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Cell membranes display a tremendous complexity of lipids and proteins designed to perform the functions cells require. To coordinate these functions, the membrane is able to laterally segregate its constituents. This capability is based on dynamic liquid-liquid immiscibility and underlies the raft concept of membrane subcompartmentalization. Lipid rafts are fluctuating nanoscale assemblies of sphingolipid, cholesterol, and proteins that can be stabilized to coalesce, forming platforms that function in membrane signaling and trafficking. Here we review the evidence for how this principle combines the potential for sphingolipid-cholesterol self-assembly with protein specificity to selectively focus membrane bioactivity.

Key Points The endoplasmic reticulum (ER) forms tight membrane contact sites (MCSs) with several organelles in animal cells and yeast. The function of MCSs between the ER and mitochondria and endosomes are summarized in this Review. Electron microscopy studies reveal that although MCSs are less than 30 nm apart, the membranes do not fuse and each organelle maintains its identity. Ribosomes are excluded from the ER membrane at MCSs, and the distance between the ER and other membranes is close enough to suggest that the two organelles are tethered together by other proteins located on apposing membranes. Live-cell fluorescence microscopy reveals that ER-organelle MCSs can remain stable while both organelles traffic through the cell on the cytoskeleton. Recently identified factors have been shown to regulate organelle trafficking through MCS formation. ER–organelle MCSs regulate the lipid environment of the organelle membrane apposed to the ER. Lipid-synthesis proteins on the ER can modify lipids on the membrane of another organelle or on protein complexes. ER MCS may also transfer lipids between membranes. ER–organelle MCSs are sites of dynamic Ca 2+ crosstalk. Organelles can sequester Ca 2+ released from the ER, which can regulate processes in these organelles. Additionally, ER Ca 2+ release may regulate protein complexes at ER MCS. Mitochondria and endosomes undergo fission and fusion to, respectively, maintain their integrity and properly sort signalling receptors in the cell. ER–organelle MCSs define the position of fission for both mitochondria and endosomes, and the ER could have a variety of roles at those specific MCSs that are destined for fission. Endoplasmic reticulum (ER) is typically associated with protein biogenesis. However, recent studies suggest that it additionally synchronizes and regulates a plethora of intracellular events owing to its ability to form tight membrane associations, so-called membrane contact sites (MCSs), with other organelles. The endoplasmic reticulum (ER) is the largest organelle in the cell, and its functions have been studied for decades. The past several years have provided novel insights into the existence of distinct domains between the ER and other organelles, known as membrane contact sites (MCSs). At these contact sites, organelle membranes are closely apposed and tethered, but do not fuse. Here, various protein complexes can work in concert to perform specialized functions such as binding, sensing and transferring molecules, as well as engaging in organelle biogenesis and dynamics. This Review describes the structure and functions of MCSs, primarily focusing on contacts of the ER with mitochondria and endosomes.

Eukaryotic cells can undergo different forms of programmed cell death, many of which culminate in plasma membrane rupture as the defining terminal event 1 – 7 . Plasma membrane rupture was long thought to be driven by osmotic pressure, but it has recently been shown to be in many cases an active process, mediated by the protein ninjurin-1 8 (NINJ1). Here we resolve the structure of NINJ1 and the mechanism by which it ruptures membranes. Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane α-helices. Filament directionality and stability is defined by two amphipathic α-helices that interlink adjacent filament subunits. The NINJ1 filament features a hydrophilic side and a hydrophobic side, and molecular dynamics simulations show that it can stably cap membrane edges. The function of the resulting supramolecular arrangement was validated by site-directed mutagenesis. Our data thus suggest that, during lytic cell death, the extracellular α-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. The membrane protein NINJ1 is therefore an interactive component of the eukaryotic cell membrane that functions as an in-built breaking point in response to activation of cell death. Structural, biochemical and mutagenesis studies indicate that, in dying cells, the membrane protein NINJ1 assembles into filaments, disrupting the cell membrane.

Key Points Cellular membranes are laterally heterogeneous and consist of transient and dynamic domains with varying properties, which prominently include ordered lipid-driven domains that are referred to as lipid (or membrane) rafts. Membrane domains can be induced and regulated by a variety of interactions, which include specific lipid–lipid and lipid–protein interactions, bulk membrane properties, and interactions between membrane components and the underlying cytoskeleton. Advanced microscopy and biochemistry techniques facilitate the study of membrane domains; however, these domains still elude direct in vivo visualization. The multiplicity of possible organizational states and their context-dependent nature most likely account for experimental inconsistencies. Membrane rafts potentially have crucial physiological roles across cell types that range from immune cells to cancer cells. Membrane domains are conserved throughout the domains of life, which supports their functional importance in biological systems. Lipid rafts are relatively ordered membrane domains that are enriched in cholesterol and saturated lipids, and selectively recruit other lipids and proteins. They are dynamic and heterogeneous in composition and are thus challenging to visualize in vivo . New technologies are providing novel insights into the formation, organization and functions of these membrane domains. Cellular plasma membranes are laterally heterogeneous, featuring a variety of distinct subcompartments that differ in their biophysical properties and composition. A large number of studies have focused on understanding the basis for this heterogeneity and its physiological relevance. The membrane raft hypothesis formalized a physicochemical principle for a subtype of such lateral membrane heterogeneity, in which the preferential associations between cholesterol and saturated lipids drive the formation of relatively packed (or ordered) membrane domains that selectively recruit certain lipids and proteins. Recent studies have yielded new insights into this mechanism and its relevance in vivo , owing primarily to the development of improved biochemical and biophysical technologies.

Microbial nucleic acids are critical for the induction of innate immune responses, a host defense mechanism against infection by microbes. Recent studies have indicated that double-stranded DNA (dsDNA) induces potent innate immune responses via the induction of type I IFN (IFN) and IFN-inducible genes. However, the regulatory mechanisms underlying dsDNA-triggered signaling are not fully understood. Here we show that the translocation and assembly of the essential signal transducers, stimulator of IFN genes (STING) and TANK-binding kinase 1 (TBK1), are required for dsDNA-triggered innate immune responses. After sensing dsDNA, STING moves from the endoplasmic reticulum (ER) to the Golgi apparatus and finally reaches the cytoplasmic punctate structures to assemble with TBK1. The addition of an ER-retention signal to the C terminus of STING dampens its ability to induce antiviral responses. We also show that STING co-localizes with the autophagy proteins, microtubule-associated protein 1 light chain 3 (LC3) and autophagy-related gene 9a (Atg9a), after dsDNA stimulation. The loss of Atg9a, but not that of another autophagy-related gene (Atg7), greatly enhances the assembly of STING and TBK1 by dsDNA, leading to aberrant activation of the innate immune response. Hence Atg9a functions as a regulator of innate immunity following dsDNA stimulation as well as an essential autophagy protein. These results demonstrate that dynamic membrane traffic mediates the sequential translocation and assembly of STING, both of which are essential processes required for maximal activation of the innate immune response triggered by dsDNA.

Cholesterol is an integral component of eukaryotic cell membranes and a key molecule in controlling membrane fluidity, organization, and other physicochemical parameters. It also plays a regulatory function in antibiotic drug resistance and the immune response of cells against viruses, by stabilizing the membrane against structural damage. While it iswell understood that, structurally, cholesterol exhibits a densification effect on fluid lipid membranes, its effects on membrane bending rigidity are assumed to be nonuniversal; i.e., cholesterol stiffens saturated lipid membranes, but has no stiffening effect on membranes populated by unsaturated lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). This observation presents a clear challenge to structure–property relationships and to our understanding of cholesterol-mediated biological functions. Here, using a comprehensive approach—combining neutron spin-echo (NSE) spectroscopy, solid-state deuterium NMR (²H NMR) spectroscopy, and molecular dynamics (MD) simulations—we report that cholesterol locally increases the bending rigidity of DOPC membranes, similar to saturated membranes, by increasing the bilayer’s packing density. All three techniques, inherently sensitive to mesoscale bending fluctuations, show up to a threefold increase in effective bending rigidity with increasing cholesterol content approaching a mole fraction of 50%. Our observations are in good agreement with the known effects of cholesterol on the area-compressibility modulus and membrane structure, reaffirming membrane structure–property relationships. The current findings point to a scale-dependent manifestation of membrane properties, highlighting the need to reassess cholesterol’s role in controlling membrane bending rigidity over mesoscopic length and time scales of important biological functions, such as viral budding and lipid–protein interactions.

The gasdermins are a family of recently identified pore-forming effector proteins that cause membrane permeabilization and pyroptosis, a lytic pro-inflammatory type of cell death. Gasdermins contain a cytotoxic N-terminal domain and a C-terminal repressor domain connected by a flexible linker. Proteolytic cleavage between these two domains releases the intramolecular inhibition on the cytotoxic domain, allowing it to insert into cell membranes and form large oligomeric pores, which disrupts ion homeostasis and induces cell death. Gasdermin-induced pyroptosis plays a prominent role in many hereditary diseases and (auto)inflammatory disorders as well as in cancer. In this Review, we discuss recent developments in gasdermin research with a focus on mechanisms that control gasdermin activation, pore formation and functional consequences of gasdermin-induced membrane permeabilization.The gasdermin family of proteins has the capacity to form pores in the membrane, causing a pro-inflammatory lytic type of cell death called pyroptosis, This Review provides a comprehensive overview of the gasdermin family, the mechanisms that control their activation and their role in inflammatory disorders and cancer.

Caspase-mediated cleavage of gasdermin D, previously shown to mediate pyroptosis, acts by inducing oligomerization and pore formation in cell membranes. Gasdermin-induced cell death Pyroptosis, an inflammatory form of programmed cell death that is part of the innate immune response, is triggered by caspase-mediated cleavage of the inflammasome protein gasdermin D. Judy Lieberman and colleagues examine the underlying molecular mechanism for gasdermin functioning in pyroptosis. They present evidence that caspase 11 cleavage of gasdermin D, previously shown to mediate pyroptosis, induces oligomerization of the N-terminal domain and pore formation. Also in this issue of Nature , Feng Shao and colleagues show that the N-terminal domains of gasdermins D, A and A3 are cytotoxic because they disrupt cell membranes in both mammalian cells and artificially transformed bacteria through the formation of membrane pores. Inflammatory caspases (caspases 1, 4, 5 and 11) are activated in response to microbial infection and danger signals. When activated, they cleave mouse and human gasdermin D (GSDMD) after Asp276 and Asp275, respectively, to generate an N-terminal cleavage product (GSDMD-NT) that triggers inflammatory death (pyroptosis) and release of inflammatory cytokines such as interleukin-1β 1 , 2 . Cleavage removes the C-terminal fragment (GSDMD-CT), which is thought to fold back on GSDMD-NT to inhibit its activation. However, how GSDMD-NT causes cell death is unknown. Here we show that GSDMD-NT oligomerizes in membranes to form pores that are visible by electron microscopy. GSDMD-NT binds to phosphatidylinositol phosphates and phosphatidylserine (restricted to the cell membrane inner leaflet) and cardiolipin (present in the inner and outer leaflets of bacterial membranes). Mutation of four evolutionarily conserved basic residues blocks GSDMD-NT oligomerization, membrane binding, pore formation and pyroptosis. Because of its lipid-binding preferences, GSDMD-NT kills from within the cell, but does not harm neighbouring mammalian cells when it is released during pyroptosis. GSDMD-NT also kills cell-free bacteria in vitro and may have a direct bactericidal effect within the cytosol of host cells, but the importance of direct bacterial killing in controlling in vivo infection remains to be determined.

Phospholipids are asymmetrically distributed in the plasma membrane. This asymmetrical distribution is disrupted during apoptosis, exposing phosphatidylserine (PtdSer) on the cell surface. Using a haploid genetic screen in human cells, we found that ATP11C (adenosine triphosphatase type 11C) and CDC50A (cell division cycle protein 50A) are required for aminophospholipid translocation from the outer to the inner plasma membrane leaflet; that is, they display flippase activity. ATP11C contained caspase recognition sites, and mutations at these sites generated caspase-resistant ATP11C without affecting its flippase activity. Cells expressing caspase-resistant ATP11C did not expose PtdSer during apoptosis and were not engulfed by macrophages, which suggests that inactivation of the flippase activity is required for apoptotic PtdSer exposure. CDC50A-deficient cells displayed PtdSer on their surface and were engulfed by macrophages, indicating that PtdSer is sufficient as an \"eat me\" signal.

Kirchhausen and colleagues show that actin is required for clathrin-mediated endocytosis at membranes under tension—such as apical surfaces of polarized cells. Actin engages with Hip1R bound to clathrin light chain to complete the deformation of a clathrin-coated pit into an endocytic vesicle. Clathrin-mediated endocytosis is independent of actin dynamics in many circumstances but requires actin polymerization in others. We show that membrane tension determines the actin dependence of clathrin-coat assembly. As found previously, clathrin assembly supports formation of mature coated pits in the absence of actin polymerization on both dorsal and ventral surfaces of non-polarized mammalian cells, and also on basolateral surfaces of polarized cells. Actin engagement is necessary, however, to complete membrane deformation into a coated pit on apical surfaces of polarized cells and, more generally, on the surface of any cell in which the plasma membrane is under tension from osmotic swelling or mechanical stretching. We use these observations to alter actin dependence experimentally and show that resistance of the membrane to propagation of the clathrin lattice determines the distinction between ‘actin dependent and ‘actin independent’. We also find that light-chain-bound Hip1R mediates actin engagement. These data thus provide a unifying explanation for the role of actin dynamics in coated-pit budding.