Stemple D L, Sweet S C, Welsh M J, McIntosh J R
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309-0347.
Cell Motil Cytoskeleton. 1988;9(3):231-42. doi: 10.1002/cm.970090305.
We have compared the exchange kinetics of fluorescein-labeled calmodulin and tubulin in the spindles of living mitotic cells at metaphase. Cultured mammalian cells in early stages of mitosis were microinjected with labeled calmodulin or tubulin and returned to an incubator to allow equilibration of the fluorescent protein with the endogenous protein pools. Calmodulin becomes concentrated in the mitotic spindle, and treatments with inhibitors of tubulin assembly show that this concentration is dependent on the presence of microtubules. The steady-state exchange rates of both tubulin and calmodulin were measured by an analysis of fluorescence redistribution after photobleaching (FRAP), using cells pre-equilibrated to either 26 +/- 2 degrees C or 36 +/- 2 degrees C. A pulse of laser light focused to a 5-microns diameter column was used to destroy the fluorescence at one pole of a metaphase mitotic spindle. Ratios of fluorescence intensity from the two half-spindles and from the two polar regions were calculated for each image in a post-bleach time series to determine the rates and extents of FRAP. For tubulin, we confirm earlier observations concerning the temperature dependence of the extent of FRAP, but our data do not show a significant temperature dependence for the rate of FRAP. We hypothesize that the reduced extent of tubulin FRAP at the lower temperatures is a result of microtubules that are stable to depolymerization at 26 degrees C and are thus less likely to exchange subunits. Calmodulin's FRAP, however, does not exhibit any of the temperature dependence observed with fluorescent tubulin. At 26 +/- 2 degrees C calmodulin exchanges rapidly with the relatively stable population of microtubules, suggesting that calmodulin is bound, either directly or indirectly, to microtubule walls.
我们比较了中期有丝分裂活细胞纺锤体中荧光素标记的钙调蛋白和微管蛋白的交换动力学。将处于有丝分裂早期的培养哺乳动物细胞显微注射标记的钙调蛋白或微管蛋白，然后放回培养箱中，以使荧光蛋白与内源性蛋白池达到平衡。钙调蛋白在有丝分裂纺锤体中聚集，用微管蛋白组装抑制剂处理表明这种聚集依赖于微管的存在。使用预先平衡到26±2℃或36±2℃的细胞，通过光漂白后荧光重新分布分析（FRAP）来测量微管蛋白和钙调蛋白的稳态交换率。一束聚焦成直径5微米柱的激光脉冲用于破坏中期有丝分裂纺锤体一极的荧光。在漂白后时间序列中，为每个图像计算来自两个半纺锤体和两个极区的荧光强度比值，以确定FRAP的速率和程度。对于微管蛋白，我们证实了先前关于FRAP程度温度依赖性的观察结果，但我们的数据未显示FRAP速率有显著的温度依赖性。我们推测，在较低温度下微管蛋白FRAP程度降低是由于微管在26℃下对解聚稳定，因此亚基交换的可能性较小。然而，钙调蛋白的FRAP没有表现出荧光微管蛋白所观察到的任何温度依赖性。在26±2℃时，钙调蛋白与相对稳定的微管群体快速交换，这表明钙调蛋白直接或间接地与微管壁结合。
