Abstract
The cleavage of nuclear proteins by caspases promotes nuclear breakdown and, therefore, plays a key role in apoptosis execution. However, the detailed molecular mechanisms of these events remain unclear. To get more insights into the mechanisms of nuclear events during apoptosis we set up a rapid fractionation protocol for the separation of the cytoplasmic and nuclear fractions of cells undergoing cisplatin-induced apoptosis. Importantly, nuclear accumulation of effector caspase-3 as well as initiator caspase-2, -8 and -9 was observed using the developed protocol and immunofluorescence microscopy. The detection of caspases and their cleavage products in the nucleus occurred within the same time interval after cisplatin treatment and took place shortly before nuclear fragmentation. The entry of initiator caspases to the nucleus was independent of caspase-3. Given that all three initiator caspases had catalytic activity in the nuclei, our findings indicate that initiator caspases might participate in the proteolysis of nuclear components during apoptosis, promoting its disintegration and apoptotic cell death.
Highlights
The cleavage of nuclear proteins by caspases promotes nuclear breakdown and, plays a key role in apoptosis execution
As well as immunofluorescence microscopy, we assessed the changes in the cellular distribution of initiator caspase-2, -8, -9 and effector caspase-3 during apoptosis
The purity of the fractions was assessed in parallel by two approaches: Western blotting (WB) and DIC/fluorescence microscopy using the staining with Hoechst33342 and ER-tracker Green
Summary
The cleavage of nuclear proteins by caspases promotes nuclear breakdown and, plays a key role in apoptosis execution. To get more insights into the mechanisms of nuclear events during apoptosis we set up a rapid fractionation protocol for the separation of the cytoplasmic and nuclear fractions of cells undergoing cisplatin-induced apoptosis. The main role in the breakdown of the nucleus and other cellular compartments during apoptosis belongs to effector caspase-3, while the role of other caspases in the process remains unclear[10,11]. We aimed to assess cellular compartmentalization of caspases in the course of DNA damage-induced apoptosis taking into account time-dependent alterations in the nuclear structure during this type of cell death. Using this method, as well as immunofluorescence microscopy, we assessed the changes in the cellular distribution of initiator caspase-2, -8, -9 and effector caspase-3 during apoptosis
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