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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 images of lens slices  showing the distribution of ER (green)  and degraded DNA (red). (A) The ER  (visualized by immunofluorescence with  a protein disulfide isomerase antibody)  is abundant in the superficial layers of  the lens but completely absent from the  well-defined central OFZ. The fiber cell  nuclear membranes are also labeled by  this antibody. Degraded DNA is localized in condensed nuclear remnants,  scattered throughout the cytoplasm of fiber cells in the OFZ. Note the gap of  ∼90 μm between the last fiber cell to  contain ER (arrow) and the first to contain degraded DNA (arrowhead). e, epithelium; ap, annular pad. (B) Apoptotic  cells are occasionally detected in the anterior epithelium of the E9 lens. At this  stage of development, the epithelial cells  (e) and all of the fiber cells ( f ) contain  abundant ER. Apoptotic nuclei (arrows)  are strongly labeled by the TdT assay,  and small apoptotic bodies are often observed in the adjacent tissue (arrowheads). Note that, in contrast to the central fiber cells shown in A, the apoptotic  nuclei of the epithelial cells are found in  cells in which the ER is still present.  Bars: (A) 100 μm; (B) 25 μm.
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Figure 7: Confocal images of lens slices showing the distribution of ER (green) and degraded DNA (red). (A) The ER (visualized by immunofluorescence with a protein disulfide isomerase antibody) is abundant in the superficial layers of the lens but completely absent from the well-defined central OFZ. The fiber cell nuclear membranes are also labeled by this antibody. Degraded DNA is localized in condensed nuclear remnants, scattered throughout the cytoplasm of fiber cells in the OFZ. Note the gap of ∼90 μm between the last fiber cell to contain ER (arrow) and the first to contain degraded DNA (arrowhead). e, epithelium; ap, annular pad. (B) Apoptotic cells are occasionally detected in the anterior epithelium of the E9 lens. At this stage of development, the epithelial cells (e) and all of the fiber cells ( f ) contain abundant ER. Apoptotic nuclei (arrows) are strongly labeled by the TdT assay, and small apoptotic bodies are often observed in the adjacent tissue (arrowheads). Note that, in contrast to the central fiber cells shown in A, the apoptotic nuclei of the epithelial cells are found in cells in which the ER is still present. Bars: (A) 100 μm; (B) 25 μm.

Mentions: From studies such as those shown in Fig. 6 A, it appeared that positively labeled nuclei (i.e., those containing degraded DNA) were only detected some distance within the OFZ. If this were true, it would suggest that extensive DNA cleavage occurs some time after the disappearance of mitochondria, endoplasmic reticulum, and other organelles. To verify that this is the correct sequence of events, we performed immunofluorescence labeling of PDI (an ER marker) on E19 lens slices that had previously been labeled with TdT. In lens slices labeled with both TdT and antiPDI, a clear gap was revealed between the border of the OFZ (delineated by the PDI immunofluorescence) and the first TdT-positive nuclei (Fig. 7 A). At E19, cells that were unstained by either treatment formed a region ∼90 μm wide. We also noted an interesting developmental sequence in slices labeled with both TdT and anti-PDI. At E12, the OFZ was already clearly delineated, as described previously (Bassnett and Beebe, 1992). At this stage of development, the OFZ contained many condensed nuclei, but there was no evidence of TdT labeling. Only 3 d later, at E15, did the condensed nuclei become labeled by the TdT assay. Thus, the spatial gap observed at E19, between the disappearance of PDI immunofluorescence and the onset of DNA degradation (Fig. 7 A), paralleled the temporal gap observed at earlier stages.


Chromatin degradation in differentiating fiber cells of the eye lens.

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

Confocal images of lens slices  showing the distribution of ER (green)  and degraded DNA (red). (A) The ER  (visualized by immunofluorescence with  a protein disulfide isomerase antibody)  is abundant in the superficial layers of  the lens but completely absent from the  well-defined central OFZ. The fiber cell  nuclear membranes are also labeled by  this antibody. Degraded DNA is localized in condensed nuclear remnants,  scattered throughout the cytoplasm of fiber cells in the OFZ. Note the gap of  ∼90 μm between the last fiber cell to  contain ER (arrow) and the first to contain degraded DNA (arrowhead). e, epithelium; ap, annular pad. (B) Apoptotic  cells are occasionally detected in the anterior epithelium of the E9 lens. At this  stage of development, the epithelial cells  (e) and all of the fiber cells ( f ) contain  abundant ER. Apoptotic nuclei (arrows)  are strongly labeled by the TdT assay,  and small apoptotic bodies are often observed in the adjacent tissue (arrowheads). Note that, in contrast to the central fiber cells shown in A, the apoptotic  nuclei of the epithelial cells are found in  cells in which the ER is still present.  Bars: (A) 100 μm; (B) 25 μm.
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Figure 7: Confocal images of lens slices showing the distribution of ER (green) and degraded DNA (red). (A) The ER (visualized by immunofluorescence with a protein disulfide isomerase antibody) is abundant in the superficial layers of the lens but completely absent from the well-defined central OFZ. The fiber cell nuclear membranes are also labeled by this antibody. Degraded DNA is localized in condensed nuclear remnants, scattered throughout the cytoplasm of fiber cells in the OFZ. Note the gap of ∼90 μm between the last fiber cell to contain ER (arrow) and the first to contain degraded DNA (arrowhead). e, epithelium; ap, annular pad. (B) Apoptotic cells are occasionally detected in the anterior epithelium of the E9 lens. At this stage of development, the epithelial cells (e) and all of the fiber cells ( f ) contain abundant ER. Apoptotic nuclei (arrows) are strongly labeled by the TdT assay, and small apoptotic bodies are often observed in the adjacent tissue (arrowheads). Note that, in contrast to the central fiber cells shown in A, the apoptotic nuclei of the epithelial cells are found in cells in which the ER is still present. Bars: (A) 100 μm; (B) 25 μm.
Mentions: From studies such as those shown in Fig. 6 A, it appeared that positively labeled nuclei (i.e., those containing degraded DNA) were only detected some distance within the OFZ. If this were true, it would suggest that extensive DNA cleavage occurs some time after the disappearance of mitochondria, endoplasmic reticulum, and other organelles. To verify that this is the correct sequence of events, we performed immunofluorescence labeling of PDI (an ER marker) on E19 lens slices that had previously been labeled with TdT. In lens slices labeled with both TdT and antiPDI, a clear gap was revealed between the border of the OFZ (delineated by the PDI immunofluorescence) and the first TdT-positive nuclei (Fig. 7 A). At E19, cells that were unstained by either treatment formed a region ∼90 μm wide. We also noted an interesting developmental sequence in slices labeled with both TdT and anti-PDI. At E12, the OFZ was already clearly delineated, as described previously (Bassnett and Beebe, 1992). At this stage of development, the OFZ contained many condensed nuclei, but there was no evidence of TdT labeling. Only 3 d later, at E15, did the condensed nuclei become labeled by the TdT assay. Thus, the spatial gap observed at E19, between the disappearance of PDI immunofluorescence and the onset of DNA degradation (Fig. 7 A), paralleled the temporal gap observed at earlier stages.

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