Garrett Sarah, Auer Kristi, Compton Duane A, Kapoor Tarun M
Laboratory of Chemistry and Cell Biology, Rockefeller University, New York, NY 10021, USA.
Curr Biol. 2002 Dec 10;12(23):2055-9. doi: 10.1016/s0960-9822(02)01277-0.
Bipolar spindle formation is essential for the accurate segregation of genetic material during cell division. Although centrosomes influence the number of spindle poles during mitosis, motor and non-motor microtubule-associated proteins (MAPs) also play key roles in determining spindle morphology. TPX2 is a novel MAP also characterized in Xenopus cell-free extracts. To examine hTPX2 (human TPX2) function in human cells, we used siRNA to knock-down its expression and found that cells lacking hTPX2 arrest in mitosis with multipolar spindles. NuMA, gamma-tubulin, and centrin localize to each pole, and nocodazole treatment of cells lacking hTPX2 demonstrates that the localization of gamma-tubulin to multiple spindle poles requires intact microtubules. Furthermore, we show that the formation of monopolar microtubule arrays in human cell extracts does not require hTPX2, demonstrating that the mechanism by which hTPX2 promotes spindle bipolarity is independent of activities focusing microtubule minus ends at spindle poles. Finally, inhibition of the kinesin Eg5 in hTPX2-depleted cells leads to monopolar spindles, indicating that Eg5 function is necessary for multipolar spindle formation in the absence of hTPX2. Our observations reveal a structural role for hTPX2 in spindles and provide evidence for a balance between microtubule-based motor forces and structural spindle components.
双极纺锤体的形成对于细胞分裂过程中遗传物质的准确分离至关重要。尽管中心体在有丝分裂期间影响纺锤体极的数量，但驱动蛋白和非驱动蛋白微管相关蛋白（MAPs）在决定纺锤体形态方面也起着关键作用。TPX2是一种新发现的MAP，在非洲爪蟾无细胞提取物中也有其特征描述。为了研究人源TPX2（hTPX2）在人类细胞中的功能，我们使用小干扰RNA（siRNA）敲低其表达，发现缺乏hTPX2的细胞在有丝分裂时停滞，形成多极纺锤体。核有丝分裂器蛋白（NuMA）、γ-微管蛋白和中心蛋白定位于每个纺锤体极，用诺考达唑处理缺乏hTPX2的细胞表明，γ-微管蛋白定位于多个纺锤体极需要完整的微管。此外，我们发现人细胞提取物中单极微管阵列的形成不需要hTPX2，这表明hTPX2促进纺锤体双极性的机制独立于将微管负端聚焦于纺锤体极的活动。最后，在缺乏hTPX2的细胞中抑制驱动蛋白Eg5会导致单极纺锤体的形成，这表明在缺乏hTPX2的情况下，Eg5的功能对于多极纺锤体的形成是必需的。我们的观察揭示了hTPX2在纺锤体中的结构作用，并为基于微管的驱动力和纺锤体结构成分之间的平衡提供了证据。
