap to a significant extent with those of minus-ends. It is this overlap that obscures the usual classification of the ends. Therefore, models describing microtubule dynamic instability need to incorporate the broad and overlapping range of properties of the two ends.
为了理解微管生长和缩短的动态不稳定性机制,需要一种可靠地确定所研究微管极性的方法。海胆精子尾部轴丝片段可使正端和负端微管都开始生长,但通过视频增强微分干涉差显微镜观察,其极性并不明显。微管的极性通常通过观察两端生长和缩短过程的速率及长度差异来评估。然而实际上,很大一部分微管群体表现出的特征介于两端的平均特征之间,从而难以分类。将这些“中间”微管排除在测量群体之外会给微管动态不稳定性的理解带来偏差。我们利用依赖微管的分子马达驱动蛋白向正端的移动来明确确定任何给定微管的极性,从而规避了这个问题。用驱动蛋白包被的羧化微球在微分干涉差显微镜下清晰可见,用于确定微管的极性,然后观察其动态变化。结果发现驱动蛋白对动态不稳定性没有显著影响。通过这项技术,我们表明描述微管动态不稳定性的特性(生长和缩短的速率及长度以及这些阶段之间相互转换的频率)在正端和负端的分布有很大程度的重叠。正是这种重叠模糊了两端通常的分类。因此,描述微管动态不稳定性的模型需要纳入两端广泛且重叠的特性范围。