Abstract
ABSTRACTThe caspase-mediated regulation of many cellular processes, including apoptosis, justifies the substantial interest in understanding all of the biological features of these enzymes. To complement functional assays, it is crucial to identify caspase-activating cells in live tissues. Our work describes novel initiator caspase reporters that, for the first time, provide direct information concerning the initial steps of the caspase activation cascade in Drosophila tissues. One of our caspase sensors capitalises on the rapid subcellular localisation change of a fluorescent marker to uncover novel cellular apoptotic events relating to the actin-mediated positioning of the nucleus before cell delamination. The other construct benefits from caspase-induced nuclear translocation of a QF transcription factor. This feature enables the genetic manipulation of caspase-activating cells and reveals the spatiotemporal patterns of initiator caspase activity. Collectively, our sensors offer experimental opportunities not available by using previous reporters and have proven useful to illuminate previously unknown aspects of caspase-dependent processes in apoptotic and non-apoptotic cellular scenarios.
Highlights
Rational design of a novel Drice-based sensor (DBS) Drice is fully activated by two sequential steps of enzymatic processing, with the first cleavage step being mediated by initiator caspases (Lannan et al, 2007)
We capitalised on this processing step to devise a reporter of initiator caspase activation, which will be hereafter referred to as the Drice-based sensor (DBS)
As confirmation that DBS-S faithfully reports on caspase activity, we found that the nuclear localisation of the Histone-GFP fragment was correlated with cleaved caspase-3 immunoreactivity (Fig. 1B)
Summary
The cysteine-dependent aspartate proteases, commonly known as caspases, are the major regulators of apoptosis, and decisively modulate other essential biological functions independent of apoptosis (e.g. cell proliferation, cell differentiation and cell migration) (Aram et al, 2017; Baena-Lopez et al, 2017; Burgon and Megeney, 2017; Ellis and Horvitz, 1986; Fogarty and Bergmann, 2017; Hollville and Deshmukh, 2017; McIlwain et al, 2013; Miura, 2012; Mukherjee and Williams, 2017; Perez-Garijo, 2017; Songane et al, 2018). Their activation requires the enzymatic activity of effector caspases, and they are not functional in biological contexts without the participation of the entire caspase pathway, a situation frequently observed in non-apoptotic scenarios (Kondo et al, 2006; Napoletano et al, 2017; Ouyang et al, 2011; Wells et al, 2006) Some of these issues have been partially overcome by two recent constructs that have incorporated a CD8 membrane retention domain and a transcriptional activator (Gal4) flanking the caspase-cleavage motif (Ding et al, 2016; Tang et al, 2015). These reporters still rely on an effector caspase cleavage motif (DQVD), and the inclusion of a Gal fragment impedes their usage in combination with pre-existing Gal lines
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