Chromatin degradation in differentiating fiber cells of the eye lens.
During development, the lens of the eye becomes transparent, in part because of the elimination of nuclei and other organelles from the central lens fiber cells by an apoptotic-like mechanism. Using confocal microscopy we showed that, at the border of the organelle-free zone (OFZ), fiber cell nuclei became suddenly irregular in shape, with marginalized chromatin. Subsequently, holes appeared in the nuclear envelope and underlying laminae, and the nuclei collapsed into condensed, spherical structures. Nuclear remnants, containing DNA, histones, lamin B2, and fragments of nuclear membrane, were detected deep in the OFZ. We used in situ electrophoresis to demonstrate that fragmented DNA was present only in cells bordering the OFZ. Confocal microscopy of terminal deoxynucleotidyl transferase (TdT)-labeled lens slices confirmed that DNA fragmentation was a relatively late event in fiber differentiation, occurring after the loss of the nuclear membrane. DNA fragments with 3'-OH or 3'-PO(4) ends were not observed elsewhere in the lens under normal conditions, although they could be produced by pretreatment with DNase I or micrococcal nuclease, respectively. Dual labeling with TdT and an antibody against protein disulfide isomerase, an ER-resident protein, revealed a distinct spatial and temporal gap between the disappearance of ER and nuclear membranes and the onset of DNA degradation. Thus, fiber cell chromatin disassembly differs significantly from classical apoptosis, in both the sequence of events and the time course of the process. The fact that DNA degradation occurs only after the disappearance of mitochondrial, ER, and nuclear membranes suggests that damage to intracellular membranes may be an initiating event in nuclear breakdown.
Bassnett
S
S
Department of Ophthalmology and Visual Sciences, Washington University Medical School, St. Louis, Missouri 63110-1093, USA. Bassnetts@am.seer.wustl.edu
Mataic
D
D
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002642:Chick Embryo; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004847:Epithelial Cells; D004848:Epithelium / Q000737:chemistry / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006657:Histones / Q000032:analysis; D007908:Lens, Crystalline / Q000166:cytology* / Q000201:enzymology*; D018613:Microscopy, Confocal; D009685:Nuclear Envelope / Q000378:metabolism
D002843:Chromatin; D006657:Histones; D004247:DNA; D004706:Endodeoxyribonucleases; C019329:deoxyribonuclease II