Functional study of RanBP1 in mitotic progression.
Li, Hoi Yeung.
Date of Issue2007
School of Biological Sciences
Accurate partitioning of chromosomes in mitosis requires that the states of mitotic spindle assembly and chromosome alignment be monitored and communicated to the cell cycle machinery so that chromosome segregation does not commence until every pair of chromosomes is attached to microtubules (MT) from opposite spindle poles (Bharadwaj and Yu, 2004; Rieder and Malato, 2004). While extensive research has firmly established the importance of the cell cycle machinery in spindle assembly and chromosome segregation, recent studies have revealed that a signaling pathway mediated by RanGTPase plays an equally important role in mitosis (Karsenti and Vernos, 200 I; Zheng, 2004). The regulators of RanGTPase are spatially separated with the nucleotide exchange factor (called RCCI) tethered to the chromatin and the proteins that activate the GTPase, such as RanGAPI and RanBPI, localized in the cytoplasm (Weis, 2002). Consequently, a high RanGTP concentration can be established on the condensed chromosomes in mitosis (Kalab et aI., 2006; Kalab et aI., 2002; Li and Zheng, 2004a; Li and Zheng, 2004b). This spatial distribution of RanGTP is required for mitotic spindle assembly (Karsenti and Vernos, 2001; Zheng, 2004) and it may also regulate spindle checkpoint (Arnaoutov and Dasso, 2003). The chromosome-based RanGTP production appears to determine the spatial distribution of activities that promote microtubule (MT) nucleation and stabilization during spindle assembly (Caudron et aI., 2005). It might also function as a sensor to communicate the progression of spindle assembly to the cell cycle machinery so that anaphase onset is coupled to proper chromosome alignment. Whereas the cyclin B-dependent CDKI appears to directly regulate RanGTP production in mitosis by phosphorylating RCCI (Li and Zheng, 2004a; Li and Zheng, 2004b), there is evidence that RanGTP may in turn influence mitotic progression by regulating the spindle checkpoint and MT-kinetochore association. In Xenopus egg extracts, increasing RanGTP concentration has been shown to cause the dissociation of a number of spindle checkpoint proteins from kinetochores and to silence the spindle checkpoint (Arnaoutov and Dasso, 2003). Since an increased association of RCC1 with mitotic chromosomes coincided with metaphaseanaphase transition in the egg extracts, Arnaoutov and Dasso proposed that elevated RanGTP levels on metaphase chromosomes might silence spindle checkpoint to allow mitosis to progress into anaphase (Arnaoutov and Dasso, 2003). Additional studies have shown that RanGTP is also required to localize both RanGAPI and RanBP2 to kinetochores in mammalian somatic cells where the latter two proteins control the MY-kinetochore attachment (Joseph et aI., 2004; Salina et aI., 2003). More recently, the nuclear export receptor Crml was shown to be a downstream effector of RanGTP in mitosis that regulates MY-kinetochore attachment through RanBP2 and RanGAPI (Arnaoutov et aI., 2005; Arnaoutov and Dasso, 2005). Together, the above studies suggest that RanGTP production and turnover during mitosis could regulate chromosome alignment and metaphase-anaphase transition. However, it remains unclear whether an elevated RanGTP level could lead to checkpoint silencing and metaphaseanaphase transition in somatic cells. In addition, it is unclear how a cell could keep a lower level of RanGTP concentration until all chromosomes have reached proper alignment at the metaphase plate. Moreover, a few misaligned chromosomes are sufficient to trigger spindle checkpoint and mitotic arrest. Therefore, it is difficult to envision how RanGTP concentration is kept low in these situations where the majority of chromosomes have reached alignment at the metaphase plate. Finally, since blocking proper spindle assembly by cithcr nocodazolc or taxol could trigger spindle checkpoint, it is unclear how RanGTP concentration could be kept low under these conditions to allow an active spindle checkpoint. We reasoned that, because RanGTP is produced on each chromosome, the overall state of chromosome alignment might have signi ficant influence on the diffusion of RanGTP and the final RanGTP concentration on chromosomes. Using a computational model, we found that the statc of chromosome alignment has a strong influence on the local concentrations of RanGTP and RanGTP-transport receptor complexes that each chromosome experiences. Aided by this model, we show that elevating the concentration of RanGTP-importin Beta(a nuclear import receptor) by down-regulating RanBP I disrupts both spindle assembly and the association of spindle checkpoint protein Mad2 to unattached kinetochores, which leads to mitotic cell death. Moreover, our studies suggest that the reduced RanGTP concentration during mitotic arrest is controlled by both the degree of chromosome alignment and the interaction of RCC1 with mitotic chromosomes.