To better understand the function of the gene products involved in these rearrangements, we have used RNA interference (RNAi) to disrupt the expression of this set of kinetochore proteins
To better understand the function of the gene products involved in these rearrangements, we have used RNA interference (RNAi) to disrupt the expression of this set of kinetochore proteins. are detectable as large flares that project out laterally from the metaphase plate. Disrupting these gene products via RNA interference results in sensitivity to checkpoint stimuli, as well as defects in the organization of chromosomes at metaphase. These phenotypes suggest that these proteins, and by extension their reorganization during mitosis, are important for mediating the checkpoint response as well as directing the assembly of the metaphase plate. INTRODUCTION The kinetochore is a dynamic structure that assembles onto mitotic chromosomes and plays multiple roles during mitosis (reviewed in Rieder and Salmon, 1998 ; Cleveland kinetochore extends the length of the chromosome (Albertson and Thomson, 1982 ; reviewed in Dernburg, 2001 ), offering an increased capacity for Rabbit Polyclonal to AurB/C (phospho-Thr236/202) cytological examination of this structure and any rearrangements it may undergo. And although holocentric chromosomes seem distinct from monocentric chromosomes on a superficial level, there are many parallels between these systems. For example, in mammalian cells, the centromeres line up in the middle of the cell at metaphase, whereas the chromosome arms freely oscillate within the cell. In chromosomes presents a magnified view of what must happen for 1alpha, 25-Dihydroxy VD2-D6 mammalian centromeres to align at metaphase. In addition, it has been proposed (Zinkowski kinetochore proteins reorganize at prometaphase, as well as in response to checkpoint stimuli. We find that these kinetochore proteins dissociate from the centromere yet remain associated with the poleward faces of metaphase chromosomes. To better understand the function of the gene products involved in these rearrangements, we have used RNA interference (RNAi) to disrupt the expression of this set of kinetochore proteins. The resulting RNAi phenotypes suggest that these kinetochore proteins, and by extension their reorganization during mitosis, are important for regulating chromosome segregation. MATERIALS AND METHODS RNA Interference We generated a polymerase chain reaction template for the production of double-stranded RNA as described previously (Moore kinetochore undergoes a structural reorganization during mitosis, we examined the localization of centromere and kinetochore components by immunofluorescence microscopy. For this work, we define proteins as being centromere components if they are constitutively associated with the chromosomes. This includes the centromeric histone HCP-3/CeCENP-A (Buchwitz kinetochore and centromere (Figure 1). Consistent with previous reports, all of these antigens were present in two parallel lines on prophase chromosomes (Figure 1, a-c; our unpublished data), and a high degree of colocalization could be detected between centromere and kinetochore proteins (Buchwitz kinetochore undergo a structural reorganization during mitosis, it could be detectable as an alteration in the distribution of these kinetochore proteins relative to the centromere. Thus, using HCP-3/CeCENP-A as a marker for the centromere, we investigated whether any kinetochore proteins exhibited a staining pattern at metaphase that was not consistent with centromere localization. We found that two previously identified kinetochore components, HCP-1/CeCENP-F and SAN-1/CeMAD3 (Moore kinetochore. The staining pattern of these antigens on the metaphase plate was very similar to that of both HCP-1/CeCENP-F and SAN-1/CeMAD3 (Figure 4, a-f). Instead of the tight, nonoverlapping lines observed for centromere proteins, HIM-10/CeNUF2 and BUB-1 seemed to assemble into a more diffuse net (Figure 4, a and d) and exhibited partial colocalization with HCP-1/CeCENP-F (Figure 4, c and f). These data lead us to conclude that like HCP-1/CeCENP-F and SAN-1/CeMAD3, HIM-10/CeNUF2 and BUB-1 also dissociate from the centromere before metaphase, yet remain associated with the metaphase 1alpha, 25-Dihydroxy VD2-D6 plate. Open in a separate window Figure 4. HIM-10/CeNUF2 and BUB-1 exhibit localization patterns similar to HCP-1/CeCENP-F. Metaphase plates from four cell embryos were stained with antibodies against HCP-1/CeCENP-F (b and c and e and f) (red), and either HIM-10/CeNUF2 (a-c) or BUB-1 (d-f) (green). The staining patterns observed for HIM-10/CeNUF2 and BUB-1 are similar to those described for HCP-1/CeCENP-F and SAN-1/CeMAD3. Furthermore, costaining experiments demonstrate that HIM-10/CeNUF2 and BUB-1 exhibit partial colocalization with the HCP-1/CeCENP-F protein. Bar, 1 m. The Role of Microtubules in Mediating Kinetochore Reorganization Because the dissociation of these kinetochore proteins from the centromere coincides with the establishment of chromosome/microtubule attachments, we wanted to investigate whether spindle microtubules are required for this reorganization. To address this question, we treated with the microtubule depolymerizing agent nocodazole and asked whether the kinetochore remained associated with the centromere in the absence of spindle microtubules. After a prolonged exposure of embryos to nocodazole (20 min at 40 g/ml), we were able to identify 1alpha, 25-Dihydroxy VD2-D6 mitotic blastomeres with no polymerized microtubules (Figure 5, a-d). We demonstrated that these cells had reached later stages of mitosis by the absence of staining with mAb414, a marker for the nuclear envelope (our unpublished data). Lacking spindle. 1alpha, 25-Dihydroxy VD2-D6
