Machine learning–driven multiscale modeling reveals lipid-dependent dynamics of RAS signaling proteins

RAS is a signaling protein associated with the cell membrane that is mutated in up to 30% of human cancers. RAS signaling has been proposed to be regulated by dynamic heterogeneity of the cell membrane. Investigating such a mechanism requires near-atomistic detail at macroscopic temporal and spatial scales, which is not possible with conventional computational or experimental techniques. We demonstrate here a multiscale simulation infrastructure that uses machine learning to create a scale-bridging ensemble of over 100,000 simulations of active wild-type KRAS on a complex, asymmetric membrane. Initialized and validated with experimental data (including a new structure of active wild-type KRAS), these simulations represent a substantial advance in the ability to characterize RAS-membrane biology. We report distinctive patterns of local lipid composition that correlate with interfacially promiscuous RAS multimerization. These lipid fingerprints are coupled to RAS dynamics, predicted to influence effector binding, and therefore may be a mechanism for regulating cell signaling cascades.

Interphase Surface Stability in Liquid-Liquid Membrane Contactors Based on Track-Etched Membranes

A promising solution for the implementation of extraction processes is liquid–liquid membrane contactors. The transfer of the target component from one immiscible liquid to another is carried out inside membrane pores. For the first time, highly asymmetric track-etched membranes made of polyethylene terephthalate (PET) of the same thickness but with different pore diameters (12.5–19 nm on one side and hundreds of nanometers on the other side) were studied in the liquid–liquid membrane contactor. For analysis of the liquid–liquid interface stability, two systems widely diverging in the interfacial tension value were used: water–pentanol and water–hexadecane. The interface stability was investigated depending on the following process parameters: the porous structure, the location of the asymmetric membrane in the contactor, the velocities of liquids, and the pressure drop between them. It was shown that the stability of the interface increases with decreasing pore size. Furthermore, it is preferable to supply the aqueous phase from the side of the asymmetric membrane with the larger pore size. The asymmetry of the porous structure of the membrane makes it possible to increase the range of pressure drop values between the phases by at least two times (from 5 to 10 kPa), which does not lead to mutual dispersion of the liquids. The liquid–liquid contactor based on the asymmetric track-etched membranes allows for the extraction of impurities from the organic phase into the aqueous phase by using a 1% solution of acetone in hexadecane as an example.

Microstructure adjustment of an asymmetric ceramic membrane with high permeation performance

An asymmetric alumina ceramic membrane was prepared by secondary dip coating. The influence of different dispersants and dip coating parameters on the microstructure of the membrane separation layer was explored. Meanwhile, the pure water fluxes of the membranes with various microstructures were also studied. The results show that a separation layer with a defect-free thickness of 16.5 μm and high surface flatness can be obtained when using polycarboxylate as a dispersant and twice dip coating within 2 s + 1 s and the pure water flux of an asymmetric membrane up to 1153 L × m-2 × h-1 × bar-1. The present work provides a simple and effective method for controlling the morphology and permeation performance of an asymmetric alumina membrane.

Live imaging and conditional disruption of native vertebrate planar cell polarity

Tissue-wide coordination of polarized cytoskeletal organization and cell behaviour, critical for organ morphogenesis and tumour progression, is controlled by asymmetric membrane localization of non-canonical Wnt/planar cell polarity (PCP) signalling components. Understanding the dynamic regulation of PCP thus requires visualization of these core proteins. In vertebrates, immunohistochemical studies have provided snapshots of PCP within tissues while exogenous, fluorescently-tagged reporters have been introduced for live imaging of cell polarity. However, the functionality and spatiotemporal relevance of static and exogenous PCP readouts remain uncertain. Here we fluorescently tag an endogenous core PCP component, Vangl2, in zebrafish. We report on the authentic regulation of vertebrate PCP, through live imaging of this native sfGFP-Vangl2 protein during embryogenesis. We couple sfGFP-Vangl2 with conditional zGrad GFP-nanobody degradation methodologies for tissue-specific interrogation of PCP function. Together, our studies provide crucial insights into the establishment and maintenance of vertebrate PCP and create a powerful experimental paradigm for future investigations.

Insights into the bilayer-mediated toppling mechanism of a folate-specific ECF transporter by cryo-EM

Energy-coupling factor (ECF)–type transporters are small, asymmetric membrane protein complexes (∼115 kDa) that consist of a membrane-embedded, substrate-binding protein (S component) and a tripartite ATP-hydrolyzing module (ECF module). They import micronutrients into bacterial cells and have been proposed to use a highly unusual transport mechanism, in which the substrate is dragged across the membrane by a toppling motion of the S component. However, it remains unclear how the lipid bilayer could accommodate such a movement. Here, we used cryogenic electron microscopy at 200 kV to determine structures of a folate-specific ECF transporter in lipid nanodiscs and detergent micelles at 2.7- and 3.4-Å resolution, respectively. The structures reveal an irregularly shaped bilayer environment around the membrane-embedded complex and suggest that toppling of the S component is facilitated by protein-induced membrane deformations. In this way, structural remodeling of the lipid bilayer environment is exploited to guide the transport process.

Ultrastructural plasma membrane asymmetries in tension and curvature promote yeast cell fusion

Cell–cell fusion is central for sexual reproduction, and generally involves gametes of different shapes and sizes. In walled fission yeast Schizosaccharomyces pombe, the fusion of h+ and h− isogametes requires the fusion focus, an actin structure that concentrates glucanase-containing vesicles for cell wall digestion. Here, we present a quantitative correlative light and electron microscopy (CLEM) tomographic dataset of the fusion site, which reveals the fusion focus ultrastructure. Unexpectedly, gametes show marked asymmetries: a taut, convex plasma membrane of h− cells progressively protrudes into a more slack, wavy plasma membrane of h+ cells. Asymmetries are relaxed upon fusion, with observations of ramified fusion pores. h+ cells have a higher exo-/endocytosis ratio than h− cells, and local reduction in exocytosis strongly diminishes membrane waviness. Reciprocally, turgor pressure reduction specifically in h− cells impedes their protrusions into h+ cells and delays cell fusion. We hypothesize that asymmetric membrane conformations, due to differential turgor pressure and exocytosis/endocytosis ratios between mating types, favor cell–cell fusion.