Apical caspase reporters uncover unknown stages of apoptosis and enable ready visualization of undead cells

The 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 critical to identify caspase-activating cells in live tissues. Our work describes new 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 has capitalized on the rapid subcellular localization change of a fluorescent marker to uncover novel cellular apoptotic events. These refer to the actin-mediated positioning of the nucleus before cell delamination. The other construct has benefited from a caspase-induced nuclear translocation of a QF transcription factor. This feature enables the genetic manipulation of caspase-activating cells, whilst showing the spatio-temporal patterns of apical caspase activation. Collectively, our sensors offer new experimental opportunities that are already illuminating unknown aspects of caspase-dependent processes in apoptotic and non-apoptotic cellular scenarios. Summary statement We describe a novel set of caspase sensors that directly detect early caspase activation. The exclusive features of our reporters uncovered unknown stages of apoptosis and properties of caspase-activating cells.


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The cysteine-dependent aspartate proteases, commonly known as caspases, are the major 40 regulators of apoptosis, but also decisively modulate other essential biological functions 41 independent of apoptosis (e.g. cell proliferation, cell differentiation and cell migration) (

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Here, we describe a new set of highly sensitive caspase-reporters that overcome all the

Rational design of a novel Drice-based sensor (DBS)
90 Drice is fully activated by two sequential steps of enzymatic processing, with the first 91 cleavage step being mediated by apical caspases (mainly by Dronc, Figs. S1A and 1A) 92 (Lannan et al., 2007). Upon this first cleavage, Drice is split into two subunits (large and 93 short), which remain strongly associated to form the active protease ( Fig. S1A) (Lannan et 94 al., 2007). We capitalised on this processing step to devise a reporter of apical caspase 95 activation, which will be hereafter referred to as the Drice-based sensor (DBS). As a 96 foundation for the construct, we used an enzymatically inactive but still cleavable version of 97 Drice; Drice C211A ( Fig. 1A) (Lannan et al., 2007). This construct configuration does not 98 compromise the apical caspase-mediated excision events but prevents undesirable 99 activation of apoptosis (Lannan et al., 2007). We then appended the transmembrane domain 100 of CD8 at the N-terminus and a Histone2Av-GFP moiety to the C-terminus ( Figure 1A).

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Following this design, we created two versions of DBS. One version included a full-length

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1B and S1B). As confirmation that DBS-S faithfully reports on caspase activity, we found 114 that the nuclear localization of the Histone-GFP fragment was correlated with caspase-3 115 immunoreactivity (Fig. 1B). Additional evidence to discard unspecific cleavage of our sensor 116 outside of the Drice template was obtained by observing that the fluorescent signal of DBS-117 FL remained attached to the membranes even in apoptotic conditions, highly likely due to 118 the strong interaction between the large and small subunits of Drice (Fig. S1C). Equivalent

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observations were made expressing the sensor under the regulation of a different promoter 120 (actin promoter; Fig. S1D). These results suggest that DBS-S is able to report on caspase 121 activation in apoptotic cells, and there is no inadvertent or unspecific cleavage of DBS-S 122 template without apoptotic stimuli.

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To investigate the activity of DBS-S in Drosophila tissues, we generated transgenic flies

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After completing the basic characterisation of DBS-S in fixed samples, we analysed the 156 performance of the sensor in live tissues. DBS-S readily identified apoptotic cells in imaginal 157 discs filmed ex-vivo upon irradiation (Movie S1). Strikingly, we observed that most of the 158 GFP positive nuclei aligned to the apical surface soon after irradiation (Figs. 2A, Figure S2C,

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Movie S1, S2 and S3). This movement was tightly correlated with the apical accumulation of

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As expected, this indicates that DBS-S is insensitive to inhibitors of the effector caspases.

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Additionally, it supports the notion that DBS-S cleavage is specifically mediated by apical                 were kept at 22°C for three days in order to prevent lethality at early developmental stages.

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Hatched larvae were then transferred to 25°C until dissection before puparium formation.

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The genotypes of the flies used in these experiments were: