The role of cytoskeletal elements in the two-phase denucleation process of mammalian erythroblasts in vitro observed by laser confocal scanning microscope.

The cytoskeletal elements in the denucleation processes were observed using immunofluorescence and laser confocal scanning microscopy in the Friend virus (FVA) infected splenic erythroblasts of BALB/c mice. When cultured in the presence of erythropoietin (EPO), it was shown that the synchronized erythroid precursor cells proceeded to an autonomous nuclear extrusion when the three types of cytoskeletal elements were observed contributing to different phases of that process. The vimentin intermediate filament (IF) was shown as the nuclear anchorage elements with binding sites anchored from the nuclear lamina to the center as well as to the plasma membrane periphery. A dense perinuclear layer of vimentin fluorescence in erythroblasts was observable during the periods of 12, 24 and 36 hrs. in vitro culture. The amount of vimentin IF per cell was higher than that of tubulin and F-actin at 12-24 hrs. culture, but the vimentin filaments were observed to brake down and decreased steadily when the cells became differentiated into late erythroblasts at 36-48 hrs. Such an attenuation of vimentin filaments may facilitate the eccentric movement of the nucleus which can be regarded as the initial step (phase) of denucleation. The fluorescent intensity of tubulin and actin exhibited a significant rise and aggregated between the extruding nucleus and the incipient reticulocyte prior to and during the processes of denucleation, what indicated that the actin filaments and microtubules may play roles in the second phase of the denucleation process, or final commitment of enucleation. The erythroid differentiation-denucleation factor (EDDF), as an intrinsic factor, involved in the denucleation events, was also discussed.