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Chromatin degradation in differentiating fiber cells of the eye lens.

Bassnett S, Mataic D - J. Cell Biol. (1997)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, Washington University Medical School, St. Louis, Missouri 63110-1093, USA. Bassnetts@am.seer.wustl.edu

ABSTRACT
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.

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Merged confocal and DIC images of lens slices after TdT labeling with fluorescein-dUTP. The DIC images are shown in green  and positively labeled nuclei (containing fragmented DNA) are shown in red. (A) At the border of the OFZ, the nuclei lose their regular shape (arrows) and collapse into condensed structures that are strongly labeled by the TdT assay (arrowheads). Positively labeled debris, resulting presumably from the disintegration of labeled nuclei, extends deep into the OFZ. (B) Cortical fiber cells from a lens slice  that was pretreated for 30 min with 50 U/ml DNase I. Note that after DNase I treatment, all nuclei are labeled by the TdT assay and that  the labeling is strongest immediately beneath the nuclear membrane. (C) Equatorial region of a lens slice that had been incubated with  CIAP before TdT labeling. None of the nuclei are labeled, indicating that the superficial fibers do not contain fragmented DNA with 3′PO4 termini (see text for details). (D) Equatorial region of a lens slice that was treated sequentially with micrococcal nuclease and CIAP  before TdT labeling. All the nuclei are labeled, demonstrating the efficacy of the CIAP technique for detecting fragmented DNA with  3′-PO4 termini. Bars: (A) 50 μm; (B) 10 μm; (C) 50 μm; (D) 50 μm.
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Figure 6: Merged confocal and DIC images of lens slices after TdT labeling with fluorescein-dUTP. The DIC images are shown in green and positively labeled nuclei (containing fragmented DNA) are shown in red. (A) At the border of the OFZ, the nuclei lose their regular shape (arrows) and collapse into condensed structures that are strongly labeled by the TdT assay (arrowheads). Positively labeled debris, resulting presumably from the disintegration of labeled nuclei, extends deep into the OFZ. (B) Cortical fiber cells from a lens slice that was pretreated for 30 min with 50 U/ml DNase I. Note that after DNase I treatment, all nuclei are labeled by the TdT assay and that the labeling is strongest immediately beneath the nuclear membrane. (C) Equatorial region of a lens slice that had been incubated with CIAP before TdT labeling. None of the nuclei are labeled, indicating that the superficial fibers do not contain fragmented DNA with 3′PO4 termini (see text for details). (D) Equatorial region of a lens slice that was treated sequentially with micrococcal nuclease and CIAP before TdT labeling. All the nuclei are labeled, demonstrating the efficacy of the CIAP technique for detecting fragmented DNA with 3′-PO4 termini. Bars: (A) 50 μm; (B) 10 μm; (C) 50 μm; (D) 50 μm.

Mentions: As the karyoskeleton was disassembled at the border of the OFZ, the integrity of the nuclear envelope was also lost. Once the nuclei collapsed, only ragged fragments of membrane remained, adhering to the naked chromatin (Fig. 5). The condition of nuclear DNA was also visualized during various stages of fiber cell differentiation using an in situ TdT assay. In this assay, a tail of fluorescein-labeled dUTP molecules is incorporated at 3′-OH ends of DNA fragments. Labeled nuclei were rarely observed in the epithelial cell layer and never in the superficial fiber cells. However, strongly labeled nuclei were always detected at the border of the OFZ in lenses from embryos E15 and older (Fig. 6 A). At earlier developmental stages, labeled nuclei were not observed in the fiber cells. If the labeling reaction was performed in the absence of the TdT enzyme, no labeling was observed (data not shown). Surprisingly, only fully condensed nuclei were labeled. Irregularly shaped nuclei with heterogeneous or marginalized chromatin were not labeled by this technique. End-labeled DNA fragments were seen to persist in the cytoplasm of fiber cells hundreds of cell widths inside the border of the OFZ. The lack of labeling in the more superficial fibers could have been due to the presence of an inhibitor of the TdT assay in these cells. To control for this, some slices were preincubated in DNase I to cause extensive DNA fragmentation in vitro. In DNase I–treated tissue, all of the nuclei were labeled by the TdT assay, including those of the epithelial cells and annular pad (data not shown) and superficial fiber cells (Fig. 6 B). The pattern of DNA damage induced by the DNase I treatment was qualitatively different from that occurring naturally at the border of the OFZ. In DNase I–treated cells, the labeling was heterogeneous and strongest immediately beneath the nuclear membrane (Fig. 6 B). In contrast, the collapsed nuclei of cells at the border of the OFZ were homogeneously stained. The fact that all nuclei in the lens were labeled after the DNase I treatment suggests that the lack of label in the outer cells of untreated lenses was not due to the presence of an inhibitor of the TdT assay in these cells.


Chromatin degradation in differentiating fiber cells of the eye lens.

Bassnett S, Mataic D - J. Cell Biol. (1997)

Merged confocal and DIC images of lens slices after TdT labeling with fluorescein-dUTP. The DIC images are shown in green  and positively labeled nuclei (containing fragmented DNA) are shown in red. (A) At the border of the OFZ, the nuclei lose their regular shape (arrows) and collapse into condensed structures that are strongly labeled by the TdT assay (arrowheads). Positively labeled debris, resulting presumably from the disintegration of labeled nuclei, extends deep into the OFZ. (B) Cortical fiber cells from a lens slice  that was pretreated for 30 min with 50 U/ml DNase I. Note that after DNase I treatment, all nuclei are labeled by the TdT assay and that  the labeling is strongest immediately beneath the nuclear membrane. (C) Equatorial region of a lens slice that had been incubated with  CIAP before TdT labeling. None of the nuclei are labeled, indicating that the superficial fibers do not contain fragmented DNA with 3′PO4 termini (see text for details). (D) Equatorial region of a lens slice that was treated sequentially with micrococcal nuclease and CIAP  before TdT labeling. All the nuclei are labeled, demonstrating the efficacy of the CIAP technique for detecting fragmented DNA with  3′-PO4 termini. Bars: (A) 50 μm; (B) 10 μm; (C) 50 μm; (D) 50 μm.
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Figure 6: Merged confocal and DIC images of lens slices after TdT labeling with fluorescein-dUTP. The DIC images are shown in green and positively labeled nuclei (containing fragmented DNA) are shown in red. (A) At the border of the OFZ, the nuclei lose their regular shape (arrows) and collapse into condensed structures that are strongly labeled by the TdT assay (arrowheads). Positively labeled debris, resulting presumably from the disintegration of labeled nuclei, extends deep into the OFZ. (B) Cortical fiber cells from a lens slice that was pretreated for 30 min with 50 U/ml DNase I. Note that after DNase I treatment, all nuclei are labeled by the TdT assay and that the labeling is strongest immediately beneath the nuclear membrane. (C) Equatorial region of a lens slice that had been incubated with CIAP before TdT labeling. None of the nuclei are labeled, indicating that the superficial fibers do not contain fragmented DNA with 3′PO4 termini (see text for details). (D) Equatorial region of a lens slice that was treated sequentially with micrococcal nuclease and CIAP before TdT labeling. All the nuclei are labeled, demonstrating the efficacy of the CIAP technique for detecting fragmented DNA with 3′-PO4 termini. Bars: (A) 50 μm; (B) 10 μm; (C) 50 μm; (D) 50 μm.
Mentions: As the karyoskeleton was disassembled at the border of the OFZ, the integrity of the nuclear envelope was also lost. Once the nuclei collapsed, only ragged fragments of membrane remained, adhering to the naked chromatin (Fig. 5). The condition of nuclear DNA was also visualized during various stages of fiber cell differentiation using an in situ TdT assay. In this assay, a tail of fluorescein-labeled dUTP molecules is incorporated at 3′-OH ends of DNA fragments. Labeled nuclei were rarely observed in the epithelial cell layer and never in the superficial fiber cells. However, strongly labeled nuclei were always detected at the border of the OFZ in lenses from embryos E15 and older (Fig. 6 A). At earlier developmental stages, labeled nuclei were not observed in the fiber cells. If the labeling reaction was performed in the absence of the TdT enzyme, no labeling was observed (data not shown). Surprisingly, only fully condensed nuclei were labeled. Irregularly shaped nuclei with heterogeneous or marginalized chromatin were not labeled by this technique. End-labeled DNA fragments were seen to persist in the cytoplasm of fiber cells hundreds of cell widths inside the border of the OFZ. The lack of labeling in the more superficial fibers could have been due to the presence of an inhibitor of the TdT assay in these cells. To control for this, some slices were preincubated in DNase I to cause extensive DNA fragmentation in vitro. In DNase I–treated tissue, all of the nuclei were labeled by the TdT assay, including those of the epithelial cells and annular pad (data not shown) and superficial fiber cells (Fig. 6 B). The pattern of DNA damage induced by the DNase I treatment was qualitatively different from that occurring naturally at the border of the OFZ. In DNase I–treated cells, the labeling was heterogeneous and strongest immediately beneath the nuclear membrane (Fig. 6 B). In contrast, the collapsed nuclei of cells at the border of the OFZ were homogeneously stained. The fact that all nuclei in the lens were labeled after the DNase I treatment suggests that the lack of label in the outer cells of untreated lenses was not due to the presence of an inhibitor of the TdT assay in these cells.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, Washington University Medical School, St. Louis, Missouri 63110-1093, USA. Bassnetts@am.seer.wustl.edu

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus