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Summary Artificial cells can shed new light on the molecular basis for life and hold potential for new chemical technologies. Inspired by how nature dynamically regulates its membrane compartments, we aim to repurpose the endosomal sorting complex required for transport (ESCRT) to generate complex membrane architectures as suitable scaffolds for artificial cells. Purified ESCRT-III components perform topological transformations on giant unilamellar vesicles to create complex “vesicles-within-a-vesicle” architectures resembling the compartmentalization in eukaryotic cells. Thus far, the proposed mechanisms for this activity are based on how assembly and disassembly of ESCRT-III on the membrane drives deformation. Here we demonstrate the existence of a negative feedback mechanism from membrane mechanics that regulates ESCRT-III remodeling activity. Intraluminal vesicle (ILV) formation removes excess membrane area, increasing tension, which in turn suppresses downstream ILV formation. This mechanism for in vitro regulation of ESCRT-III activity may also have important implications for its in vivo functions., Graphical Abstract, Highlights • ESCRT proteins are used to create compartmentalized artificial cell architectures • In vitro ESCRT activity is weakly dependent on the stoichiometry of Vps20 or Vps24 • ESCRT function is strongly regulated by membrane tension • Membrane tension provides a negative feedback mechanism to attenuate remodeling, Biochemistry; Bioengineering; Cell Biology; Biophysics

Thaller et al. demonstrate that direct binding between phosphatidic acid (PA) and the ESCRT Chm7 is required for nuclear envelope surveillance; PA also accumulates at nuclear envelope herniations. Thus, tight control of PA metabolism is required for nuclear envelope homeostasis., Mechanisms that control nuclear membrane remodeling are essential to maintain the integrity of the nucleus but remain to be fully defined. Here, we identify a phosphatidic acid (PA)–binding capacity in the nuclear envelope (NE)–specific ESCRT, Chm7, in budding yeast. Chm7’s interaction with PA-rich membranes is mediated through a conserved hydrophobic stretch of amino acids, which confers recruitment to the NE in a manner that is independent of but required for Chm7’s interaction with the LAP2-emerin-MAN1 (LEM) domain protein Heh1 (LEM2). Consistent with the functional importance of PA binding, mutation of this region abrogates recruitment of Chm7 to membranes and abolishes Chm7 function in the context of NE herniations that form during defective nuclear pore complex (NPC) biogenesis. In fact, we show that a PA sensor specifically accumulates within these NE herniations. We suggest that local control of PA metabolism is important for ensuring productive NE remodeling and that its dysregulation may contribute to pathologies associated with defective NPC assembly.

Mechanisms that control nuclear membrane remodeling are essential to maintain the integrity of the nucleus but remain to be fully defined. Here, we identify a phosphatidic acid (PA)-binding activity in the nuclear envelope-specific ESCRT, Chm7, in budding yeast. PA-binding is mediated through a conserved hydrophobic stretch of amino acids, which confers specific binding to the inner nuclear membrane (INM). This INM-binding is independent but nonetheless required for interaction with the LAP2-emerin-MAN1 (LEM) domain protein, Heh1 (LEM2). Consistent with the functional importance of PA-binding, mutation of this region inhibits recruitment of Chm7 to the INM and abolishes Chm7 function in the context of nuclear envelope herniations or “blebs” that form during defective nuclear pore complex (NPC) biogenesis. In fact, we show that PA accumulates at nuclear envelope herniations. We suggest that local control of PA metabolism is important for ensuring productive nuclear envelope remodeling and that its dysregulation may contribute to pathologies associated with defective NPC assembly.

The endosomal sorting complex required for transport (ESCRT) organises in supramolecular structures on the surface of lipid bilayers to drive membrane invagination and scission of intraluminal vesicles (ILVs), a process also controlled by membrane mechanics. However, ESCRT association with the membrane is also mediated by electrostatic interactions with anionic phospholipids. Phospholipid distribution within natural biomembranes is inhomogeneous due to, for example, the formation of lipid rafts and curvature-driven lipid sorting. Here, we have used phase-separated giant unilamellar vesicles (GUVs) to investigate the link between phosphatidylserine (PS)-rich lipid domains and ESCRT activity. We employ GUVs composed of phase separating lipid mixtures, where unsaturated DOPS and saturated DPPS lipids are incorporated individually or simultaneously to enhance PS localisation in liquid disordered (Ld) and/or liquid ordered (Lo) domains, respectively. PS partitioning between the coexisting phases is confirmed by a fluorescent Annexin V probe. Ultimately, we find that ILV generation promoted by ESCRTs is significantly enhanced when PS lipids localise within Ld domains. However, the ILVs that form are rich in Lo lipids. We interpret this surprising observation as preferential recruitment of the Lo phase beneath the ESCRT complex due to its increased rigidity, where the Ld phase is favoured in the neck of the resultant buds to facilitate the high membrane curvature in these regions of the membrane during the ILV formation process. Ld domains offer lower resistance to membrane bending, demonstrating a mechanism by which the composition and mechanics of membranes can be coupled to regulate the location and efficiency of ESCRT activity.

Artificial cells can shed new light on the molecular basis for life and hold potential for new chemical technologies. Inspired by how nature dynamically regulates its membrane compartments, we aim to repurpose the endosomal sorting complex required for transport (ESCRT) to generate complex membrane architectures as suitable scaffolds for artificial cells. Purified ESCRT-III components perform topological transformations on giant unilamellar vesicles (GUVs) to create complex “vesicles-within-a-vesicle” architectures resembling the compartmentalisation in eukaryotic cells. Thus far, the proposed mechanisms for this activity are based on how assembly and disassembly of ESCRT-III on the membrane drives deformation. Here we demonstrate the existence of a negative feedback mechanism from membrane mechanics that regulates ESCRT-III activity. ILV formation removes excess membrane area, increasing tension, which in turn suppresses downstream ILV formation. This mechanism for in vitro regulation of ESCRT-III activity may also have important implications for its in vivo functions.

The complexity of eukaryotic cells is underscored by the compartmentalization of chemical signals by phospholipid membranes. A grand challenge of synthetic biology is building life from the ‘bottom-up’, for the purpose of generating systems simple enough to precisely interrogate biological pathways or for adapting biology to perform entirely novel functions. Achieving compartmentalization of chemistries in an addressable manner is a task exquisitely refined by nature and embodied in a unique membrane remodelling machinery that pushes membranes away from the cytosol, the ESCRT-III (endosomal sorting complex required for transport-III) complex. Here, we show efforts to engineer a single ESCRT-III protein merging functional features from its different components. The activity of such a designed ESCRT-III is shown by its ability to drive the formation of compartments encapsulating fluorescent cargo. It appears that the modular nature of ESCRT-III allows its functional repurposing into a minimal machinery that performs sophisticated membrane remodelling, therefore enabling its use to create eukaryotic-like multi-compartment architectures.