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The nuclear envelope (NE) is a spherical double membrane with elastic properties. How NE shape and elasticity are regulated by lipid chemistry is unknown. Here we discover lipid acyl chain unsaturation as essential for NE and nuclear pore complex (NPC) architecture and function. Increased lipid saturation rigidifies the NE and the endoplasmic reticulum into planar, polygonal membranes, which are fracture prone. These membranes exhibit a micron-scale segregation of lipids into ordered and disordered phases, excluding NPCs from the ordered phase. Balanced lipid saturation is required for NPC integrity, pore membrane curvature and nucleocytoplasmic transport. Oxygen deprivation amplifies the impact of saturated lipids, causing NE rigidification and rupture. Conversely, lipid droplets buffer saturated lipids to preserve NE architecture. Our study uncovers a fundamental link between lipid acyl chain structure and the integrity of the cell nucleus with implications for nuclear membrane malfunction in ischaemic tissues.

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The inner nuclear membrane has an expanded role in lipid metabolism.

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The cell nucleus is surrounded by a double membrane. The lipid packing and viscosity of membranes is critical for their function and is tightly controlled by lipid saturation. Circuits regulating the lipid saturation of the outer nuclear membrane (ONM) and contiguous endoplasmic reticulum (ER) are known. However, how lipid saturation is controlled in the inner nuclear membrane (INM) has remained enigmatic. Using INM biosensors and targeted genetic manipulations, we show that increased lipid unsaturation causes a reprogramming of lipid storage metabolism across the nuclear envelope (NE). Cells induce lipid droplet (LD) formation specifically from the distant ONM/ER, whereas LD formation at the INM is suppressed. In doing so, unsaturated fatty acids are shifted away from the INM. We identify the transcription circuits that topologically reprogram LD synthesis and identify seipin and phosphatidic acid as critical effectors. Our study suggests a detoxification mechanism protecting the INM from excess lipid unsaturation.

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The conserved yeast E3 ubiquitin ligase Bre1 and its partner, the E2 ubiquitin-conjugating enzyme Rad6, monoubiquitinate histone H2B across gene bodies during the transcription cycle1. Although processive ubiquitination might-in principle-arise from Bre1 and Rad6 travelling with RNA polymerase II2, the mechanism of H2B ubiquitination across genic nucleosomes remains unclear. Here we implicate liquid-liquid phase separation3 as the underlying mechanism. Biochemical reconstitution shows that Bre1 binds the scaffold protein Lge1, which possesses an intrinsically disordered region that phase-separates via multivalent interactions. The resulting condensates comprise a core of Lge1 encapsulated by an outer catalytic shell of Bre1. This layered liquid recruits Rad6 and the nucleosomal substrate, which accelerates the ubiquitination of H2B. In vivo, the condensate-forming region of Lge1 is required to ubiquitinate H2B in gene bodies beyond the +1 nucleosome. Our data suggest that layered condensates of histone-modifying enzymes generate chromatin-associated 'reaction chambers', with augmented catalytic activity along gene bodies. Equivalent processes may occur in human cells, and cause neurological disease when impaired.

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The inner nuclear membrane (INM) encases the genome and is fused with the outer nuclear membrane (ONM) to form the nuclear envelope. The ONM is contiguous with the endoplasmic reticulum (ER), the main site of phospholipid synthesis. In contrast to the ER and ONM, evidence for a metabolic activity of the INM has been lacking. Here, we show that the INM is an adaptable membrane territory capable of lipid metabolism. S. cerevisiae cells target enzymes to the INM that can promote lipid storage. Lipid storage involves the synthesis of nuclear lipid droplets from the INM and is characterized by lipid exchange through Seipin-dependent membrane bridges. We identify the genetic circuit for nuclear lipid droplet synthesis and a role of these organelles in regulating this circuit by sequestration of a transcription factor. Our findings suggest a link between INM metabolism and genome regulation and have potential relevance for human lipodystrophy.

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BACKGROUND AND OBJECTIVES: Adipose-derived mesenchymal stem cells (ADSCs) are promising candidates in regenerative medicine. The need for in vitro propagation to obtain therapeutic quantities of the cells imposes a risk of impaired functionality due to cellular senescence. The aim of the study was to analyze in vitro senescence of previously cryopreserved human ADSCs subjected to serial passages in cell culture. METHODS AND RESULTS: ADSC cultures from 8 donors were cultivated until proliferation arrest was reached. A gradual decline of ADSC fitness was observed by altered cell morphology, loss of proliferative, clonogenic and differentiation abilities and increased ß-galactosidase expression all of which occurred in a donor-specific manner. Relative telomere length (RTL) analysis revealed that only three tested cultures encountered replicative senescence. The presence of two ADSC subsets with significantly different RTL and cell size was discovered. The heterogeneity of ADSC cultures was supported by the intermittent nature of aging seen in tested samples. CONCLUSIONS: We conclude that the onset of in vitro senescence of ADSCs is a strongly donor-specific process which is complicated by the intricate dynamics of cell subsets present in ADSC population. This complexity needs to be carefully considered when elaborating protocols for personalized cellular therapy.

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Nuclear pore complexes (NPCs) are selective transport channels embedded in the nuclear envelope. The cylindrical NPC core forms a protein coat lining a highly curved membrane opening and has a basket-like structure appended to the nucleoplasmic side. How NPCs interact with lipids, promoting membrane bending and NPC integrity, is poorly understood. Here we show that the NPC basket proteins Nup1 and Nup60 directly induce membrane curvature by amphipathic helix insertion into the lipid bilayer. In a cell-free system, both Nup1 and Nup60 transform spherical liposomes into highly curved membrane structures. In vivo, high levels of the Nup1/Nup60 amphipathic helices cause deformation of the yeast nuclear membrane, whereas adjacent helical regions contribute to anchoring the basket to the NPC core. Basket amphipathic helices are functionally linked to distinct transmembrane nucleoporins of the NPC core, suggesting a key contribution to the membrane remodeling events that underlie NPC assembly.

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