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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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In situ electrophoresis of an E15 lens slice. (A) Low-magnification image of an ethidium-stained lens slice after electrophoresis. The slice is embedded in a block of 0.3% agarose. The dark space between the two bright arms (arrowheads) represents the OFZ.  The arms are formed by the ethidium-stained fiber cell nuclei and are continuous with the band of annular pad and epithelial nuclei seen  dimly in the background. The uneven bright strip extending across the image is a reflection from the edge of the agarose block. The arrow depicts the direction of the electrical field. (B) Intermediate-magnification view of a lens slice that was pretreated with DNase I before electrophoresis to cause fragmentation of fiber cell DNA. Note the diffuse clouds of ethidium-stained material emanating from all  of the fiber cell nuclei. (C) Untreated lens slice showing two streams of ethidium-stained material emanating from nuclei immediately  adjacent to the OFZ. (D) High-magnification image of cells at the border of the OFZ. Brightly stained individual nuclei are visible. A  stream of ethidium-stained material can be seen issuing from cells immediately adjacent to the OFZ. Bars: (A) 500 μm; (B) 250 μm; (C)  100 μm; (D) 25 μm.
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Figure 9: In situ electrophoresis of an E15 lens slice. (A) Low-magnification image of an ethidium-stained lens slice after electrophoresis. The slice is embedded in a block of 0.3% agarose. The dark space between the two bright arms (arrowheads) represents the OFZ. The arms are formed by the ethidium-stained fiber cell nuclei and are continuous with the band of annular pad and epithelial nuclei seen dimly in the background. The uneven bright strip extending across the image is a reflection from the edge of the agarose block. The arrow depicts the direction of the electrical field. (B) Intermediate-magnification view of a lens slice that was pretreated with DNase I before electrophoresis to cause fragmentation of fiber cell DNA. Note the diffuse clouds of ethidium-stained material emanating from all of the fiber cell nuclei. (C) Untreated lens slice showing two streams of ethidium-stained material emanating from nuclei immediately adjacent to the OFZ. (D) High-magnification image of cells at the border of the OFZ. Brightly stained individual nuclei are visible. A stream of ethidium-stained material can be seen issuing from cells immediately adjacent to the OFZ. Bars: (A) 500 μm; (B) 250 μm; (C) 100 μm; (D) 25 μm.

Mentions: We also used a novel nonenzymatic technique for visualizing the condition of the chromatin during fiber cell differentiation. In our in situ electrophoresis assay, a lens slice was embedded in agarose, permeabilized, and oriented perpendicular to a weak electric field. Fragments of DNA that were sufficiently small to be electrophoretically mobile were driven from the lens cells into the agarose, where they were stained with ethidium bromide. A diagram showing the orientation of the lens slice and the expected appearance of the tissue under the confocal microscope is shown in Fig. 8. The optical-sectioning capability of the confocal microscope enabled us to position the focal plane at the level of the fiber cell nuclei. With this optical arrangement, the OFZ appeared as a dark gap between two arms of strongly fluorescent nuclei (Fig. 9 A). If the lens slice was pretreated with DNase I before electrophoresis, two diffuse clouds of ethidium-stained material were observed emanating from the fiber cells (Fig. 9 B). The clouds of DNA appeared to come from all the annular pad and fiber cells. In contrast, in non–DNase I–treated slices, ethidium-stained material was only observed emanating from cells at the immediate border of the OFZ (Fig. 9, C and D). We assume that, in each case, the ethidium-stained material emanating from the lens slices represents low–molecular weight DNA because cellular RNA (which would also be weakly stained by ethidium bromide) is degraded under the extremely alkaline conditions of the assay.


Chromatin degradation in differentiating fiber cells of the eye lens.

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

In situ electrophoresis of an E15 lens slice. (A) Low-magnification image of an ethidium-stained lens slice after electrophoresis. The slice is embedded in a block of 0.3% agarose. The dark space between the two bright arms (arrowheads) represents the OFZ.  The arms are formed by the ethidium-stained fiber cell nuclei and are continuous with the band of annular pad and epithelial nuclei seen  dimly in the background. The uneven bright strip extending across the image is a reflection from the edge of the agarose block. The arrow depicts the direction of the electrical field. (B) Intermediate-magnification view of a lens slice that was pretreated with DNase I before electrophoresis to cause fragmentation of fiber cell DNA. Note the diffuse clouds of ethidium-stained material emanating from all  of the fiber cell nuclei. (C) Untreated lens slice showing two streams of ethidium-stained material emanating from nuclei immediately  adjacent to the OFZ. (D) High-magnification image of cells at the border of the OFZ. Brightly stained individual nuclei are visible. A  stream of ethidium-stained material can be seen issuing from cells immediately adjacent to the OFZ. Bars: (A) 500 μm; (B) 250 μm; (C)  100 μm; (D) 25 μm.
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Figure 9: In situ electrophoresis of an E15 lens slice. (A) Low-magnification image of an ethidium-stained lens slice after electrophoresis. The slice is embedded in a block of 0.3% agarose. The dark space between the two bright arms (arrowheads) represents the OFZ. The arms are formed by the ethidium-stained fiber cell nuclei and are continuous with the band of annular pad and epithelial nuclei seen dimly in the background. The uneven bright strip extending across the image is a reflection from the edge of the agarose block. The arrow depicts the direction of the electrical field. (B) Intermediate-magnification view of a lens slice that was pretreated with DNase I before electrophoresis to cause fragmentation of fiber cell DNA. Note the diffuse clouds of ethidium-stained material emanating from all of the fiber cell nuclei. (C) Untreated lens slice showing two streams of ethidium-stained material emanating from nuclei immediately adjacent to the OFZ. (D) High-magnification image of cells at the border of the OFZ. Brightly stained individual nuclei are visible. A stream of ethidium-stained material can be seen issuing from cells immediately adjacent to the OFZ. Bars: (A) 500 μm; (B) 250 μm; (C) 100 μm; (D) 25 μm.
Mentions: We also used a novel nonenzymatic technique for visualizing the condition of the chromatin during fiber cell differentiation. In our in situ electrophoresis assay, a lens slice was embedded in agarose, permeabilized, and oriented perpendicular to a weak electric field. Fragments of DNA that were sufficiently small to be electrophoretically mobile were driven from the lens cells into the agarose, where they were stained with ethidium bromide. A diagram showing the orientation of the lens slice and the expected appearance of the tissue under the confocal microscope is shown in Fig. 8. The optical-sectioning capability of the confocal microscope enabled us to position the focal plane at the level of the fiber cell nuclei. With this optical arrangement, the OFZ appeared as a dark gap between two arms of strongly fluorescent nuclei (Fig. 9 A). If the lens slice was pretreated with DNase I before electrophoresis, two diffuse clouds of ethidium-stained material were observed emanating from the fiber cells (Fig. 9 B). The clouds of DNA appeared to come from all the annular pad and fiber cells. In contrast, in non–DNase I–treated slices, ethidium-stained material was only observed emanating from cells at the immediate border of the OFZ (Fig. 9, C and D). We assume that, in each case, the ethidium-stained material emanating from the lens slices represents low–molecular weight DNA because cellular RNA (which would also be weakly stained by ethidium bromide) is degraded under the extremely alkaline conditions of the assay.

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