Figure 8

Plasma membrane permeabilization causes rapid cytosolic depletion of XIAP and re-establishes Smac release. (a) Representative images of the response of an untreated MCF-7 Smac-YFP cell to digitonin-based plasma membrane permeabilization. Upon permeabilization, C-XIAP-C fluorescence was lost from the cytosol. Mitochondrial Smac-YFP fluorescence did not noticeably change. Scale bar=10 μm. (b) Representative images of the response of a TRAIL/CHX pre-treated MCF-7 Smac-YFP cell to digitonin-based plasma membrane permeabilization. Upon permeabilization, C-XIAP-C fluorescence was lost from the cytosol. Mitochondrial Smac-YFP was lost in parallel. Scale bar=10 μm. (c, d) Temporal profiles of Smac-YFP and C-XIAP-C release in untreated (c) or TRAIL/CHX pre-treated (d) MCF-7 Smac-YFP cells. Smac-YFP release was plotted as the change in cellular fluorescence S.D., C-XIAP-C loss was plotted as the change in cellular fluorescence intensity. Two representative cells are shown each. Release onset was set to time zero. A total of n=4 untreated cells or n=8 pre-treated cells from three experiments each were analysed. (e) Comparison of amounts of Smac-YFP released after permeabilization of untreated or TRAIL/CHX pre-treated cells expressing C-XIAP-C. Cellular fluorescence S.D. for 4 and 8 cells per group were compared at 5 min after onset of C-XIAP-C loss. Error bars represent S.E.M. ^*P<0.05 (Student's t-test). (f) Mitochondrial Smac retention cannot be detected by biochemical fractionation. MCF-7 cells transfected to express Cerulean or C-XIAP-C were treated with TRAIL/CHX for the indicated times and subjected to digitonin-based biochemical fractionation. Smac release was detected by immunoblotting. Longer exposures were used to detect signals corresponding to poly-ubiquitinated Smac (calculated molecular weights of 44 and 52.5 kDa, respectively). Porin and α-tubulin served as loading controls. Experiment was repeated eight times with similar results