Figure 1.
Figure 1. Mouse FL erythroblast enucleation is characterized by dumbbell-shaped nuclear morphology and prominent F-actin foci at the cell surface constriction and at the rear of the translocating nucleus. (A) Extended-focus projections of confocal Z-stacks of mouse FL erythroblasts at various stages of enucleation immunostained for Ter119, phalloidin for F-actin, and Hoechst for nuclei. Arrows, bright F-actin spot in polarized cells and at the rear of the translocating nucleus opposite its membrane point of contact. Open arrowheads, F-actin cap at the site of nuclear contact with the membrane in a polarized cell. Closed arrowheads, F-actin foci at the cell surface constriction in enucleating cells, connecting to fine F-actin cables extending over the nuclear protrusion. Dashed lines, Ter119 membrane sorting boundary. Bar, 4 µm. (B) Schematic representation of quantitative image analysis of nuclear and cell surface constriction geometry in mouse FL enucleating erythroblasts. (C) Scatter plot of cell surface constriction diameter plotted as a function of nuclear protrusion for erythroblasts at all stages of nuclear expulsion. Parabolic regression was applied to the data (P < .0001). (D) Bar graph of nuclear long axis plotted as a function of nuclear protrusion for erythroblasts binned according to extent of enucleation. ***P < .001. (E) Bar graph of the cell surface constriction diameter size for erythroblasts binned according to extent of enucleation. *P < .05; ***P < .001. Images for quantification of FL erythroblasts were obtained from 133 confocal Z-stacks of cells obtained from 23 embryos (12 different litters). A total of 128 polarized or enucleating cells were analyzed. One hundred two 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) mouse FL erythroblasts at various stages of enucleation immunostained for Ter119, phalloidin for F-actin, and Hoechst for nuclei. Also shown is F-actin at the Ter119-sorting boundary, defined by yellow brackets, of (a′-f′) 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 in the neck region. In erythroblasts beginning to enucleate or nearly completely enucleated with smaller constriction diameters, F-actin completely surrounds the nucleus beneath the cell surface constriction (b′, e′). In erythroblasts with larger constriction diameters, F-actin exhibits small gaps (c′) and/or large F-actin–free regions (d′). 3D grid dimensions are as indicated on each image for A-F.

Mouse FL erythroblast enucleation is characterized by dumbbell-shaped nuclear morphology and prominent F-actin foci at the cell surface constriction and at the rear of the translocating nucleus. (A) Extended-focus projections of confocal Z-stacks of mouse FL erythroblasts at various stages of enucleation immunostained for Ter119, phalloidin for F-actin, and Hoechst for nuclei. Arrows, bright F-actin spot in polarized cells and at the rear of the translocating nucleus opposite its membrane point of contact. Open arrowheads, F-actin cap at the site of nuclear contact with the membrane in a polarized cell. Closed arrowheads, F-actin foci at the cell surface constriction in enucleating cells, connecting to fine F-actin cables extending over the nuclear protrusion. Dashed lines, Ter119 membrane sorting boundary. Bar, 4 µm. (B) Schematic representation of quantitative image analysis of nuclear and cell surface constriction geometry in mouse FL enucleating erythroblasts. (C) Scatter plot of cell surface constriction diameter plotted as a function of nuclear protrusion for erythroblasts at all stages of nuclear expulsion. Parabolic regression was applied to the data (P < .0001). (D) Bar graph of nuclear long axis plotted as a function of nuclear protrusion for erythroblasts binned according to extent of enucleation. ***P < .001. (E) Bar graph of the cell surface constriction diameter size for erythroblasts binned according to extent of enucleation. *P < .05; ***P < .001. Images for quantification of FL erythroblasts were obtained from 133 confocal Z-stacks of cells obtained from 23 embryos (12 different litters). A total of 128 polarized or enucleating cells were analyzed. One hundred two 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) mouse FL erythroblasts at various stages of enucleation immunostained for Ter119, phalloidin for F-actin, and Hoechst for nuclei. Also shown is F-actin at the Ter119-sorting boundary, defined by yellow brackets, of (a′-f′) 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 in the neck region. In erythroblasts beginning to enucleate or nearly completely enucleated with smaller constriction diameters, F-actin completely surrounds the nucleus beneath the cell surface constriction (b′, e′). In erythroblasts with larger constriction diameters, F-actin exhibits small gaps (c′) and/or large F-actin–free regions (d′). 3D grid dimensions are as indicated on each image for A-F.

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