The M protein of coronavirus plays a central role in virus assembly, turning cellular membranes into workshops where virus and host factors come together to make new virus particles. We investigated ...how M structure and organization is related to virus shape and size using cryo-electron microscopy, tomography and statistical analysis. We present evidence that suggests M can adopt two conformations and that membrane curvature is regulated by one M conformer. Elongated M protein is associated with rigidity, clusters of spikes and a relatively narrow range of membrane curvature. In contrast, compact M protein is associated with flexibility and low spike density. Analysis of several types of virus-like particles and virions revealed that S protein, N protein and genomic RNA each help to regulate virion size and variation, presumably through interactions with M. These findings provide insight into how M protein functions to promote virus assembly.
Abstract
In the course of cancer progression tumor cells undergo morphological changes that lead to increased motility and invasiveness thus promoting formation of metastases. This process called ...epithelial to mesenchymal transition (EMT) is triggered by transforming growth factor (TGFβ) but for gaining the full invasive potential an interplay between signaling of TGFβ and Ras GTPases is required. Ras proteins possess a lipidated domain that mediates Ras association with the plasma membrane, which is essential for Ras biological functions. Type and number of the lipid anchors are the main difference among three Ras variants—H-ras, N-ras and K-ras. The lipid anchors determine membrane partitioning of lipidated proteins into membrane areas of specific physico-chemical properties and curvature. In this study, we investigated the effect of TGFβ treatment on the subcellular localization of H-ras and K-ras. We show that TGFβ increases positive plasma membrane curvature, which is subsequently sensed by H-ras, leading to its elevated plasma membrane localization and activation. This observation suggests the existence of a novel positive feedback loop whereby the increased level of plasma membrane curvature during TGFβ induced EMT attracts more Ras molecules to the plasma membrane resulting in increased Ras activity which in turn promotes further EMT and thus ultimately enables the acquisition of full invasive potential.
Irradiated metallic nanoparticles hold great promise as heat transducers in photothermal applications such as drug delivery assays or photothermal therapy. We quantify the temperature increase of ...individual gold nanoparticles trapped in three dimensions near lipid vesicles exhibiting temperature sensitive permeability. The surface temperature can increase by hundreds of degrees Celsius even at moderate laser powers. Also, there are significant differences of the heat profiles in two-dimensional and three-dimensional trapping assays.
Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction for both types of proteins is mediated by 11 amino acid repeats that form ...amphipathic helices. This similarity suggests that synucleins and apolipoproteins might have comparable effects on lipid membranes, but this has not been shown directly. Here, we find that α-synuclein, β-synuclein, and apolipoprotein A-1 have the conserved functional ability to induce membrane curvature and to convert large vesicles into highly curved membrane tubules and vesicles. The resulting structures are morphologically similar to those generated by amphiphysin, a curvature-inducing protein involved in endocytosis. Unlike amphiphysin, however, synucleins and apolipoproteins do not require any scaffolding domains and curvature induction is mediated by the membrane insertion and wedging of amphipathic helices alone. Moreover, we frequently observed that α-synuclein caused membrane structures that had the appearance of nascent budding vesicles. The ability to function as a minimal machinery for vesicle budding agrees well with recent findings that α-synuclein plays a role in vesicle trafficking and enhances endocytosis. Induction of membrane curvature must be under strict regulation in vivo; however, as we find it can also cause disruption of membrane integrity. Because the degree of membrane curvature induction depends on the concerted action of multiple proteins, controlling the local protein density of tubulating proteins may be important. How cellular safeguarding mechanisms prevent such potentially toxic events and whether they go awry in disease remains to be determined.
The importance of curvature as a structural feature of biological membranes has been recognized for many years and has fascinated scientists from a wide range of different backgrounds. On the one ...hand, changes in membrane morphology are involved in a plethora of phenomena involving the plasma membrane of eukaryotic cells, including endo- and exocytosis, phagocytosis and filopodia formation. On the other hand, a multitude of intracellular processes at the level of organelles rely on generation, modulation, and maintenance of membrane curvature to maintain the organelle shape and functionality. The contribution of biophysicists and biologists is essential for shedding light on the mechanistic understanding and quantification of these processes. Given the vast complexity of phenomena and mechanisms involved in the coupling between membrane shape and function, it is not always clear in what direction to advance to eventually arrive at an exhaustive understanding of this important research area. The 2018 Biomembrane Curvature and Remodeling Roadmap of Journal of Physics D: Applied Physics addresses this need for clarity and is intended to provide guidance both for students who have just entered the field as well as established scientists who would like to improve their orientation within this fascinating area.
Liposomes, or vesicles, have been studied extensively both as models of biological membranes and as drug delivery vehicles. Typically it is assumed that all liposomes within the same preparation are ...identical. Here by employing pairs of fluorescently labeled lipids we demonstrated an up to 10-fold variation in the relative lipid composition of individual liposomes with diameters between 50 nm and 15 μm. Since the physicochemical properties of liposomes are directly linked to their composition, a direct consequence of compositional inhomogeneities is a polydispersity in the properties of the individual liposomes in an ensemble.
The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most ...abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of “open” clathrin-coated pits (CCPs) to “necked”/“closed” CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake.
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•CALM loss increases size and frequency of early endocytic clathrin-coated structures•Depletion of CALM slows endocytic clathrin-coated pit maturation and endocytic rate•CALM possesses an N-terminal, membrane-curvature-sensing/driving amphipathic helix•Clathrin-coated pit maturation is regulated by CALM’s N-terminal amphipathic helix
Miller et al. demonstrate that depletion of the abundant endocytic protein CALM increases the diameter and percentage of early endocytic clathrin-coated structures (CCSs) while delaying CCS maturation and reducing endocytic rates. CALM’s ability to influence these CCS properties depends on a membrane-inserting amphipathic helix, which senses and promotes membrane curvature.
The targeted spatial organization (sorting) of Gprotein-coupled receptors (GPCRs) is essential for their biological function and often takes place in highly curved membrane compartments such as ...filopodia, endocytic pits, trafficking vesicles or endosome tubules. However, the influence of geometrical membrane curvature on GPCR sorting remains unknown. Here we used fluorescence imaging to establish a quantitative correlation between membrane curvature and sorting of three prototypic class A GPCRs (the neuropeptide Y receptor Y2, the β
adrenergic receptor and the β
adrenergic receptor) in living cells. Fitting of a thermodynamic model to the data enabled us to quantify how sorting is mediated by an energetic drive to match receptor shape and membrane curvature. Curvature-dependent sorting was regulated by ligands in a specific manner. We anticipate that this curvature-dependent biomechanical coupling mechanism contributes to the sorting, trafficking and function of transmembrane proteins in general.
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