Cell cycle-dependent changes in the dynamics of MAP 2 and MAP 4 in cultured cells.
Cell cycle-dependent changes in the dynamics of MAP 2 and MAP 4 in cultured cells.
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DOI：
10.1083/jcb.109.1.211
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发表时间：
1989-07
期刊：
影响因子：
--
通讯作者：
McIntosh JR
中科院分区：
文献类型：
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作者：
Olmsted JB;Stemple DL;Saxton WM;Neighbors BW;McIntosh JR
为了研究微管相关蛋白（MAP）在活细胞中的行为，用6-碘乙酰胺荧光素对MAP 4和MAP 2进行了衍生化，并利用微光录像系统和光漂白后的荧光再分布分析了显微注射MAP的分布。显微注射荧光标记的MAP 4或MAP 2后1分钟内，在间期或有丝分裂的PtK 1细胞中可见荧光微管阵列。冷处理后的荧光MAP 2-含有细胞（3小时，4摄氏度），微管荧光消失，和唯一的荧光高于背景位于中心体;微管模式返回升温后。诺考多唑处理后微管免疫荧光的丧失在MAP注射的细胞和对照细胞中相似，表明注射的荧光素标记的MAP 2不能稳定微管. FRAP进一步检查了MAP的动态。间期细胞的FRAP分析表明，MAP 2重新分布的半衰期（60 +/- 25 s）略长于MAP 4（44 +/- 20 s），但这两种类型的MAP结合微管在体内交换可溶性MAP的速度超过微管蛋白的周转率。这些数据表明，微管在间期细胞组装不断交换人口的MAP。37 ℃或26 ℃下的中期细胞显示MAP 2和MAP 4的平均再分布半衰期相似;这些比间期速率快3-4倍（MAP 2，t1/2 = 14 +/- 6 s; MAP 4，t1/2 = 17 +/- 5 s）。在26或37 ℃下，注射MAP的细胞中纺锤体荧光的恢复程度为84-94%。虽然大多数中期微管蛋白，如地图，在生理条件下迅速和完全翻转，已发表的工作表明，在26摄氏度的营业额的速度或程度降低，这表明快速有丝分裂MAP交换不仅仅是因为快速微管蛋白营业额。MAP 4结合到末期中间体的交换与中期纺锤体中观察到的动力学相当（t1/2 =约27 s），而中间体微管蛋白交换缓慢（大于300 s）。这些数据表明微管上MAP交换的速率是细胞周期中时间的函数。
To examine the behavior of microtubule-associated proteins (MAPs) in living cells, MAP 4 and MAP 2 have been derivatized with 6- iodoacetamido-fluorescein, and the distribution of microinjected MAP has been analyzed using a low light level video system and fluorescence redistribution after photobleaching. Within 1 min following microinjection of fluoresceinated MAP 4 or MAP 2, fluorescent microtubule arrays were visible in interphase or mitotic PtK1 cells. After cold treatment of fluorescent MAP 2-containing cells (3 h, 4 degrees C), microtubule fluorescence disappeared, and the only fluorescence above background was located at the centrosomes; microtubule patterns returned upon warming. Loss of microtubule immunofluorescence after nocodozole treatment was similar in MAP- injected and control cells, suggesting that injected fluorescein- labeled MAP 2 did not stabilize microtubules. The dynamics of the MAPs were examined further by FRAP. FRAP analysis of interphase cells demonstrated that MAP 2 redistributed with half-times slightly longer (60 +/- 25 s) than those for MAP 4 (44 +/- 20 s), but both types of MAPs bound to microtubules in vivo exchanged with soluble MAPs at rates exceeding the rate of tubulin turnover. These data imply that microtubules in interphase cells are assembled with constantly exchanging populations of MAP. Metaphase cells at 37 degrees C or 26 degrees C showed similar mean redistribution half-times for both MAP 2 and MAP 4; these were 3-4 fold faster than the interphase rates (MAP 2, t1/2 = 14 +/- 6 s; MAP 4, t1/2 = 17 +/- 5 s). The extent of recovery of spindle fluorescence in MAP-injected cells was to 84-94% at either 26 or 37 degrees C. Although most metaphase tubulin, like the MAPs, turns over rapidly and completely under physiologic conditions, published work shows either reduced rates or extents of turnover at 26 degrees C, suggesting that the fast mitotic MAP exchange is not simply because of fast tubulin turnover. Exchange of MAP 4 bound to telophase midbodies occurred with dynamics comparable to those seen in metaphase spindles (t1/2 = approximately 27 s) whereas midbody tubulin exchange was slow (greater than 300 s). These data demonstrate that the rate of MAP exchange on microtubules is a function of time in the cell cycle.