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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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Confocal micrographs of lamin B2 immunofluorescence in an E17 lens slice after high-temperature antigen retrieval (see text  for details). (A) Fiber cells in the peripheral cortex. The immunofluorescence is restricted to the lamina of the fiber cell nuclei. (B) Lowmagnification view of cells near the border of the OFZ. Note that even nuclear remnants (arrowhead) are positive for lamin B2 immunofluorescence. (C) High-magnification view of cells immediately adjacent to the OFZ (B, arrow). In many of these cells, the nuclear  lamina has become distorted, and the profiles of the nuclei are irregular (arrows), although some nuclei still retain a more normal appearance (arrowhead). (D) High-magnification view of cells immediately within the OFZ (B, arrowhead). At this point in the denucleation process, the fiber nuclei have collapsed into small spherical structures (arrows), and rents are apparent in the nuclear lamina.  Bars: (A) 25 μm; (B) 50 μm; (C) 10 μm; (D) 5 μm.
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Figure 3: Confocal micrographs of lamin B2 immunofluorescence in an E17 lens slice after high-temperature antigen retrieval (see text for details). (A) Fiber cells in the peripheral cortex. The immunofluorescence is restricted to the lamina of the fiber cell nuclei. (B) Lowmagnification view of cells near the border of the OFZ. Note that even nuclear remnants (arrowhead) are positive for lamin B2 immunofluorescence. (C) High-magnification view of cells immediately adjacent to the OFZ (B, arrow). In many of these cells, the nuclear lamina has become distorted, and the profiles of the nuclei are irregular (arrows), although some nuclei still retain a more normal appearance (arrowhead). (D) High-magnification view of cells immediately within the OFZ (B, arrowhead). At this point in the denucleation process, the fiber nuclei have collapsed into small spherical structures (arrows), and rents are apparent in the nuclear lamina. Bars: (A) 25 μm; (B) 50 μm; (C) 10 μm; (D) 5 μm.

Mentions: Confocal microscopy and immunofluorescence were used to visualize the fate of two nuclear components, lamin B2 (Fig. 3) and histone proteins (Fig. 4), in fiber cells near the border of the OFZ. Lamin B2 is a member of a family of intermediate filament proteins that together form the karyoskeleton and, as such, are usually distributed immediately beneath the nuclear membrane. In the superficial cortex of the lens, the lamin B2 immunofluorescence specifically delineates the fiber cell nuclear membrane (Fig. 3 A). In these experiments, omission of the primary antibody, or its substitution by ascites fluid, resulted in complete absence of nuclear fluorescence, demonstrating the specificity of the antibody (data not shown). At the border of the OFZ, the nuclei collapsed and lamin B2–positive nuclear debris persisted for hundreds of cell widths into the OFZ (Fig. 3 B). Immediately before nuclear collapse (Fig. 3 B, arrow), lamin B2 immunofluorescence became uneven and the nuclear profile was irregular (Fig. 3 C). Two nuclei in the early stages of nuclear collapse are indicated by arrows in Fig. 3 C. An adjacent nucleus, with a relatively intact lamina, is indicated by an arrowhead. Soon after the collapse of the nuclei (Fig. 3, B, arrowhead), rents were evident in the nuclear lamina (Fig. 3, D).


Chromatin degradation in differentiating fiber cells of the eye lens.

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

Confocal micrographs of lamin B2 immunofluorescence in an E17 lens slice after high-temperature antigen retrieval (see text  for details). (A) Fiber cells in the peripheral cortex. The immunofluorescence is restricted to the lamina of the fiber cell nuclei. (B) Lowmagnification view of cells near the border of the OFZ. Note that even nuclear remnants (arrowhead) are positive for lamin B2 immunofluorescence. (C) High-magnification view of cells immediately adjacent to the OFZ (B, arrow). In many of these cells, the nuclear  lamina has become distorted, and the profiles of the nuclei are irregular (arrows), although some nuclei still retain a more normal appearance (arrowhead). (D) High-magnification view of cells immediately within the OFZ (B, arrowhead). At this point in the denucleation process, the fiber nuclei have collapsed into small spherical structures (arrows), and rents are apparent in the nuclear lamina.  Bars: (A) 25 μm; (B) 50 μm; (C) 10 μm; (D) 5 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139849&req=5

Figure 3: Confocal micrographs of lamin B2 immunofluorescence in an E17 lens slice after high-temperature antigen retrieval (see text for details). (A) Fiber cells in the peripheral cortex. The immunofluorescence is restricted to the lamina of the fiber cell nuclei. (B) Lowmagnification view of cells near the border of the OFZ. Note that even nuclear remnants (arrowhead) are positive for lamin B2 immunofluorescence. (C) High-magnification view of cells immediately adjacent to the OFZ (B, arrow). In many of these cells, the nuclear lamina has become distorted, and the profiles of the nuclei are irregular (arrows), although some nuclei still retain a more normal appearance (arrowhead). (D) High-magnification view of cells immediately within the OFZ (B, arrowhead). At this point in the denucleation process, the fiber nuclei have collapsed into small spherical structures (arrows), and rents are apparent in the nuclear lamina. Bars: (A) 25 μm; (B) 50 μm; (C) 10 μm; (D) 5 μm.
Mentions: Confocal microscopy and immunofluorescence were used to visualize the fate of two nuclear components, lamin B2 (Fig. 3) and histone proteins (Fig. 4), in fiber cells near the border of the OFZ. Lamin B2 is a member of a family of intermediate filament proteins that together form the karyoskeleton and, as such, are usually distributed immediately beneath the nuclear membrane. In the superficial cortex of the lens, the lamin B2 immunofluorescence specifically delineates the fiber cell nuclear membrane (Fig. 3 A). In these experiments, omission of the primary antibody, or its substitution by ascites fluid, resulted in complete absence of nuclear fluorescence, demonstrating the specificity of the antibody (data not shown). At the border of the OFZ, the nuclei collapsed and lamin B2–positive nuclear debris persisted for hundreds of cell widths into the OFZ (Fig. 3 B). Immediately before nuclear collapse (Fig. 3 B, arrow), lamin B2 immunofluorescence became uneven and the nuclear profile was irregular (Fig. 3 C). Two nuclei in the early stages of nuclear collapse are indicated by arrows in Fig. 3 C. An adjacent nucleus, with a relatively intact lamina, is indicated by an arrowhead. Soon after the collapse of the nuclei (Fig. 3, B, arrowhead), rents were evident in the nuclear lamina (Fig. 3, D).

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