Christodoulou Andri, Lederer Carsten W, Surrey Thomas, Vernos Isabelle, Santama Niovi
Department of Biological Sciences, University of Cyprus and Cyprus Institute of Neurology and Genetics, PO Box 20537, 1678 Nicosia, Cyprus.
J Cell Sci. 2006 May 15;119(Pt 10):2035-47. doi: 10.1242/jcs.02922. Epub 2006 Apr 25.
Inhibition of motor protein activity has been linked with defects in the formation of poles in the spindle of dividing cells. However, the molecular mechanisms underlying the functional relationship between motor activity and centrosome dynamics have remained uncharacterised. Here, we characterise KIFC5A, a mouse kinesin-like protein that is highly expressed in dividing cells and tissues, and is subject to developmental and cell-type-specific regulation. KIFC5A is a minus-end-directed, microtubule-dependent motor that produces velocities of up to 1.26 microm minute(-1) in gliding assays and possesses microtubule bundling activity. It is nuclear in interphase, localises to the centre of the two microtubule asters at the beginning of mitosis, and to spindle microtubules in later mitotic phases. Overexpression of KIFC5A in mouse cells causes the formation of aberrant, non-separated microtubule asters and mitotic arrest in a prometaphase-like state. KIFC5A knockdown partly rescues the phenotype caused by inhibition of plus-end-directed motor Eg5 by monastrol on the mitotic spindle, indicating that it is involved in the balance of forces determining bipolar spindle assembly and integrity. Silencing of KIFC5A also results in centrosome amplification detectable throughout the cell cycle. Supernumerary centrosomes arise primarily as a result of reduplication and partly as a result of cytokinesis defects. They contain duplicated centrioles and have the ability to organise microtubule asters, resulting in the formation of multipolar spindles. We show that KIFC5A interacts with nucleotide-binding proteins 1 and 2 (Nubp1 and Nubp2), which have extensive sequence similarity to prokaryotic division-site-determining protein MinD. Nubp1 and Nubp2 also interact with each other. Knockdown of Nubp1 or double knockdown of Nubp1 and Nubp2 (Nubp1&Nubp2) both phenocopy the KIFC5A silencing effect. These results implicate KIFC5A and the Nubp proteins in a common regulatory pathway involved in the control of centrosome duplication in mammalian cells.
运动蛋白活性的抑制与分裂细胞纺锤体两极形成缺陷有关。然而，运动活性与中心体动力学之间功能关系的分子机制仍不清楚。在这里，我们对KIFC5A进行了表征，它是一种小鼠驱动蛋白样蛋白，在分裂细胞和组织中高度表达，并受到发育和细胞类型特异性调控。KIFC5A是一种向负端移动、依赖微管的运动蛋白，在滑行试验中产生的速度高达1.26微米/分钟，并具有微管束集活性。它在间期位于细胞核中，在有丝分裂开始时定位于两个微管星状体的中心，在有丝分裂后期定位于纺锤体微管。在小鼠细胞中过表达KIFC5A会导致异常的、未分离的微管星状体形成，并使有丝分裂停滞在前中期样状态。敲低KIFC5A部分挽救了由单星孢菌素抑制有丝分裂纺锤体上的正端驱动运动蛋白Eg5所导致的表型，表明它参与了决定双极纺锤体组装和完整性的力的平衡。沉默KIFC5A还会导致在整个细胞周期中都能检测到中心体扩增。多余的中心体主要是由于复制产生的，部分是由于胞质分裂缺陷产生的。它们含有复制的中心粒，并有能力组织微管星状体，从而导致多极纺锤体的形成。我们发现KIFC5A与核苷酸结合蛋白1和2（Nubp1和Nubp2）相互作用，它们与原核生物分裂位点决定蛋白MinD具有广泛的序列相似性。Nubp1和Nubp2也相互作用。敲低Nubp1或同时敲低Nubp1和Nubp2（Nubp1&Nubp2）均模拟了KIFC5A沉默效应。这些结果表明，KIFC5A和Nubp蛋白参与了哺乳动物细胞中控制中心体复制的共同调控途径。
