Huang J, Raff J W
Wellcome/CRC Institute and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK.
EMBO J. 1999 Apr 15;18(8):2184-95. doi: 10.1093/emboj/18.8.2184.
We have followed the behaviour of a cyclin B-green fluorescent protein (GFP) fusion protein in living Drosophila embryos in order to study how the localization and destruction of cyclin B is regulated in space and time. We show that the fusion protein accumulates at centrosomes in interphase, in the nucleus in prophase, on the mitotic spindle in prometaphase and on the microtubules that overlap in the middle of the spindle in metaphase. In cellularized embryos, toward the end of metaphase, the spindle-associated cyclin B-GFP disappears from the spindle in a wave that starts at the spindle poles and spreads to the spindle equator; when the cyclin B-GFP on the spindle is almost undetectable, the chromosomes enter anaphase, and any remaining cytoplasmic cyclin B-GFP then disappears over the next few minutes. The endogenous cyclin B protein appears to behave in a similar manner. These findings suggest that the inactivation of cyclin B is regulated spatially in Drosophila cells. We show that the anaphase-promoting complex/cyclosome (APC/C) specifically interacts with microtubules in embryo extracts, but it is not confined to the spindle in mitosis, suggesting that the spatially regulated disappearance of cyclin B may reflect the spatially regulated activation of the APC/C.
为了研究细胞周期蛋白B的定位和降解在空间和时间上是如何调控的，我们追踪了细胞周期蛋白B-绿色荧光蛋白（GFP）融合蛋白在活的果蝇胚胎中的行为。我们发现，融合蛋白在间期聚集在中心体，前期在细胞核中，前中期在有丝分裂纺锤体上，中期在纺锤体中部重叠的微管上。在细胞化胚胎中，接近中期结束时，与纺锤体相关的细胞周期蛋白B-GFP以一种从纺锤体极开始并蔓延到纺锤体赤道的波的形式从纺锤体上消失；当纺锤体上的细胞周期蛋白B-GFP几乎检测不到时，染色体进入后期，随后任何剩余的细胞质细胞周期蛋白B-GFP在接下来的几分钟内消失。内源性细胞周期蛋白B蛋白似乎也有类似的行为。这些发现表明，果蝇细胞中细胞周期蛋白B的失活在空间上受到调控。我们发现后期促进复合体/细胞周期体（APC/C）在胚胎提取物中特异性地与微管相互作用，但在有丝分裂时并不局限于纺锤体，这表明细胞周期蛋白B在空间上受调控的消失可能反映了APC/C在空间上受调控的激活。
