Regulation of nuclear envelope permeability in cell death and survival

Abstract

The nuclear pore complex (NPC) mediates macromolecular exchange between nucleus and cytoplasm. It is a regulated channel whose functional properties are modulated in response to the physiological status of the cell. Identifying the factors responsible for regulating NPC activity is crucial to understand how intracellular signaling cues are integrated at the level of this channel to control nucleocytoplasmic trafficking. For proteins lacking active translocation signals the NPC acts as a molecular sieve limiting passage across the nuclear envelope (NE) to proteins with a MW below ~40 kD. Here, we investigate how this permeability barrier is altered in paradigms of cell death and cell survival, i.e., apoptosis induction via staurosporine, and enhanced viability via overexpression of Bcl-2. We monitor dynamic changes of the NPC’s size-exclusion limit for passive diffusion by confocal time-lapse microscopy of cells undergoing apoptosis, and use different diffusion markers to determine how Bcl-2 expression affects steady-state NE permeability. We show that staurosporine triggers an immediate and gradual leakiness of the NE preceding the appearance of apoptotic hallmarks. Bcl-2 expression leads to a constitutive increase in NE permeability, and its localization at the NE is sufficient for the effect, evincing a functional role for Bcl-2 at the nuclear membrane. In both settings, NPC leakiness correlates with reduced Ca²⁺ in internal stores, as demonstrated by fluorometric measurements of ER/NE Ca²⁺ levels. By comparing two cellular models with opposite outcome these data pinpoint ER/NE Ca²⁺ as a general and physiologically relevant regulator of the permeability barrier function of the NPC.

Keywords: :

• Bcl-2
• calcium
• Nup153
• nuclear pore complex
• staurosporine
• apoptosis
• caspases
• calpains
• confocal microscopy

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Christoph Borner (University of Freiburg) for the Bcl-2 expression plasmids and cell lines, Luca Scorrano (University of Padova) for pSERCA2, Jan Ellenberg (EMBL, Heidelberg) for pNup153-GFP, Akis Karakesisoglou (University of Durham) for pEGFP-C1-Tm-Nesprin-2, Henning Walczak (Imperial College London) for lz-TRAIL and Thomas Meergans for HeLa Tet-Off cells. We are grateful to Felix Schönenberger for support in image analysis, and to Anja Holtz for helpful discussions and sequence analysis of the 4xCherry expression plasmid.

Supplemental Materials

Supplemental materials may be found here: http://www.landesbioscience.com/journals/nucleus/article/21982/