Inhibition of tubulin polymerization or depolymerization impedes enucleation by inhibition of erythroblast polarization. (A) Effect of an inhibitor of tubulin polymerization (colchicine) or an inhibitor of tubulin depolymerization (taxol) on the enucleating efficiency of WT erythroblasts in fast enucleation assay, normalized as percentile of the control sample (mean ± SEM; n = 3). *P < .05 for 5μM colchicine-treated sample versus control. ‡P < .005 for 10 and 20μM taxol-treated samples vs control. The absolute enucleation efficiency of the control sample in this experiment was 47.9% ± 4.6%. (B) Polarized microtubule formation is visible in control WT erythroblasts (stained with β-tubulin–AlexaFluor-488 and the nuclear stain Draq5), whereas β-tubulin is diffusely stained in the erythroblasts incubated with colchicine (5μM) for 6 hours in the fast in vitro enucleation assay. Images were obtained with a 40× objective lens by Imagestreamx. (C) Confocal microscopy images of WT erythroblasts incubated without and with colchicine or taxol. β-tubulin was labeled with AlexaFluor-488 (green) and nucleus with DAPI (blue). Z-stack images were obtained with a 63× oil-immersed objective lens, numerical aperture 1.45, and processed using Volocity Version 4.1 software to produce a 3-dimensional reconstruction of the cells visualized; 1 unit represents 6.8 μm. WT orthochromatic erythroblasts demonstrate a unipolar microtubule assembly embracing the nucleus that appears to be pushed through as the cell elongates. Inhibition of microtubule polymerization with colchicine caused a diffuse staining for β-tubulin, whereas inhibition of microtubule depolymerization with taxol produced thickened microtubule bundles (previously seen by Koury et al6 ) that appear to maintain a grip around the nucleus. (D) Microtubules radiate from a γ-tubulin–rich area in orthochromatic erythroblasts to induce cell polarization. β-tubulin was labeled with AlexaFluor-488 (green), γ-tubulin with AlexaFluor-555 (red), and nucleus with DAPI (blue), and images were obtained with a 60× objective lens by Imagestreamx. (E) Inhibition of tubulin polymerization by colchicine inhibited cell polarization, as demonstrated from the distribution of the parameter Delta Centroid BF-DRQ5, which measures the distance between the center of the cell body as seen in bright-field and the center of the nuclear staining achieved with Draq5; median and mean values of the Delta Centroid BF-DRQ5 of control and colchicine-treated WT orthochromatic erythroblasts are shown, and the difference of the 2 samples is statistically significant (P < .001).
