Survivin localization during endomitosis of high ploidy mouse megakaryocytes.

During polyploidization the megakaryocyte (MK) undergoes endomitosis, which involves skipping late anaphase and cytokinesis.1,2 As a regulator of anaphase and cytokinesis, our laboratory implicated survivin in this process due to the absence of survivin protein in endomitotic mouse MKs,3 which are typically of high ploidy. Gurbuxani et al4 also noted survivin’s down-regulation at the mRNA level as well as absence at the protein level in mouse MKs. In contrast, Geddis et al5 and Lordier et al6 reported normal midzone localization of survivin during anaphase in human MKs (typically of low ploidy). Upon further investigation of survivin in the MK lineage in vivo,7 we re-examined survivin expression and localization using a new antibody and a more sensitive technique. Although the intensity of staining (reflective of survivin expression) was variable in MKs of the same preparations (from dim to strong), survivin was clearly observed on regions of microtubule attachment to chromosomes throughout endomitosis of murine MKs, but seldom localized to midzone microtubule spindles of high-ploidy MKs (Figure 1A-B). Remaining discrepancies in survivin localization during MK endomitosis in mouse and human cultures may be attributed to inherent differences between these systems, such as their different ploidy levels at which they are being reported, and/or less pronounced midzone territories during anaphase in mouse MKs compared with human MKs. Human survivin has been shown to be a critical component of midzone assembly/stabilization in normally dividing cells,8 and several signals may regulate its localization. Survivin is clearly localized to the chromosomes in a majority of the high-ploidy (8N and higher) mouse MKs in anaphase. As to low-ploidy MKs, we noted this mislocalization in rare 4N cells (Figure 1C), but not enough cells were visualized at this state to drive a concrete conclusion. Proper staining of survivin was detected at midzones in control mouse NIH 3T3 diploid cells (Figure 1D). Hence, we conclude that survivin fails to fully translocate into the midzone in most mouse MKs. This failure may lead to its gradual degradation and low-level detection. Forced expression of survivin in the MK lineage in mouse did not result in decreased MK polyploidy7; however, in these studies ectopically expressed survivin was not observed to localize to midzone territories (D.J.M. and K.R., unpublished data, October 2007). Together, these findings support the notion that regulation of survivin during MK endomitosis may be critically controlled at the localization level, as also indicated by Vong et al,9 who detail a highly dynamic and balanced mechanism for localization of survivin to the centromeres during prophase and metaphase in human and mouse cell lines. Improper survivin localization during anaphase could be a primary or secondary regulatory point, restricting complete midzone formation and cytokinesis in polyploidizing MKs.