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.
Highlights
Apoptosis entails a complex set of events, which must have evolved to provide a specific advantage to the individual
Fluorescence recovery after photobleaching (FRAP) 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
We confirmed, using FRAP, that in living HeLa cells the recovery of High Mobility Group Box 1 (HMGB1)-GFP fluorescence is complete and fast; 80% recovery is obtained in about 1.5 seconds (Figure 1C)
Summary
Apoptosis entails a complex set of events, which must have evolved to provide a specific advantage to the individual. While the ultimate advantage of cell apoptosis (favouring in some way the other cells of the organism of the population) is clear, less clear is the advantage provided by each of the events occurring during apoptosis, including chromatin condensation, DNA laddering and nuclear fragmentation. Besides CAD, Endonuclease G released from the mitochondria and more than 20 other enzymatic activities have been implicated in apoptotic DNA cleavage [3]. Only two proteins appear to be immobilized in condensed apoptotic nuclei: CAD [5] and High Mobility Group Box 1 (HMGB1) [4]. We have proposed that apoptotic cells actively modify their chromatin to retain HMGB1 and avoid issuing an inflammatory signal [8]
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