To the editor:
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in these studies ectopically expressed survivin was not observed to localize to midzone territories (D.J.M. and K.R., unpublished data, October 2007).
![Figure 1. Localization of survivin in mouse MKs in anaphase. Mouse bone marrow cells were cultured in IMDM supplemented with 10% FBS, 1% penicillin/streptomycin, and 25 ng/mL thrombopoietin for 3 days. For immunostaining, cytospun cells were fixed in 4% fresh paraformaldehyde at 4°C overnight, washed with buffer and permeabilized with 0.25% Triton X-100 in PBS for 20 minutes at room temperature. Slides were blocked with 3% bovine serum albumin in PBS for 60 minutes at room temperature followed by overnight incubation with primary antibodies (full-length polyclonal antibody to survivin, sc-10811, 1:250 dilution; α-tubulin [Sigma-Aldrich], 1:600 dilution) in staining buffer (0.5% BSA, PBS, 0.25% Triton X-100) at 4°C. After 3 washes with 0.25% Triton X-100 in PBS, slides were incubated with appropriate secondary antibodies (red signal for survivin and green for α-tubulin), washed, and then stained with DAPI (blue) to visualize chromosomes. (A) Shown is a representative 16N MK analyzed of 50 MKs in anaphase examined. The intensity of staining was variable (from dim to strong), but all showed staining. (B) An 8N MK in anaphase. In 4% of MKs (2 of 50 MKs in anaphase), a portion of survivin translocated from the chromosomes to the microtubules as indicated by the orange color. White arrows point to distinct overlap of survivin (red) and midzone microtubules (green) in upper left expanded area. However, the majority of survivin remains associated with DNA (yellow arrows) as indicated by the pink color (red and blue overlap, expanded area in lower right corner). (C) A low-ploidy (4N) MK in anaphase; 6 4N MKs showed a similar profile. (D) A diploid NIH 3T3 cell in anaphase demonstrates typical midzone localization of survivin. Magnification, 900×.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/116/12/10.1182_blood-2010-04-280420/4/m_zh89991056290001.jpeg?Expires=1735457172&Signature=eIpgBV3im3AML9~E5PehUbIIyVVCNT9BTJhrgGYMOeCSjPCqYgRpSN5WvM8EwJj6RtocZDiEDSEPVunVRRUiL6ssuMiAceRX3DjzapqLlr-~27vDLV6EmqXWhvLxl8Spu3~auIwv0H6KFodnKft0KdHdGFM8nmncMgj-rRkFWsc0SYbqTEz4zZ-4WmeISyKHm1IsU~ERbjDF~zAUFc7WwQATssQC2~GJipyM5z4snwDqGWSDcYvEw6R768ryy7ZWLrKQHfCmEjxGwZeY20FaBQ9d2Xqkqbm057PSSucyXFSwVCIBrBZAn57o25JrLrVUjjLwOEEwZ5RPLsEfjHKGDA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Localization of survivin in mouse MKs in anaphase. Mouse bone marrow cells were cultured in IMDM supplemented with 10% FBS, 1% penicillin/streptomycin, and 25 ng/mL thrombopoietin for 3 days. For immunostaining, cytospun cells were fixed in 4% fresh paraformaldehyde at 4°C overnight, washed with buffer and permeabilized with 0.25% Triton X-100 in PBS for 20 minutes at room temperature. Slides were blocked with 3% bovine serum albumin in PBS for 60 minutes at room temperature followed by overnight incubation with primary antibodies (full-length polyclonal antibody to survivin, sc-10811, 1:250 dilution; α-tubulin [Sigma-Aldrich], 1:600 dilution) in staining buffer (0.5% BSA, PBS, 0.25% Triton X-100) at 4°C. After 3 washes with 0.25% Triton X-100 in PBS, slides were incubated with appropriate secondary antibodies (red signal for survivin and green for α-tubulin), washed, and then stained with DAPI (blue) to visualize chromosomes. (A) Shown is a representative 16N MK analyzed of 50 MKs in anaphase examined. The intensity of staining was variable (from dim to strong), but all showed staining. (B) An 8N MK in anaphase. In 4% of MKs (2 of 50 MKs in anaphase), a portion of survivin translocated from the chromosomes to the microtubules as indicated by the orange color. White arrows point to distinct overlap of survivin (red) and midzone microtubules (green) in upper left expanded area. However, the majority of survivin remains associated with DNA (yellow arrows) as indicated by the pink color (red and blue overlap, expanded area in lower right corner). (C) A low-ploidy (4N) MK in anaphase; 6 4N MKs showed a similar profile. (D) A diploid NIH 3T3 cell in anaphase demonstrates typical midzone localization of survivin. Magnification, 900×.
Localization of survivin in mouse MKs in anaphase. Mouse bone marrow cells were cultured in IMDM supplemented with 10% FBS, 1% penicillin/streptomycin, and 25 ng/mL thrombopoietin for 3 days. For immunostaining, cytospun cells were fixed in 4% fresh paraformaldehyde at 4°C overnight, washed with buffer and permeabilized with 0.25% Triton X-100 in PBS for 20 minutes at room temperature. Slides were blocked with 3% bovine serum albumin in PBS for 60 minutes at room temperature followed by overnight incubation with primary antibodies (full-length polyclonal antibody to survivin, sc-10811, 1:250 dilution; α-tubulin [Sigma-Aldrich], 1:600 dilution) in staining buffer (0.5% BSA, PBS, 0.25% Triton X-100) at 4°C. After 3 washes with 0.25% Triton X-100 in PBS, slides were incubated with appropriate secondary antibodies (red signal for survivin and green for α-tubulin), washed, and then stained with DAPI (blue) to visualize chromosomes. (A) Shown is a representative 16N MK analyzed of 50 MKs in anaphase examined. The intensity of staining was variable (from dim to strong), but all showed staining. (B) An 8N MK in anaphase. In 4% of MKs (2 of 50 MKs in anaphase), a portion of survivin translocated from the chromosomes to the microtubules as indicated by the orange color. White arrows point to distinct overlap of survivin (red) and midzone microtubules (green) in upper left expanded area. However, the majority of survivin remains associated with DNA (yellow arrows) as indicated by the pink color (red and blue overlap, expanded area in lower right corner). (C) A low-ploidy (4N) MK in anaphase; 6 4N MKs showed a similar profile. (D) A diploid NIH 3T3 cell in anaphase demonstrates typical midzone localization of survivin. Magnification, 900×.
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.
Authorship
Acknowledgments: This work was supported by National Heart, Lung, and Blood Institute grant HL80442 (K.R.). K.R. is an Established Investigator with the American Heart Association. IACUC approval at Boston University was attained for all mice used in this study.
Contribution: D.J.M. designed the study, performed the experiments, analyzed the data, and drafted the manuscript; and K.R. designed the study, analyzed the data, and drafted the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Dr Katya Ravid, Boston University School of Medicine, 700 Albany St W-601, Boston, MA 02118; e-mail: kravid@bu.edu.
References
National Institutes of Health
