We employed microinjection of an NIR dye caspase substrate to measure caspase activity in individual, living Xenopus oocytes and early embryos in real-time. This assay provides quantitation of caspase activity that can be obtained rapidly (and repeatedly over time), and is easily distinguishable from oocyte maturation-induced GVBD. Relative to the traditional Xenopus cell-free system, this approach enables evaluation of individual cells, and has the unique strength of being able to monitor caspase activity and morphological apoptotic death in oocytes and developing embryos as separable entities (; and ).
The Xenopus oocyte is easily microinjected and NIR imaging can be performed immediately following exposure to cytosolic cytochrome c
. This enables us to address questions that have been technically impractical, a strength that is illustrated by our studies probing caspase activation kinetics. These experiments confirm that caspases become activated extremely rapidly in response to cytochrome c
(in under five minutes). Additionally, we investigated whether continued caspase activity is required to cause apoptosis. Our data indicate that even short-term activation of caspases (as brief as 10 minutes of exposure to cytosolic cytochrome c
) causes an oocyte to die via apoptosis, suggesting that only caspase substrates cleaved within this timeframe are essential for cell dismantling. In light of the vast number of identified caspase substrates31,32
, and the uncertainty of precisely how caspase-mediated proteolysis translates into organized cellular breakdown, this result yields insight into how caspase substrates might be prioritized for further functional characterization. Very recently, Edgington et al. reported the development of activity-based fluorescent probes for imaging the kinetics of caspase activation in vivo33
. It would be of considerable interest to compare these reagents to the substrate used here for their resolution and sensitivity in our single cell analyses.
In addition to the cytochrome c
-induced caspase activity described, we also observed significant fluorescence of embryos microinjected with IRDye alone by nineteen hours after fertilization (). Sublethal caspase activity has been reported in Xenopus oocytes as well as in human preimplantation embryos34,35
, and we hypothesize that our observations reflect the accumulation of minimal amounts of active caspases present at any given time that become significant only in summation. It is unclear whether these minimal amounts of caspase activity are important for development or are simply an inexorable consequence of rapid cell division that is tolerated by the embryo. Regardless, this observation prompted us reflect upon our IAP studies, where we saw that Smac addition, which we believe serves to neutralize IAPs in the oocyte, decreases the amount of cytochrome c
required to activate caspases. Perhaps the levels of IAPs endowed to an oocyte determine whether an embryo will be able to limit the amount of caspase activation to within a tolerable range. One intriguing possibility warranting further investigation is that IAP levels decrease with age, making older oocytes more prone to apoptotic death following fertilization.
A series of studies performed in Xenopus embryos refer to the presence of cytoplasmic bridges between daughter cells26,27,28
. Our studies in which we microinject cytochrome c
into individual blastomeres and cause entire embryo apoptosis support this idea. It is well known that checkpoint activation does not occur in embryos until after the mid-blastula transition36
; one important reason for such a delay might be to prevent the triggering of apoptosis by minor cellular damage during a time when death of an individual blastomere would lead to death of the entire embryo. Conversely, in the presence of a cell-damaging stimulus sufficiently strong to warrant induction of cell death in a single cell, it would likely be advantageous to rapidly induce death of the entire embryo to avoid significant developmental anomalies.
Although our experiments with microinjected cytochrome c and Emi2 strongly support the notion of communicating cytoplasms, we were unable to image this phenomenon using fluorescent dyes. So although this assay offers a powerful means for detecting caspase activity within a single cell, it may be that this imaging modality lacks the sensitivity required to detect a very weak signal when in direct proximity to a very strong one.
Although clearly advantageous for monitoring apoptosis as part of an inquiry into basic cellular and developmental processes, the technique presented here might also be employed as a screening modality to identify apoptotic regulators. The 96-well format combined with the ease of oocyte microinjection make this system poised for either medium-throughput or secondary screens. Given our previous reports suggesting that an apoptosome-stimulating therapeutic might be useful in the treatment of breast and brain tumors37,38
, it may prove worthwhile to employ this assay to screen small-molecule libraries for a cytochrome c
mimetic. Moreover, Xenopus oocytes have been used to good effect in screening small pool cDNA libraries for novel cell cycle regulators39
. A similar approach, using the NIR dye as an apoptotic indicator, may allow identification of novel cell death-regulating proteins.