I am using active caspase 3 to detect apoptosis by immunofluorescence. In the last experiment, we got some positive signals in the cytoplasm. Can this be indicative of caspase activity? Thank you
Hello Angel Martin Bastida
Caspase-3 exists in the form of pro-enzyme and is located predominantly in the cytoplasm of cells. During apoptosis, caspase-3 is cleaved and activated by upstream caspases and is translocated into the nucleus to cleave its nuclear substrates resulting in characteristic apoptotic nuclear changes such as DNA fragmentation, chromatin condensation and nuclear disruption.
Positive signal is mostly found in nuclei of morphologically identifiable apoptotic cells. However, active caspase 3 can also be detected as nuclear and cytoplasmic staining in cells without morphological signs of apoptosis.
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Caspase is the highly specialized family of cysteinyl-aspartate proteases, which is present as procaspase-3 (as zymogen forms) in the cell cytoplasm. The conversion from procaspase-3 to active caspase-3 (cleaved caspase-3) occurs very rapidly and almost in parallel to mitochondrial membrane depolarization and the release of cytochrome c. The cytochrome c elevation in the cytoplasm activates upstream caspases that results in the rapid conversion of pro- to active caspase-3. Once activated in the cytoplasm, caspase-3 moves to nucleus with the help of carrier protein wherein it starts PARP cleavage, considered to be a hallmark of apoptosis. This event is rapidly followed by the disruption cellular histo-architecture by caspase-3 that results in membrane blabbing, cytoplasmic fragmentation etc. morphological features characteristics of apoptosis. Indeed, the reasons why a cell can apparently tolerate an apoptotic stimulus for several hours before this rapid chain of events occurs remains to be investigated.
The biochemical machinery during apoptosis and the role of active caspse-3 has been updated in mammalian cells by our team.
Hope our publication on the mechanism of apoptosis could be useful:
Apoptosis in mammalian oocytes: a review. . 2015 Aug;20(8):1019-25.Apoptosis
doi: 10.1007/s10495-015-1136-y.
https://pubmed.ncbi.nlm.nih.gov/25958165/
Germ cell depletion from mammalian ovary: possible involvement of apoptosis and autophagy. . J Biomed Sci 2018 Apr 23;25(1):36. doi: 10.1186/s12929-018-0438-0.
https://pubmed.ncbi.nlm.nih.gov/29681242/
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