Presence of a defect in karyokinesis during megakaryocyte endomitosis

Abstract

Megakaryocyte is the naturally polyploid cell that gives rise to platelets. Polyploidization occurs by endomitosis, a process corresponding to a late failure of cytokinesis with a backward movement of the daughter cells. Generally, a pure defect in cytokinesis produces a multinucleated cell, but megakaryocytes are characterized by a single polylobulated nucleus with a 2^N ploidy. Here, we show the existence of a defect in karyokinesis during the endomitotic process. From late telophase until the reversal of cytokinesis, some dipolar mitosis/endomitosis and most multipolar endomitosis present a thin DNA link between the segregated chromosomes surrounded by an incomplete nuclear membrane formation, which implies that sister chromatid separation is not complete. This observation may explain why polyploid megakaryocytes display a single polylobulated nucleus along with an increase in ploidy.

Introduction

Platelets are continuously produced from megakaryocytes (MK) localized in the bone marrow. During differentiation, instead of dividing one cell to two cells, MKs replicate their DNA content without cell division and become polyploid by a process called endomitosis. Previous works showed that endomitosis corresponded to a failure of late cytokinesis.^1,2 A simple defect in cytokinesis generally induces multinucleated cells, whereas MK possess a single polylobulated nucleus with a 2^N DNA content, which suggests there exists also a karyokinesis defect. By analyzing the nuclear kinetics during endomitosis, we observed the presence of nucleoplasmic bridges (NPM) in most multipolar endomitosis from telophase until the cytokinesis failure. These results suggest MK endomitosis presents a karyokinesis defect that probably favors a single polylobulated nucleus formation.

Results

Recent studies have shown that MK endomitosis is an incomplete mitosis characterized by a cytokinesis failure^1,2 related to both a defect in Rho activation^2,3 and the absence of Myosin IIB accumulation in the contractile ring.⁴ In this work, we tried to understand if a defect in karyokinesis was also present. We studied the chromosome kinetics by DNA staining and nuclear membrane dynamics by laminA/C immunostaining. LaminA/C is one important component of the nuclear envelope, which is disassembled during the first stage of mitosis and reassembled onto the chromosomes at the last stages of mitosis.⁵ During MK endomitosis, the nuclear kinetics is identical to what is observed during a classical mitosis until telophase; nuclear membrane breakdown began in prometaphase of endomitosis, as previously described.^6,7 The nuclear membrane remained disassembled until anaphase and then started to reassemble around the segregated chromosomes at late telophase (Fig. 1A,I–VI). However, the lamin signal remained very weak until the end of endomitosis when compared with the control cells in interphase (Fig. 1A, VII–IX). The nuclear membrane assembly was incomplete in agreement with the presence of condensed chromosomes, which explains why the nuclear membrane could still fusion to form a single polylobulated nucleus despite the late failure of cytokinesis (Fig. 1B). Some (15%) dipolar mitosis/endomitosis and most multipolar endomitosis (73%) exhibited a thin extension of lamin between segregated chromosomes from the beginning of nuclear membrane formation until the end of endomitosis (149 dipolar and 20 multipolar cells examined in three independent experiments) (Figs. 1A , B and 2B; see also Fig. S1). The percentage of MK presenting lamin extension in multipolar endomitosis was much higher than in the erythro-megakaryocytic cell line UT-7 (6%), which does not polyploidize in presence of GM-CSF.

Figure 1. Presence of a defect in karyokinesis during endomitosis. (A)I–III: Until anaphase, lamin immunostaining shows that the nuclear membrane of endomitotic MK remains disassembled. IV–VI: At late telophase, the nuclear membrane starts to reassemble. IV–IX: The nuclear membrane is not complete at the end of endomitosis. White arrows, Lamin between segregated chromosomes; yellow arrow, one control cell not in mitosis. (B) A typical polyploid MK, which possesses a single polylobulated nucleus after endomitosis. I, TOTO; II, lamin A/C; III, lamin A/C (green) and TOTO (blue); white arrows, DNA and lamin link between segregated chromosomes.

Figure 2. Presence of nucleoplasmic bridges (NPM) during endomitosis. (A) Presence of a DNA link between segregated chromosomes. (A and B) Tubulin α, red; lamin A/C, green; TOTO, blue. Bar, 10 μM. (B) Percentage of mitosis/endomitosis exhibits NPM. (C) Phospho-H3 is present in the NPM. Phospho-H3, red; Tubulin, green; TOTO, blue. Bar, 5 μM. (D) Co-localization of Phospho-H3 and lamin around segregated chromosomes during MK telophase. Phospho-H3 3, red; lamin A/C, green; TOTO, blue. Bar, 5 μM.

This abnormality corresponds to nucleoplasmic bridges (NPM), as staining with TOTO revealed the presence of DNA. Sometimes, the connection was so thin that TOTO staining was not obvious but could be observed after overexposing the images (Fig. 2A). This presence of DNA was confirmed by the presence of phospho-histone 3 (phospho-H3) in the bridges. The histone 3 phosphorylation is spatially and temporally coordinated with mitotic chromosome condensation.⁸ The presence of phospho-H3 in the bridges confirmed the existence of DNA linking among the separating chromosomes (Fig. 2C and D). Significant 4N MKs in interphase presented two nuclei (19.5–46.6% in six experiments) suggesting in this case a pure cytokinesis defect. In contrast, the great majority of 8N MKs presented a single polylobulated nucleus (> 85% in six experiments). These results parallel the differences observed in the percentage of NPM in dipolar and multipolar endomitosis (Fig. 2B).

As Fanconi proteins and BLM play an important role in resolving these DNA links,^9,10 we therefore studied whether they were expressed in MKs. The western blot revealed BLM expression in MKs (Fig. 3A). Furthermore, FANCD2 and BLM were detected in NPM during MKs endomitosis as in classic mitosis (Fig. 3B and C). This pathway hence seems to be at least partially functional in MKs.

Figure 3. FANCD2 and BLM are present normally during MK endomitosis. (A) Western blot reveals BLM expression increased by proteasome inhibition. (B)I–III: BLM (green) localized to ultrafine anaphase bridges. IV–VI: FANCD2 (red) localized as spots on mitotic chromosomes. VII–IX: The arrow indicates one FANCD2 (green) spot on separating chromosomes during anaphase of endomitosis. (C) The arrow indicates a subset of BLM-stained bridges (green) connected to FANCD2 (red) spots on separating chromosome sets at anaphase. Bars, 10 μM.

Altogether, we have demonstrated a karyokinesis defect during MK endomitosis characterized by the presence of NPM, which are not completely resolved at the end of telophase. This defect increases with ploidy and may be involved in a single multilobulated nucleus formation in polyploid megakaryocytes. Finally, it will be important to determine the precise mechanisms responsible for the presence of these NPM in MK endomitosis.

Discussion

This work revealed the persistence of NPM at telophase in most multipolar MKs endomitosis, but only in a few dipolar mitosis/endomitosis. In addition, a large percentage of 4N MKs was truly binucleated, whereas the majority of higher ploidy MKs was mononucleated. This is in agreement with previous observations showing the presence of small binucleated MKs in cord blood cultures.¹¹ After 4N, an increased defect in karyokinesis was seen with the presence of NPM in the great majority of endomitotic MKs. This may be related to the absence of a true anaphase B with a limited endomitotic spindle elongation^6,7 and the asymmetrical chromosome segregation in multipolar endomitosis.¹² The presence of unresolved NPM may also favor the cytokinesis defect, because abscission cannot occur when DNA linkage persists, which could explain why polyploid MKs may rarely return to mitotic cycles.^13,14 In addition, it could also explain why polyploid MKs have a single nucleus, because if the NPM did not resolve, the nuclear membrane is reassembled around unseparated DNA masses to form the link between segregated chromosomes. In human epithelial cells, it has been observed that cells showing chromatin bridges exhibited different fate: in most cases, bridge breakage occurred and cells eventually completed division; in contrast, most cells with unbroken bridges did not complete abscission, but resulted in two daughter cells connected with an ultra-fine DNA string, and the remaining cell became tetraploid with two nuclei (binucleated) coupled with an unbroken chromatin bridge.¹⁵ The latter possibility is similar to what is observed in MKs. Several pathways are implicated in resolution of DNA bridges at the end of mitosis. The pathway best described concerns Aurora B activation. This kinase remains activated when chromatin is trapped between diving cells. This activation promotes proper chromosome segregation by delaying abscission and prevents tetraploidization. If Aurora B activation is inhibited before the bridge resolved, furrow regression is observed, leading to cells with two nuclei connected by DNA bridges.¹⁶ In MKs Aurora B activity could be observed at the end of MK endomitosis, but we cannot exclude that its activation is not sufficiently sustained to resolve the DNA bridges during most MK endomitosis or that another pathway is indispensable.¹⁷ In favor of this last hypothesis, it has been recently suggested that sister chromatid disjunction at the NPM origin is frequently incomplete in human cells, but Fanconi proteins and BLM play a cooperative role in resolving these DNA links.^9,10 However, this pathway seems to be at least partially functional in MKs, as both proteins were present in NPM. As NPM are induced by replicative stress, it is possible that polyploidization is associated to a replicative stress, such as c-myc activation,¹⁸ leading to increased DNA linkage, which cannot be completely resolved by Aurora B or the FANC and BLM pathways. However, we cannot exclude that in some endomitotic MK, the DNA bridge could be finally resolved because it was reported that polyploid MK could occasionally complete cytokinesis and divided into separated polyploid daughter cells.^13,14

Altogether, we have demonstrated a defect in karyokinesis during MK endomitosis characterized by the presence of NPM, which are not completely resolved at the end of telophase. This defect increases with ploidy and may be involved in the formation of a single multilobulated nucleus in polyploid megakaryocytes. Finally, it will be important to determine the precise mechanisms responsible for the presence of these NPM in MK endomitosis.

Materials and Methods

In vitro culture of megakaryocytes derived from human CD34⁺ cells

CD34⁺ cells were isolated by an immunomagnetic technique (Miltenyi Biotec) from the bone marrow of healthy subjects and cultured as described.²

Separation of 4N and 8N MKs

Hoechst 33342 (10 μg/mL; Sigma) was added in MKs culture for 1h at 37°C. Cells were stained with the anti-CD41APC monoclonal antibody (MoAb) (PharMingen) for 30 min at 4°C. 4N and 8N CD41⁺ cells were individually sorted as described.²

Immunofluorescence

Fixation and immunofluorescence were performed on CD41⁺ MKs sorted at day 6 and cultured overnight before the experiments, as described previously.² Cells were examined under a LSM 510 (Carl Zeiss) confocal microscopy with a X63/1.4NA oil objective or an Olympus Atimes X70 fluorescence microscope with a X100/1.3 oil objective. The following antibodies were used: rabbit anti-α tubulin (1:100, ABR, CO), mouse anti-lamin A/C (1:100), mouse anti-phospho histone H3 (Ser10) (1:100, Millipore), mouse anti-α tubulin and mouse anti-β tubulin MoAbs (Sigma, Saint Quentin Fallavier), rabbit anti-FANCD2 (1:1,000, Abcam) and goat anti-Bloom protein (BLM) (1:150, Santa Cruz). Appropriate secondary antibodies were conjugated with Alexa 488, Alexa 546 or Alexa 592 (Molecular Probes, Invitrogen). TOTO-3 iodide or DAPI (Molecular Probes) was applied for nucleus staining. Three-dimensional images analyses were performed with Zeiss Image Examiner software.

Cell line

UT-7 cells were cultured in DMEM medium (Invitrogen) supplemented with 10% FBS and 2.5 ng/mL of hGM-CSF (Leucomax).

Western blot

Western blot was performed on CD41⁺ and CD41^- cells sorted at day 6 of MK culture as described.³ MG132 (10 μM) was added to some samples before cell collection.

Glossary

Abbreviations:

MK

megakaryocyte

NPM

nucleoplasmic bridges

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.