THE ROLE OF CYTOSKELETAL ELEMENTS IN THE 2-PHASE DENUCLEATION PROCESSOF 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. Whe n 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 o bserved contributing to different phases of that process. The vimentin intermediate filament (IF) was shown as the nuclear anchorage element s with binding sites anchored from the nuclear lamina to the center as well as to the plasma membrane periphery. A dense perinuclear layer o f vimentin fluorescence in erythroblasts was observable during the per iods 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. cu lture, but the vimentin filaments were observed to brake down and decr eased steadily when the cells became differentiated into late erythrob lasts at 36-48 hrs. Such an attenuation of vimentin filaments may faci litate 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 dur ing the processes of denucleation, what indicated that the actin filam ents and microtubules may play roles in the second phase of the denucl eation process, or final commitment of enucleation. The erythroid diff erentiation-denucleation factor (EDDF), as an intrinsic factor, involv ed in the denucleation events, was also discussed.