Dhamodharan R, Wadsworth P
Molecular and Cellular Biology Program, University of Massachusetts at Amherst 01003, USA.
J Cell Sci. 1995 Apr;108 ( Pt 4):1679-89. doi: 10.1242/jcs.108.4.1679.
Heat-stable brain microtubule associated proteins (MAPs) and purified microtubule associated protein 2 (MAP-2) were microinjected into cultured BSC-1 cells which had been previously injected with rhodamine-labeled tubulin. The dynamic instability behavior of individual microtubules was then examined using low-light-level fluorescence microscopy and quantitative microtubule tracking methods. Both MAP preparations suppressed microtubule dynamics in vivo, by reducing the average rate and extent of both growing and shortening events. The average duration of growing events was not affected. When measured as events/unit time, heat-stable MAPs and MAP-2 did not significantly alter the frequency of rescue; the frequency of catastrophe was decreased approximately two-fold by heat-stable MAPs and MAP-2. When transition frequencies were calculated as events/unit distance, both MAP preparations increased the frequency of rescue, without altering the frequency of catastrophe. The percentage of total time spent in the phases of growth, shrink and pause was determined. Both MAP-2 and heat-stable MAPs decreased the percentage of time spent shortening, increased the percentage of time spent paused, and had no effect on percentage of time spent growing. Heat-stable MAPs increased the average pause duration, decreased the average number of events per minute per microtubule and increased the probability that a paused microtubule would switch to growing rather than shortening. The results demonstrate that addition of MAPs to living cells reduces the dynamic behavior of individual microtubules primarily by suppressing the magnitude of dynamic events and increasing the time spent in pause, where no change in the microtubule length can be detected. The results further suggest that the expression of MAPs directly contributes to cell type-specific microtubule dynamic behavior.
将热稳定脑微管相关蛋白（MAPs）和纯化的微管相关蛋白2（MAP - 2）显微注射到先前已注射罗丹明标记微管蛋白的培养BSC - 1细胞中。然后使用低光水平荧光显微镜和定量微管追踪方法检查单个微管的动态不稳定性行为。两种MAP制剂均通过降低生长和缩短事件的平均速率和程度来抑制体内微管动力学。生长事件的平均持续时间不受影响。当以事件/单位时间来衡量时，热稳定MAPs和MAP - 2并未显著改变挽救频率；热稳定MAPs和MAP - 2使灾难频率降低了约两倍。当将转变频率计算为事件/单位距离时，两种MAP制剂均增加了挽救频率，而不改变灾难频率。确定了在生长、收缩和暂停阶段所花费的总时间百分比。MAP - 2和热稳定MAPs均降低了收缩所花费的时间百分比，增加了暂停所花费的时间百分比，并且对生长所花费的时间百分比没有影响。热稳定MAPs增加了平均暂停持续时间，降低了每个微管每分钟的平均事件数，并增加了暂停微管转向生长而非缩短的概率。结果表明，向活细胞中添加MAPs主要通过抑制动态事件的幅度并增加暂停所花费的时间来降低单个微管的动态行为，在此期间无法检测到微管长度的变化。结果进一步表明，MAPs的表达直接促成了细胞类型特异性的微管动态行为。
