Nuclear pores dilate and constrict in cellulo

Description Mechanosensitive nuclear pores The nucleus of eukaryotic cells is enclosed by the nuclear envelope, a double membrane punctuated with nuclear pore complexes (NPCs). These giant channels in the nuclear envelope mediate nucleocytoplasmic exchange. Zimmerli et al. show that the mechanical status of the nuclear membranes controls their nuclear pore diameter. Pulling forces imposed through nuclear membranes lead to stretching of NPCs and dilation of their diameter, whereas relief of such forces causes NPC constriction. Thus, the control of nuclear size and shape is functionally linked with NPC conformation and nucleocytoplasmic transport activity. —SMH Nuclear pore complexes are mechanosensitive and their diameter is regulated by nuclear envelope tension. INTRODUCTION The nucleus harbors and protects the genetic information in eukaryotes. It is surrounded by the nuclear envelope, which separates the nucleoplasm from the cytosol. The double-membrane system of the nuclear envelope is physically connected to the cytoskeleton and chromatin on either side. It senses and signals mechanical stimuli. Nuclear pore complexes (NPCs) mediate nucleocytoplasmic exchange. They fuse the inner and outer nuclear membranes of the nuclear envelope to form an aqueous central channel. Their intricate cylindrical architecture consists of ~30 nucleoporins. The nucleoporin scaffold embraces the membrane fusion topology in a highly dynamic fashion. Variations in NPC diameter have been reported, but the physiological circumstances and the molecular details remain unknown. RATIONALE To investigate the dynamics of NPC conformation in vivo, we structurally analyzed NPCs in Schizosaccharomyces pombe cells. We used cryo–electron tomography with subsequent subtomogram averaging to quantify the diameter of NPCs in cells exposed to different stress stimuli. We found a dilated NPC scaffold in exponentially growing cells that constricted under conditions of energy depletion and hyperosmotic shock. Structural analysis in combination with integrative structural modeling revealed that the NPC scaffold underwent large-scale movements and bending during constriction. Thereby, the volume of the central channel was lessened to about one-half. Reduced nucleocytoplasmic diffusion was apparent. Scaffold nucleoporins do not have any known motor activity that would allow them to exert forces. Thus, it appears likely that the observed dynamics of the diameter of the NPC were the result of forces that are laterally applied within the nuclear envelope. Under conditions of both energy depletion and hyperosmotic shock, cellular and nuclear volumes were reduced such that nuclear shrinkage led to an excess of nuclear membranes. We therefore hypothesized that the NPC scaffold is mechanosensitive. We surmised that a reduction of nuclear envelope membrane tension resulted in a consequent NPC constriction into a conformational ground state. RESULTS We outline several predictions of a conceptual model in which nuclear envelope membrane tension regulates NPC diameter. In such a scenario, nuclear volume and NPC diameter should be dependent on each other. The rigidity of the NPC scaffold should counteract its dilation. On the basis of membrane elastic theory, we predicted that osmotic pressure in the nuclear envelope lumen and membrane tension in the nuclear envelope act equivalently. As a consequence, the distance between the inner and outer nuclear membranes should grow linearly with both. We set out to test those predictions experimentally. We found that nuclear shrinkage correlated with NPC constriction. Recovery experiments established that this behavior was reversible. Constricted NPCs dilated when shifted back to normal medium. Nuclear volumes recovered, and cells remained viable. In exponentially growing cells, genetic perturbation of the NPC scaffold resulted in further dilation. We segmented the inner and outer nuclear membranes in cryo–electron tomograms and quantified their distance. As expected, the diameter of the NPC directly correlated with the distance between the inner and outer nuclear membranes. CONCLUSION Our data strongly suggest that the NPC scaffold is mechanosensitive and that membrane tension regulates its diameter. The data link the conformation of the NPC to the mechanical status of the nuclear envelope. This finding has implications for various cellular processes during which the nucleus may be exposed to mechanical forces. This includes cell differentiation and migration, nuclear envelope maintenance in mechanically active tissues, the import of very large cargos such as viral capsids, metastasis, and changing osmotic conditions. Under such conditions, nuclear envelope membrane tension will change globally or locally. The resulting changes in the diameter of the NPC may have functional consequences that remain to be investigated further in the future. NPCs constrict under conditions of cellular energy depletion and hyperosmotic shock. Cryo–electron microscopy maps of different NPC conformations are shown isosurface rendered as seen from the cytosol in exponentially growing (light gray), hyperosmotically shocked (yellow), and energy-depleted cells (blue) (top). Nuclear membranes are shown in dark gray. Cartoons indicate the respective conformational changes. Upon exposure to hyperosmotic shock and energy depletion, NPCs constrict to smaller central channel diameters. The nuclear volume is decreased, and the distance between the inner and outer nuclear membranes is reduced (bottom), which is indicative of a loss of nuclear envelope membrane tension. INM, inner nuclear membrane; ONM, outer nuclear membrane. In eukaryotic cells, nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes and mediate nucleocytoplasmic exchange. They are made of 30 different nucleoporins and form a cylindrical architecture around an aqueous central channel. This architecture is highly dynamic in space and time. Variations in NPC diameter have been reported, but the physiological circumstances and the molecular details remain unknown. Here, we combined cryo–electron tomography with integrative structural modeling to capture a molecular movie of the respective large-scale conformational changes in cellulo. Although NPCs of exponentially growing cells adopted a dilated conformation, they reversibly constricted upon cellular energy depletion or conditions of hypertonic osmotic stress. Our data point to a model where the nuclear envelope membrane tension is linked to the conformation of the NPC.