Cell membrane tension affects and is affected by many fundamental cellular processes, yet it is poorly understood. Recent experiments show that membrane tension can propagate at vastly different ...speeds in different cell types, reflecting physiological adaptations. Here we briefly review the current knowledge about membrane tension gradients, membrane flows, and their physiological context.
Cell-free reconstitution of membrane traffic reactions and the morphological characterization of membrane intermediates that accumulate under these conditions have helped to elucidate the physical ...and molecular mechanisms involved in membrane transport. To gain a better understanding of endocytosis, we have reconstituted vesicle budding and fission from isolated plasma membrane sheets and imaged these events. Electron and fluorescence microscopy, including subdiffraction-limit imaging by stochastic optical reconstruction microscopy (STORM), revealed F-BAR (FBP17) domain coated tubules nucleated by clathrin-coated buds when fission was blocked by GTPgammaS. Triggering fission by replacing GTPgammaS with GTP led not only to separation of clathrin-coated buds, but also to vesicle formation by fragmentation of the tubules. These results suggest a functional link between FBP17-dependent membrane tubulation and clathrin-dependent budding. They also show that clathrin spatially directs plasma membrane invaginations that lead to the generation of endocytic vesicles larger than those enclosed by the coat.
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.
Cell membranes contain a large variety of lipid types and are crowded with proteins, endowing them with the plasticity needed to fulfill their key roles in cell functioning. The compositional ...complexity of cellular membranes gives rise to a heterogeneous lateral organization, which is still poorly understood. Computational models, in particular molecular dynamics simulations and related techniques, have provided important insight into the organizational principles of cell membranes over the past decades. Now, we are witnessing a transition from simulations of simpler membrane models to multicomponent systems, culminating in realistic models of an increasing variety of cell types and organelles. Here, we review the state of the art in the field of realistic membrane simulations and discuss the current limitations and challenges ahead.
Ca²⁺ influx through plasma membrane lesions triggers a rapid repair process that was previously shown to require the exocytosis of lysosomal organelles (Reddy, A., E. Caler, and N. Andrews. 2001. ...CELL: 106:157-169). However, how exocytosis leads to membrane resealing has remained obscure, particularly for stable lesions caused by pore-forming proteins. In this study, we show that Ca²⁺-dependent resealing after permeabilization with the bacterial toxin streptolysin O (SLO) requires endocytosis via a novel pathway that removes SLO-containing pores from the plasma membrane. We also find that endocytosis is similarly required to repair lesions formed in mechanically wounded cells. Inhibition of lesion endocytosis (by sterol depletion) inhibits repair, whereas enhancement of endocytosis through disruption of the actin cytoskeleton facilitates resealing. Thus, endocytosis promotes wound resealing by removing lesions from the plasma membrane. These findings provide an important new insight into how cells protect themselves not only from mechanical injury but also from microbial toxins and pore-forming proteins produced by the immune system.
In Gram-negative bacteria, a synergistic relationship between slow passive uptake of antibiotics across the outer membrane and active efflux transporters creates a permeability barrier, which ...efficiently reduces the effective concentrations of antibiotics in cells and, hence, their activities. To analyze the relative contributions of active efflux and the passive barrier to the activities of antibiotics, we constructed Escherichia coli strains with controllable permeability of the outer membrane. The strains expressed a large pore that does not discriminate between compounds on the basis of their hydrophilicity and sensitizes cells to a variety of antibacterial agents. We found that the efficacies of antibiotics in these strains were specifically affected by either active efflux or slow uptake, or both, and reflect differences in the properties of the outer membrane barrier, the repertoire of efflux pumps, and the inhibitory activities of antibiotics. Our results identify antibiotics which are the best candidates for the potentiation of activities through efflux inhibition and permeabilization of the outer membrane.
Lipid Rafts As a Membrane-Organizing Principle Lingwood, Daniel; Simons, Kai
Science (American Association for the Advancement of Science),
2010, 20100101, 2010-Jan-01, 2010-01-00, Volume:
327, Issue:
5961
Journal Article
Peer reviewed
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.
Inflammatory caspases cleave the gasdermin D (GSDMD) protein to trigger pyroptosis, a lytic form of cell death that is crucial for immune defences and diseases. GSDMD contains a functionally ...important gasdermin-N domain that is shared in the gasdermin family. The functional mechanism of action of gasdermin proteins is unknown. Here we show that the gasdermin-N domains of the gasdermin proteins GSDMD, GSDMA3 and GSDMA can bind membrane lipids, phosphoinositides and cardiolipin, and exhibit membrane-disrupting cytotoxicity in mammalian cells and artificially transformed bacteria. Gasdermin-N moved to the plasma membrane during pyroptosis. Purified gasdermin-N efficiently lysed phosphoinositide/cardiolipin-containing liposomes and formed pores on membranes made of artificial or natural phospholipid mixtures. Most gasdermin pores had an inner diameter of 10–14 nm and contained 16 symmetric protomers. The crystal structure of GSDMA3 showed an autoinhibited two-domain architecture that is conserved in the gasdermin family. Structure-guided mutagenesis demonstrated that the liposome-leakage and pore-forming activities of the gasdermin-N domain are required for pyroptosis. These findings reveal the mechanism for pyroptosis and provide insights into the roles of the gasdermin family in necrosis, immunity and diseases.
Dietary fat promotes pathological insulin resistance through chronic inflammation. The inactivation of inflammatory proteins produced by macrophages improves diet-induced diabetes, but how ...nutrient-dense diets induce diabetes is unknown. Membrane lipids affect the innate immune response, which requires domains that influence high-fat-diet-induced chronic inflammation and alter cell function based on phospholipid composition. Endogenous fatty acid synthesis, mediated by fatty acid synthase (FAS), affects membrane composition. Here we show that macrophage FAS is indispensable for diet-induced inflammation. Deleting Fasn in macrophages prevents diet-induced insulin resistance, recruitment of macrophages to adipose tissue and chronic inflammation in mice. We found that FAS deficiency alters membrane order and composition, impairing the retention of plasma membrane cholesterol and disrupting Rho GTPase trafficking-a process required for cell adhesion, migration and activation. Expression of a constitutively active Rho GTPase, however, restored inflammatory signalling. Exogenous palmitate was partitioned to different pools from endogenous lipids and did not rescue inflammatory signalling. However, exogenous cholesterol, as well as other planar sterols, did rescue signalling, with cholesterol restoring FAS-induced perturbations in membrane order. Our results show that the production of endogenous fat in macrophages is necessary for the development of exogenous-fat-induced insulin resistance through the creation of a receptive environment at the plasma membrane for the assembly of cholesterol-dependent signalling networks.
Most antifungal peptides (AFPs), if not all, have membrane activity, while some also have alternative targets. Fungal membranes share many characteristics with mammalian membranes with only a few ...differences, such as differences in sphingolipids, phosphatidylinositol (PI) content and the main sterol is ergosterol. Fungal membranes are also more negative and a better target for cationic AFPs. Targeting just the fungal membrane lipids such as phosphatidylinositol and/or ergosterol by AFPs often translates into mammalian cell toxicity. Conversely, a specific AFP target in the fungal pathogen, such as glucosylceramide, mannosyldiinositol phosphorylceramide or a fungal protein target translates into high pathogen selectivity. However, a lower target concentration, absence or change in the specific fungal target can naturally lead to resistance, although such resistance in turn could result in reduced pathogen virulence. The question is then to be or not to be membrane active - what is the best choice for a successful AFP? In this review we deliberate on this question by focusing on the recent advances in our knowledge on how natural AFPs target fungi.
•Is membrane activity the best option for antifungal peptides (AFPs)?•Fungal membranes have unique lipids that are targeted by AFPs.•Targeting just fungal membrane lipids can translate into mammalian cell toxicity.•If an AFP only has one specific fungal target there is some potential for resistance.•Many AFPs target the fungal membrane, as well as other fungal components/systems.
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