Localization, Dynamics, and Function of Survivin Revealed by Expression of Functional SurvivinDsRed Fusion Proteins in the Living Cell

Survivin, a member of the inhibitor of apoptosis protein family, has attracted growing attention due to its expression in various tumors and its potential application in tumor therapy. However, its subcellular localization and function have remained controversial: Recent studies revealed that survivin is localized at the mitotic spindle, binds caspases, and could thus protect cells from apoptosis. The cell cycle-dependent expression of survivin and its antiapoptotic function led to the hypothesis that survivin connects the cell cycle with apoptosis, thus providing a death switch for the termination of defective mitosis. In other studies, survivin was detected at kinetochores, cleavage furrow, and midbody, localizations being characteristic for chromosomal passenger proteins. These proteins are involved in cytokinesis as inferred from the observation that RNA interference and expression of mutant proteins led to cytokinesis defects without an increase in apoptosis. To remedy these discrepancies, we analyzed the localizations of a survivinDsRed fusion protein in HeLa cells by using confocal laser scanning microscopy and time-lapse video imaging. SurvivinDsRed was excluded from the interphase nucleus and was detected in centrosomes and at kinetochores. It dissociated from chromosomes at the anaphase/telophase transition and accumulated at the ends of polar microtubuli where it was immediately condensed to the midbody. Overexpression of both survivinDsRed and of a phosphorylation-defective mutant conferred resistance against apoptosis-inducing reagents, but only the overexpressed mutant protein caused an aberrant cytokinesis. These data characterize in detail the dynamics of survivin in vertebrate cells and confirm that survivin represents a chromosomal passenger protein

Post–Cytochrome c Protection from Apoptosis Conferred by a MAPK Pathway in Xenopus Egg Extracts

In response to many different apoptotic stimuli, cytochrome c is released from the intermembrane space of the mitochondria into the cytoplasm, where it serves as a cofactor in the activation of procaspase 9. Inhibition of this process can occur either by preventing cytochrome c release or by blocking caspase activation or activity. Experiments involving in vitro reconstitution of apoptosis in cell-free extracts of Xenopus laevis eggs have suggested that extracts arrested in interphase are susceptible to an endogenous apoptotic program leading to caspase activation, whereas extracts arrested in meiotic metaphase are not. We report here that Mos/MEK/MAPK pathways active in M phase–arrested eggs are responsible for rendering them refractory to apoptosis. Interestingly, M phase–arrested extracts are competent to release cytochrome c, yet still do not activate caspases. Concomitantly, we have also demonstrated that recombinant Mos, MEK, and ERK are sufficient to block cytochrome c–dependent caspase activation in purified Xenopus cytosol, which lacks both transcription and translation. These data indicate that the MAP kinase pathway can target and inhibit post–cytochrome c release apoptotic events in the absence of new mRNA/protein synthesis and that this biochemical pathway is responsible for the apoptotic inhibition observed in meiotic X. laevis egg extracts