Human bone marrow erythroblast enucleation is characterized by spherical nuclear morphology and prominent F-actin spots at the rear of the translocating nucleus. (A) Extended-focus projections of confocal Z-stacks of human bone marrow erythroblasts at various stages of enucleation were immunostained for GPA, phalloidin for F-actin, and Hoechst for nuclei. Arrows, prominent cytoplasmic F-actin spots. Arrowheads, small F-actin spot at GPA sorting boundary. Bar, 4 µm. (B) Schematic representation of quantitative image analysis of the geometry of the cell and nucleus at the GPA-sorting boundary. (C) Scatterplot of cell diameter at GPA-sorting boundary plotted as a function of nuclear protrusion for erythroblasts at all stages of nuclear expulsion. No significant correlation was observed. (D) Bar graph of nuclear long axis plotted as a function of extent of nuclear protrusion past the GPA-sorting boundary for erythroblasts binned according to extent of enucleation. The long axis of the nucleus remains unchanged during enucleation. (E) Bar graph of cell diameter at GPA-sorting boundary plotted as a function of nuclear protrusion for erythroblasts binned according to extent of enucleation. Diameter of the GPA-sorting membrane boundary remains constant and is the size of the nucleus. The nucleus does not need to deform to pass through this opening during expulsion from human erythroblasts. Images for quantification of human erythroblasts were obtained from 73 confocal Z-stacks of cells obtained from 6 separate human bone marrow samples. A total of 71 polarized or enucleating cells were analyzed. Twenty-four enucleating cells with protruding nuclei were analyzed in panel C and separated into mid, early, and late enucleation stages for panels D-E. Numbers (n) of polarized or enucleating (mid, early, late) cells or pyrenocytes indicated on bars in panels D and E. (F) 3D reconstructions of confocal Z-stacks of (a-f) human bone marrow erythroblasts at various stages of enucleation immunostained for GPA, phalloidin for F-actin, and Hoechst for nuclei. Also shown is F-actin in regions, defined by yellow brackets, of (a′-f′) human bone marrow erythroblasts rotated 90° around the axis running perpendicular to the direction of nuclear expulsion, providing a view through the “neck” of the enucleating erythroblast. Dashed yellow lines demarcate location of the nuclear perimeter at the GPA-sorting boundary. Few, small puncta of F-actin are associated with the sorting boundaries of the enucleating erythroblasts. 3D grid dimensions are as indicated.