O'Brien E T, Salmon E D, Walker R A, Erickson H P
Department of Cell Biology, Duke University, Durham, North Carolina 27710.
Biochemistry. 1990 Jul 17;29(28):6648-56. doi: 10.1021/bi00480a014.
We investigated the effect of magnesium ion (Mg) on the parameters of dynamic instability of individual porcine brain microtubules. Rates of elongation and rapid shortening were measured by using video-enhanced DIC light microscopy and evaluated by using computer-generated plots of microtubule length vs time. Increasing [Mg] from 0.25 to 6 mM increased the second-order association rate constant for elongation about 25% at each end. At plus ends, this resulted in a 1.5-2-fold increase in elongation rates over the tubulin concentrations explored. Rapid shortening rates were more dramatically affected by Mg. As [Mg] was increased from 0.25 to 6 mM, the average rate of rapid shortening increased about 3-fold at plus ends and 4-5-fold at minus ends. The ends had roughly equivalent average rates at low [Mg], of 30-45 microns/min. At any Mg concentration, rates of disassembly varied from one microtubule to another, and often an individual microtubule would exhibit more than one rate during a single shortening phase. Individual rates at 6 mM Mg varied from 12 to 250 microns/min. Over the concentration range explored, Mg affected the frequencies of transition from elongation to shortening and back only at minus ends. Minus ends were relatively stable at low [Mg], having 4 times the frequency of rescue than at high [Mg], and a lower frequency of catastrophe (particularly evident at low tubulin concentrations). Plus ends, surprisingly, were highly unstable at all Mg concentrations investigated, having about the same transition frequencies as did the least stable (high Mg) minus ends. Our results have implications for models of the GTP cap, again emphasizing that GTP caps cannot build up in proportion to elongation rate, and must be constrained to the tips of growing microtubules.
我们研究了镁离子（Mg）对单个猪脑微管动态不稳定性参数的影响。通过视频增强型微分干涉相差（DIC）光学显微镜测量伸长率和快速缩短率，并使用计算机生成的微管长度与时间的曲线图进行评估。将[Mg]从0.25 mM增加到6 mM，使两端的二阶伸长缔合速率常数增加约25%。在正端，这导致在所研究的微管蛋白浓度范围内伸长率增加1.5至2倍。快速缩短率受Mg的影响更为显著。随着[Mg]从0.25 mM增加到6 mM，正端的快速缩短平均速率增加约3倍，负端增加4至5倍。在低[Mg]时，两端的平均速率大致相当，为30至45微米/分钟。在任何Mg浓度下，不同微管的解聚速率各不相同，并且在单个缩短阶段，单个微管通常会表现出不止一种速率。在6 mM Mg时，个体速率在12至250微米/分钟之间变化。在所研究的浓度范围内，Mg仅在负端影响从伸长到缩短以及再回到伸长的转变频率。在低[Mg]时，负端相对稳定，拯救频率是高[Mg]时的4倍，灾难频率较低（在低微管蛋白浓度下尤为明显）。令人惊讶的是，在所有研究的Mg浓度下，正端都高度不稳定，其转变频率与最不稳定（高Mg）的负端大致相同。我们的结果对GTP帽模型具有启示意义，再次强调GTP帽不能与伸长率成比例积累，并且必须局限于生长中的微管尖端。
