Caspase-9-dependent decrease of nuclear pore channel hydrophobicity is accompanied by nuclear envelope leakiness

Nuclear envelope permeability increases remarkably 2.5 hours after cytochrome c injection into oocytes. Passive diffusion of 70-kDa FITC-dextran into isolated oocyte nuclei at different points in time after cytochrome c injection. Controls were injected with nuclear isolation medium. The small inset shows schematically the measurement setup.

Nuclear barrier leakiness is independent of caspase-3 but is modulated by caspase-9. Passive diffusion of 70-kDa FITC-dextran into isolated oocyte nuclei after incubation in apoptotic cytosolic extracts in presence of caspase inhibitors. Pretreatment of nuclei with importin-β 45-462 prevents the permeability increase almost completely. Error bars represent SEM values.

Initial rates of diffusion of 70-kDa FITC-dextran after incubation of nuclei in apoptotic extracts. (A, B) Pretreatment of NPCs with importin-β 45-462 leads to a blockage of the NPC channel, which prevents apoptotic nuclear barrier leakiness as shown in C. (C) Initial rates of diffusion after preincubation with importin-β 45-462 and incubation in an apoptotic extract. (D) Rates of diffusion after incubation of nuclei in an apoptotic extract in presence of caspase inhibitors. Asterisks indicate significantly different from “apoptotic extract” condition. ^⁎⁎P < 0.05; ^⁎⁎⁎P < 0.001.

Schematic interaction between high-aspect-ratio atomic force microscope tip and nuclear pore channel (true to size comparison). NPC model modified from Patel et al.¹¹ Small inset: SEM image of a high-aspect-ratio tip (image taken from: http://www.nano-tools.com).

Nuclear envelope (NE) surface hydrophobicity maps. (A, B) Height images of the cytoplasmic face of the NE before and after incubation with an apoptotic cytosolic extract. (C, D) Corresponding hydrophobicity maps. Each white pixel represents an adhesive event between the hydrophobic tip and the sample. (E, F) Overlay of the height images and the hydrophobicity maps. Images are 1.2 × 1.2 μm each.

The density of hydrophobic spots in the nuclear pore channel (NPC) central channel is decreased after incubation of the nuclear envelope in an apoptotic cytosolic extract. Hydrophobic spot density is measured as a fraction of pixels showing adhesion peaks divided by the entity of pixels in the area of the NPC central channels. Error bars represent SEM values. Upper right inset: Representative adhesion measurement. The hydrophobic atomic force microscope tip is brought into contact with the sample. Upon retraction of the tip an adhesive force is felt by the cantilever, which results in an adhesion peak (dashed circle).