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Several nuclear events during apoptosis depend on caspase-3 activation but do not constitute a common pathway.

Trisciuoglio L, Bianchi ME - PLoS ONE (2009)

Bottom Line: A number of nuclear events occur during apoptosis, including DNA laddering, nuclear lamina breakdown, phosphorylation of histones H2B and histone H2AX, and the tight binding to chromatin of HMGB1 and CAD, the nuclease responsible for DNA laddering.We find that all depend directly or indirectly on caspase-3 activation.CAD activation, H2AX phosphorylation and DNA laddering cluster together into a pathway, but all other events appear to be independent of each other downstream of caspase-3, and likely evolved subject to different functional pressures.

View Article: PubMed Central - PubMed

Affiliation: Chromatin Dynamics Unit, San Raffaele Scientific Institute, Milano, Italy.

ABSTRACT
A number of nuclear events occur during apoptosis, including DNA laddering, nuclear lamina breakdown, phosphorylation of histones H2B and histone H2AX, and the tight binding to chromatin of HMGB1 and CAD, the nuclease responsible for DNA laddering. We have performed an epistasis analysis to investigate whether these events cluster together in pathways. We find that all depend directly or indirectly on caspase-3 activation. CAD activation, H2AX phosphorylation and DNA laddering cluster together into a pathway, but all other events appear to be independent of each other downstream of caspase-3, and likely evolved subject to different functional pressures.

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Related in: MedlinePlus

The entire HMGB1 protein is required for its immobilization onto apoptotic chromatin.(A) Schematic representation of the GFP fusion proteins. (B) A typical FRAP from one cell. Fluorescence intensity in the bleached spot is plotted as a function of time. The typical parameters of a FRAP experiment are shown. (C) FRAP experiments performed in proliferating HeLa cells transiently expressing the various GFP fusion proteins. Each line corresponds to the average of fluorescence recovery in at least 20 cells. The standard deviation is about 5% over most data points, and is not shown to avoid clutter. (D) FRAP experiments performed in apoptotic HeLa cells transiently expressing the various GFP fusion proteins. Each line corresponds to the fluorescence recovery measured in a different cell. Individual cell traces are shown, rather than their average, because of the high variation among apoptotic cells.
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pone-0006234-g001: The entire HMGB1 protein is required for its immobilization onto apoptotic chromatin.(A) Schematic representation of the GFP fusion proteins. (B) A typical FRAP from one cell. Fluorescence intensity in the bleached spot is plotted as a function of time. The typical parameters of a FRAP experiment are shown. (C) FRAP experiments performed in proliferating HeLa cells transiently expressing the various GFP fusion proteins. Each line corresponds to the average of fluorescence recovery in at least 20 cells. The standard deviation is about 5% over most data points, and is not shown to avoid clutter. (D) FRAP experiments performed in apoptotic HeLa cells transiently expressing the various GFP fusion proteins. Each line corresponds to the fluorescence recovery measured in a different cell. Individual cell traces are shown, rather than their average, because of the high variation among apoptotic cells.

Mentions: The immobilization of HMGB1 and CAD during apoptosis has been demonstrated using GFP fusion proteins and laser photobleaching [4], [5]. FRAP (fluorescence recovery after photobleaching) entails photobleaching of a small spot in the nucleus, followed by repeated imaging of the photobleached spot to measure the recovery of fluorescence within it (and thus the mixing of non-photobleached and photobleached but fully functional molecules from adjacent areas in the nucleus). FRAP is a time-lapse experiment: the fluorescence in the bleach region is monitored during a pre-bleach period to determine the initial fluorescence intensity F(initial), immediately after the bleaching F(bleached) and until it reaches a final value F(final), when no further increase can be detected (Figure 1B).


Several nuclear events during apoptosis depend on caspase-3 activation but do not constitute a common pathway.

Trisciuoglio L, Bianchi ME - PLoS ONE (2009)

The entire HMGB1 protein is required for its immobilization onto apoptotic chromatin.(A) Schematic representation of the GFP fusion proteins. (B) A typical FRAP from one cell. Fluorescence intensity in the bleached spot is plotted as a function of time. The typical parameters of a FRAP experiment are shown. (C) FRAP experiments performed in proliferating HeLa cells transiently expressing the various GFP fusion proteins. Each line corresponds to the average of fluorescence recovery in at least 20 cells. The standard deviation is about 5% over most data points, and is not shown to avoid clutter. (D) FRAP experiments performed in apoptotic HeLa cells transiently expressing the various GFP fusion proteins. Each line corresponds to the fluorescence recovery measured in a different cell. Individual cell traces are shown, rather than their average, because of the high variation among apoptotic cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2713420&req=5

pone-0006234-g001: The entire HMGB1 protein is required for its immobilization onto apoptotic chromatin.(A) Schematic representation of the GFP fusion proteins. (B) A typical FRAP from one cell. Fluorescence intensity in the bleached spot is plotted as a function of time. The typical parameters of a FRAP experiment are shown. (C) FRAP experiments performed in proliferating HeLa cells transiently expressing the various GFP fusion proteins. Each line corresponds to the average of fluorescence recovery in at least 20 cells. The standard deviation is about 5% over most data points, and is not shown to avoid clutter. (D) FRAP experiments performed in apoptotic HeLa cells transiently expressing the various GFP fusion proteins. Each line corresponds to the fluorescence recovery measured in a different cell. Individual cell traces are shown, rather than their average, because of the high variation among apoptotic cells.
Mentions: The immobilization of HMGB1 and CAD during apoptosis has been demonstrated using GFP fusion proteins and laser photobleaching [4], [5]. FRAP (fluorescence recovery after photobleaching) entails photobleaching of a small spot in the nucleus, followed by repeated imaging of the photobleached spot to measure the recovery of fluorescence within it (and thus the mixing of non-photobleached and photobleached but fully functional molecules from adjacent areas in the nucleus). FRAP is a time-lapse experiment: the fluorescence in the bleach region is monitored during a pre-bleach period to determine the initial fluorescence intensity F(initial), immediately after the bleaching F(bleached) and until it reaches a final value F(final), when no further increase can be detected (Figure 1B).

Bottom Line: A number of nuclear events occur during apoptosis, including DNA laddering, nuclear lamina breakdown, phosphorylation of histones H2B and histone H2AX, and the tight binding to chromatin of HMGB1 and CAD, the nuclease responsible for DNA laddering.We find that all depend directly or indirectly on caspase-3 activation.CAD activation, H2AX phosphorylation and DNA laddering cluster together into a pathway, but all other events appear to be independent of each other downstream of caspase-3, and likely evolved subject to different functional pressures.

View Article: PubMed Central - PubMed

Affiliation: Chromatin Dynamics Unit, San Raffaele Scientific Institute, Milano, Italy.

ABSTRACT
A number of nuclear events occur during apoptosis, including DNA laddering, nuclear lamina breakdown, phosphorylation of histones H2B and histone H2AX, and the tight binding to chromatin of HMGB1 and CAD, the nuclease responsible for DNA laddering. We have performed an epistasis analysis to investigate whether these events cluster together in pathways. We find that all depend directly or indirectly on caspase-3 activation. CAD activation, H2AX phosphorylation and DNA laddering cluster together into a pathway, but all other events appear to be independent of each other downstream of caspase-3, and likely evolved subject to different functional pressures.

Show MeSH
Related in: MedlinePlus