STS-induced alterations of nuclear envelope permeability are

VI.1.6.1. STS-induced early nuclear leakage is abolished by addition of the Ca²⁺ chelator BAPTA/AM

As all tested protease inhibitors had no diminishing effect on apoptotic nuclear envelope permeabilization, another, non-protease mechanism was

Mooren et al. 2004; Enns et al., 2003). The effect of Ca²⁺ on nuclear envelope permeability was investigated by confocal live cell imaging of HeLa cells in the presence of the Ca²⁺ chelator BAPTA/AM. Therefore, the cells were co-transfected with Nup153-GFP and 4xCherry and 40 min prior to STS addition, the cells were incubated with BAPTA/AM.

Addition of BAPTA/AM completely abolishes early nuclear leakage in STS-treated cells (Figure 23, blue trace). Caspase-activation, chromatin condensation, nuclear pore cleavage and the subsequent loss of nuclear permeability barrier function are unaltered in the presence of BAPTA/AM (Figure 23 and Figure 24).

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Figure 23: Ca²⁺ mediates early STS-induced nuclear leakage. Averaged traces of at least 13 cells from different, independent experiments, in the presence (blue trace) or absence (red trace) of BAPTA/AM. Addition of BAPTA/AM completely abolishes early STS-induced nuclear leakage. Loss of permeability barrier function at the time point of chromatin condensation (dotted line) due to caspase activation still occurs in the presence of BAPTA/AM, visualized by the abrupt increase in nuclear 4xCherry fluorescence. Error bars present SEM

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Figure 24: Caspase activation and apoptosis occur in the presence of BAPTA/AM. HeLa 229 cells were treated with STS for the indicated time points in the presence (grey bars) or absence of BAPTA/AM (red bars). A) Caspase activity was measured by cleavage of DEVD-AFC. No differences in caspase actvation could be detected. zVAD addition completely abolishes caspase activity (black bar).

B) In the same lysates used for caspase activity measurement, the percentage of apoptotic cells was determined by counting condensed nuclei. Error bars show SEM.

VI.1.6.2. STS reduces ER Ca²⁺ level

The amount of Ca²⁺ in the ER has been shown to alter nuclear permeability (Enns et al., 2003; Mooren et al., 2004, Scorrano et al., 2003). To further specify the role of Ca²⁺ in STS-induced increase in nuclear envelope permeability, Ca²⁺ - loading of the ER was determined in control cells, STS- and TRAIL- treated HeLa 229 cells. The apoptosis inducer TRAIL was used as a reference stimulus. TRAIL does not elicit early nuclear leakage and should therefore not elicit ER Ca²⁺ -level alterations. Endplasmic reticulum Ca²⁺-level were assessed with the fluorescent Ca²⁺ sensor Fluo-4. After treatment with either STS or TRAIL for 1h, ER Ca²⁺-level were determined with a fluorescence plate reader. Therefore, Thapsigargin, which is an inhibitor of the sarcoplasmic/endoplasmic calcium-ATPase SERCA was added and the amount of released Ca²⁺ from internal stores was assessed.

The Thapsigargin-induced increase in Fluo-4 fluorescence is a measure for the amount of Ca²⁺ released from the ER.

HeLa 229 cells treated with STS for 1h show reduced ER Ca²⁺ level, whereas cells treated with TRAIL for 1h have unaltered ER Ca²⁺ level (Figure

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Figure 25: STS reduces ER Ca²⁺ level. A) Representative traces of HeLa 229 cells incubated with Fluo-4 and treated with 0.5µM STS or 300ng/ml TRAIL. The basal Ca²⁺ level of control - (black trace), STS-treated - (red trace), and TRAIL-treated cells (grey trace) was detected by a fluorescence plate reader. The increase in Fluo-4 fluorescence after thapsigargin addition was detected. B) Quantification of three independent experiments. All experiments were performed in triplicates. The difference between basal level and peak level corresponds to the amount of released ER Ca²⁺. The released Ca²⁺ was expressed as percent of the Ca²⁺ amount released by control cells. Statistics: one way ANOVA compared to control with Bonferroni post-test. *** p < 0.001

VI.1.7. Active nuclear transport is unaffected by alteration of ER Ca²⁺

level.

Alterations in ER Ca²⁺ not only influence passive diffusion but also have an impact on active transport (Greber and Gerace, 1995). To investigate, whether altered Ca²⁺ content and the early increase in nuclear envelope permeability affect active nuclear transport in STS-induced cell death, Hela 229 cells were transfected with the permeability marker 4xCherry and a marker for active nuclear transport: GFP-NLS. GFP-NLS is a construct of about 24 kDa which is small enough to pass the nuclear envelope by passive diffusion, but due to its nuclear localization signal (NLS) is actively imported into the nucleus in healthy cells, resulting in a exclusively nuclear localization.

If active nuclear transport is impaired, GFP-NLS is no longer imported into the nucleus and distributes over the whole cell. Confocal live cell imaging of cells treated with STS was performed and the GFP-NLS signal in the cytoplasm was measured. As a comparison cells were treated with TRAIL.

Since in this model nuclear envelope permeability and Ca²⁺ content are

unchanged until final caspase-mediated execution of apoptosis, no alteration in active transport functionality was expected (Figure 26).

Active nuclear transport is functional in both models of apoptosis until caspase activation, chromatin condensation and loss of nuclear envelope permeability. Only directly before caspase activation and final disruption of the NPC does increased leakage of GFP-NLS into the cytosol occur in STS treated cells. This indicates that at this stage the rate of actively imported GFP-NLS molecules no longer compensates for the the rate of passive efflux. Although early nuclear leakage and decreased ER Ca²⁺ level occur in STS-induced apoptosis, active nuclear transport is largely unaffected. These results lead to the conclusion that active and passive transport are largely uncoupled.

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Figure 26: Active nuclear transport is unaffected by alterations of ER Ca²⁺. Cytosolic GFP-NLS fluorescence in cells treated with TRAIL (black trace) and STS (red trace) was detected and plotted against time. For each apoptotic stimulus, 15 cells from different, independent experiments were evaluated and averaged. Error bars show SEM