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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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The fate of the nuclear membrane and DNA in fiber cells near the  border of the OFZ. The lens slice was  stained with DiOC6 (for membranes)  and SYTO 17 (for DNA) and viewed  with a confocal microscope. Cortical fiber cells are shown in A–C, cells just outside the border of the OFZ are shown in  D–F, cells immediately within the OFZ  are shown in G–I, and cells in the center  of the lens are shown in J–L. A, D, G,  and J are differential interference contrast images; B, E, H, and K, are confocal images of the DiOC6 fluorescence;  and C, F, I, and L are the corresponding  confocal images of the SYTO 17 fluorescence. Note the sudden change in nuclear morphology at the border of the  OFZ and the loss of the nuclear membrane (D–F). The last nucleus to possess  a nuclear membrane is indicated by the  arrow in D–F. Collapsed nuclei are evident in cells within the OFZ and SYTO  17–stained nuclear remnants (L, arrows)  can be discerned, even in the most central fiber cells. Bar, 25 μm.
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Figure 2: The fate of the nuclear membrane and DNA in fiber cells near the border of the OFZ. The lens slice was stained with DiOC6 (for membranes) and SYTO 17 (for DNA) and viewed with a confocal microscope. Cortical fiber cells are shown in A–C, cells just outside the border of the OFZ are shown in D–F, cells immediately within the OFZ are shown in G–I, and cells in the center of the lens are shown in J–L. A, D, G, and J are differential interference contrast images; B, E, H, and K, are confocal images of the DiOC6 fluorescence; and C, F, I, and L are the corresponding confocal images of the SYTO 17 fluorescence. Note the sudden change in nuclear morphology at the border of the OFZ and the loss of the nuclear membrane (D–F). The last nucleus to possess a nuclear membrane is indicated by the arrow in D–F. Collapsed nuclei are evident in cells within the OFZ and SYTO 17–stained nuclear remnants (L, arrows) can be discerned, even in the most central fiber cells. Bar, 25 μm.

Mentions: Cut open along the optic axis, an E15 embryonic chicken lens has the cellular organization shown in diagrammatic form in Fig. 1. Confocal microscopic images of the changes in nuclear organization in cells near the border of the OFZ are shown in Fig. 2. Lens slices were stained with both DiOC6 and SYTO 17, enabling the simultaneous visualization of the fiber cell membrane systems (Fig. 2, B, E, H, and K) and DNA (Fig. 2, C, F, I, and L), respectively, in addition to differential interference contrast (DIC) images (Fig. 2, A, D, G, and J). In the outer cortex of the lens, the leaf-shaped fiber cell nuclei were the only prominent subcellular feature of the DIC images (Fig. 2 A). The corresponding confocal image obtained with DiOC6 (Fig. 2 C) showed labeling of the fiber cell membrane systems, including the plasma membrane and the nuclear envelope. The distribution of DNA was visualized simultaneously using SYTO 17. The fiber cell nuclei were evenly stained by SYTO 17, with prominent nucleoli. In fiber cells near the border of the OFZ, a change in the organization of the nuclei was apparent (Fig. 2, D–F). In the space of a few cell layers, nuclei passed through distinct stages. In the first stage, the leaf-shaped nuclei characteristic of the outer cell layers became irregular in shape with an undulating nuclear membrane. At this point, the DNA staining was uneven, with marginalization of the chromatin. Subsequently, the nuclei collapsed into small spherical structures that were visible under DIC. Although the collapsed nuclei were intensely stained by the SYTO 17, the chromatin now appeared “naked” (i.e., no longer contained within a nuclear membrane). The last nucleus with an intact nuclear membrane is indicated by the arrow in Fig. 2 E. Fiber cells containing collapsed nuclei extend for 100–200 μm into the OFZ. Even in cells at the very center of the lens, small, positively stained nuclear remnants could be discerned (Fig. 2 L) in the otherwise featureless fiber cell cytoplasm.


Chromatin degradation in differentiating fiber cells of the eye lens.

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

The fate of the nuclear membrane and DNA in fiber cells near the  border of the OFZ. The lens slice was  stained with DiOC6 (for membranes)  and SYTO 17 (for DNA) and viewed  with a confocal microscope. Cortical fiber cells are shown in A–C, cells just outside the border of the OFZ are shown in  D–F, cells immediately within the OFZ  are shown in G–I, and cells in the center  of the lens are shown in J–L. A, D, G,  and J are differential interference contrast images; B, E, H, and K, are confocal images of the DiOC6 fluorescence;  and C, F, I, and L are the corresponding  confocal images of the SYTO 17 fluorescence. Note the sudden change in nuclear morphology at the border of the  OFZ and the loss of the nuclear membrane (D–F). The last nucleus to possess  a nuclear membrane is indicated by the  arrow in D–F. Collapsed nuclei are evident in cells within the OFZ and SYTO  17–stained nuclear remnants (L, arrows)  can be discerned, even in the most central fiber cells. Bar, 25 μm.
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Figure 2: The fate of the nuclear membrane and DNA in fiber cells near the border of the OFZ. The lens slice was stained with DiOC6 (for membranes) and SYTO 17 (for DNA) and viewed with a confocal microscope. Cortical fiber cells are shown in A–C, cells just outside the border of the OFZ are shown in D–F, cells immediately within the OFZ are shown in G–I, and cells in the center of the lens are shown in J–L. A, D, G, and J are differential interference contrast images; B, E, H, and K, are confocal images of the DiOC6 fluorescence; and C, F, I, and L are the corresponding confocal images of the SYTO 17 fluorescence. Note the sudden change in nuclear morphology at the border of the OFZ and the loss of the nuclear membrane (D–F). The last nucleus to possess a nuclear membrane is indicated by the arrow in D–F. Collapsed nuclei are evident in cells within the OFZ and SYTO 17–stained nuclear remnants (L, arrows) can be discerned, even in the most central fiber cells. Bar, 25 μm.
Mentions: Cut open along the optic axis, an E15 embryonic chicken lens has the cellular organization shown in diagrammatic form in Fig. 1. Confocal microscopic images of the changes in nuclear organization in cells near the border of the OFZ are shown in Fig. 2. Lens slices were stained with both DiOC6 and SYTO 17, enabling the simultaneous visualization of the fiber cell membrane systems (Fig. 2, B, E, H, and K) and DNA (Fig. 2, C, F, I, and L), respectively, in addition to differential interference contrast (DIC) images (Fig. 2, A, D, G, and J). In the outer cortex of the lens, the leaf-shaped fiber cell nuclei were the only prominent subcellular feature of the DIC images (Fig. 2 A). The corresponding confocal image obtained with DiOC6 (Fig. 2 C) showed labeling of the fiber cell membrane systems, including the plasma membrane and the nuclear envelope. The distribution of DNA was visualized simultaneously using SYTO 17. The fiber cell nuclei were evenly stained by SYTO 17, with prominent nucleoli. In fiber cells near the border of the OFZ, a change in the organization of the nuclei was apparent (Fig. 2, D–F). In the space of a few cell layers, nuclei passed through distinct stages. In the first stage, the leaf-shaped nuclei characteristic of the outer cell layers became irregular in shape with an undulating nuclear membrane. At this point, the DNA staining was uneven, with marginalization of the chromatin. Subsequently, the nuclei collapsed into small spherical structures that were visible under DIC. Although the collapsed nuclei were intensely stained by the SYTO 17, the chromatin now appeared “naked” (i.e., no longer contained within a nuclear membrane). The last nucleus with an intact nuclear membrane is indicated by the arrow in Fig. 2 E. Fiber cells containing collapsed nuclei extend for 100–200 μm into the OFZ. Even in cells at the very center of the lens, small, positively stained nuclear remnants could be discerned (Fig. 2 L) in the otherwise featureless fiber cell cytoplasm.

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