DeLuca Jennifer G, Moree Ben, Hickey Jennifer M, Kilmartin John V, Salmon E D
University of North Carolina at Chapel Hill, Department of Biology, Chapel Hill, NC 27599, USA.
J Cell Biol. 2002 Nov 25;159(4):549-55. doi: 10.1083/jcb.200208159. Epub 2002 Nov 18.
Identification of proteins that couple kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in kinetochore-microtubule attachment and suggest that hNuf2 is part of a molecular linker between the kinetochore attachment site and tubulin subunits within the lattice of attached plus ends.
鉴定将动粒与纺锤体微管相连的蛋白质对于理解有丝分裂过程中如何实现精确的染色体分离至关重要。在此我们表明，蛋白质hNuf2在HeLa细胞中特异性地在动粒处发挥作用，以实现微管的稳定附着。当通过RNA干扰使hNuf2缺失时，随着细胞进入有丝分裂，纺锤体正常形成，但动粒无法与纺锤体微管形成附着，细胞在前中期停滞，纺锤体检查点处于激活状态。缺失hNuf2的动粒保留了微管马达蛋白CENP - E和胞质动力蛋白，这些蛋白之前被认为参与募集动粒微管。动粒也保留了可检测水平的纺锤体检查点蛋白Mad2和BubR1，这与未附着的动粒激活纺锤体检查点的预期相符。此外，hNuf2缺失所产生的细胞周期阻滞诱导有丝分裂细胞发生细胞死亡。这些数据突出了hNuf2在动粒 - 微管附着中的特定作用，并表明hNuf2是动粒附着位点与附着正端晶格内微管蛋白亚基之间分子连接体的一部分。
