GLUT4 localisation with the plasma membrane is unaffected by an increase in plasma free fatty acid availability.

BACKGROUND: Insulin-stimulated glucose uptake into skeletal muscle occurs via translocation of GLUT4 from intracellular storage vesicles to the plasma membrane. Elevated free fatty acid (FFA) availability via a lipid infusion reduces glucose disposal, but this occurs in the absence of impaired proximal insulin signalling. Whether GLUT4 localisation to the plasma membrane is subsequently affected by elevated FFA availability is not known. METHODS: Trained (n = 11) and sedentary (n = 10) individuals, matched for age, sex and body mass index, received either a 6 h lipid or glycerol infusion in the setting of a concurrent hyperinsulinaemic-euglycaemic clamp. Sequential muscle biopsies (0, 2 and 6 h) were analysed for GLUT4 membrane localisation and microvesicle size and distribution using immunofluorescence microscopy. RESULTS: At baseline, trained individuals had more small GLUT4 spots at the plasma membrane, whereas sedentary individuals had larger GLUT4 spots. GLUT4 localisation with the plasma membrane increased at 2 h (P = 0.04) of the hyperinsulinemic-euglycemic clamp, and remained elevated until 6 h, with no differences between groups or infusion type. The number of GLUT4 spots was unchanged at 2 h of infusion. However, from 2 to 6 h there was a decrease in the number of small GLUT4 spots at the plasma membrane (P = 0.047), with no differences between groups or infusion type. CONCLUSION: GLUT4 localisation with the plasma membrane increases during a hyperinsulinemic-euglycemic clamp, but this is not altered by elevated FFA availability. GLUT4 appears to disperse from small GLUT4 clusters located at the plasma membrane to support glucose uptake during a hyperinsulinaemic-euglycaemic clamp.

Computational and experimental identification of keystone interactions in Ebola virus matrix protein VP40 dimer formation.

The Ebola virus (EBOV) is a lipid-enveloped virus with a negative sense RNA genome that can cause severe and often fatal viral hemorrhagic fever. The assembly and budding of EBOV is regulated by the matrix protein, VP40, which is a peripheral protein that associates with anionic lipids at the inner leaflet of the plasma membrane. VP40 is sufficient to form virus-like particles (VLPs) from cells, which are nearly indistinguishable from authentic virions. Due to the restrictions of studying EBOV in BSL-4 facilities, VP40 has served as a surrogate in cellular studies to examine the EBOV assembly and budding process from the host cell plasma membrane. VP40 is a dimer where inhibition of dimer formation halts budding and formation of new VLPs as well as VP40 localization to the plasma membrane inner leaflet. To better understand VP40 dimer stability and critical amino acids to VP40 dimer formation, we integrated computational approaches with experimental validation. Site saturation/alanine scanning calculation, combined with molecular mechanics-based generalized Born with Poisson-Boltzmann surface area (MM-GB/PBSA) method and molecular dynamics simulations were used to predict the energetic contribution of amino acids to VP40 dimer stability and the hydrogen bonding network across the dimer interface. These studies revealed several previously unknown interactions and critical residues predicted to impact VP40 dimer formation. In vitro and cellular studies were then pursued for a subset of VP40 mutations demonstrating reduction in dimer formation (in vitro) or plasma membrane localization (in cells). Together, the computational and experimental approaches revealed critical residues for VP40 dimer stability in an alpha-helical interface (between residues 106-117) as well as in a loop region (between residues 52-61) below this alpha-helical region. This study sheds light on the structural origins of VP40 dimer formation and may inform the design of a small molecule that can disrupt VP40 dimer stability.

Tumor-associated antigen-specific cell imaging based on upconversion luminescence and nucleic acid rolling circle amplification.

Tumor-associated antigen (TAA)-based diagnosis has gained prominence for early tumor screening, treatment monitoring, prognostic assessment, and minimal residual disease detection. However, limitations such as low sensitivity and difficulty in extracting non-specific binding membrane proteins still exist in traditional detection methods. Upconversion luminescence (UCL) exhibits unique physical and chemical properties under wavelength near-infrared light excitation. Rolling circle amplification (RCA) is an efficient DNA amplification technique with amplification factors as high as 105. Therefore, the above two excellent techniques can be employed for highly accurate imaging analysis of tumor cells. Herein, we developed a novel nanoplatform for TAA-specific cell imaging based on UCL and RCA technology. An aptamer-primer complex selectively binds to Mucin 1 (MUC1), one of TAA on cell surface, to trigger RCA reaction, generating a large number of repetitive sequences. These sequences provide lots of binding sites for complementary signal probes, producing UCL from lanthanide-doped upconversion nanoparticles (UCNPs) after releasing quencher group. The experimental results demonstrate the specific attachment of upconversion nanomaterials to cancer cells which express a high level of MUC1, indicating the potential of UCNPs and RCA in tumor imaging.

Meet the authors: Lori Emert-Sedlak and Tom Smithgall.

In an interview with Samantha Nelson, a scientific editor of Cell Chemical Biology, the first and corresponding authors of the research article entitled "PROTAC-mediated degradation of HIV-1 Nef efficiently restores cell-surface CD4 and MHC-I expression and blocks HIV-1 replication" share insights on their paper and life as scientists.

A comprehensive N-glycome map of porcine sperm membrane before and after capacitation.

Mapping the N-glycome of porcine sperm before and after sperm capacitation is important for understanding the rearrangement of glycoconjugates during capacitation. In this work, we characterized the N-glycome on the membranes of 18 pairs of fresh porcine sperm before capacitation and porcine sperm after capacitation by MALDI-MS (Matrix-assisted laser desorption/ionization-mass spectrometry). A total of 377 N-glycans were detected and a comprehensive N-glycome map of porcine sperm membranes before and after capacitation was generated, which presents the largest N-glycome dataset of porcine sperm cell membranes. Statistical analysis revealed a significantly higher level of high mannose glycosylation and a significantly lower level of fucosylation, galactosylation, and α-2,6-NeuAc after capacitation, which is further verified by flow cytometry and lectin blotting. This research reveals new insights into the relationship between N-glycosylation variations and sperm capacitation, including the underlying mechanisms of the capacitation process.

Endocytic vesicles act as vehicles for glucose uptake in response to growth factor stimulation.

Glycolysis is a fundamental cellular process, yet its regulatory mechanisms remain incompletely understood. Here, we show that a subset of glucose transporter 1 (GLUT1/SLC2A1) co-endocytoses with platelet-derived growth factor (PDGF) receptor (PDGFR) upon PDGF-stimulation. Furthermore, multiple glycolytic enzymes localize to these endocytosed PDGFR/GLUT1-containing vesicles adjacent to mitochondria. Contrary to current models, which emphasize the importance of glucose transporters on the cell surface, we find that PDGF-stimulated glucose uptake depends on receptor/transporter endocytosis. Our results suggest that growth factors generate glucose-loaded endocytic vesicles that deliver glucose to the glycolytic machinery in proximity to mitochondria, and argue for a new layer of regulation for glycolytic control governed by cellular membrane dynamics.

Stable Super-Resolution Imaging of Cell Membrane Nanoscale Subcompartment Dynamics with a Buffering Cyanine Dye.

Super-resolution fluorescence imaging is a crucial method for visualizing the dynamics of the cell membrane involved in various physiological and pathological processes. This requires bright fluorescent dyes with excellent photostability and labeling stability to enable long-term imaging. In this context, we introduce a buffering-strategy-based cyanine dye, SA-Cy5, designed to identify and label carbonic anhydrase IX (CA IX) located in the cell membrane. The unique feature of SA-Cy5 lies in its ability to overcome photobleaching. When the dye on the cell membrane undergoes photobleaching, it is rapidly replaced by an intact probe from the buffer pool outside the cell membrane. This dynamic replacement ensures that the fluorescence intensity on the cell membrane remains stable over time. Under the super-resolution structured illumination microscopy (SIM), the cell membrane can be continuously imaged for 60 min with a time resolution of 20 s. This extended imaging period allows for the observation of substructural dynamics of the cell membrane, including the growth and fusion of filamentous pseudopodia and the fusion of vesicles. Additionally, this buffering strategy introduces a novel approach to address the issue of poor photostability associated with the cyanine dyes.

Functional Plasmonic Microscope: Characterizing the Metabolic Activity of Single Cells via Sub-nm Membrane Fluctuations.

Metabolic abnormalities are at the center of many diseases, and the capability to film and quantify the metabolic activities of a single cell is important for understanding the heterogeneities in these abnormalities. In this paper, a functional plasmonic microscope (FPM) is used to image and measure metabolic activities without fluorescent labels at a single-cell level. The FPM can accurately image and quantify the subnanometer membrane fluctuations with a spatial resolution of 0.5 µm in real time. These active cell membrane fluctuations are caused by metabolic activities across the cell membrane. A three-dimensional (3D) morphology of the bottom cell membrane was imaged and reconstructed with FPM to illustrate the capability of the microscope for cell membrane characterization. Then, the subnanometer cell membrane fluctuations of single cells were imaged and quantified with the FPM using HeLa cells. Cell metabolic heterogeneity is analyzed based on membrane fluctuations of each individual cell that is exposed to similar environmental conditions. In addition, we demonstrated that the FPM could be used to evaluate the therapeutic responses of metabolic inhibitors (glycolysis pathway inhibitor STF 31) on a single-cell level. The result showed that the metabolic activities significantly decrease over time, but the nature of this response varies, depicting cell heterogeneity. A low-concentration dose showed a reduced fluctuation frequency with consistent fluctuation amplitudes, while the high-concentration dose showcased a decreasing trend in both cases. These results have demonstrated the capabilities of the functional plasmonic microscope to measure and quantify metabolic activities for drug discovery.

Blood cellular membrane-coated Au/polydopamine nanoparticle-targeted NIR-II antibacterial therapy.

Bacterial infections are the primary causes of infectious diseases in humans. In recent years, the abuse of antibiotics has led to the widespread enhancement of bacterial resistance. Concerns have been raised about the identification of a common treatment platform for bacterial infections. In this study, a composite nanomaterial was used for near-infrared II (NIR-II) photothermal antibacterial treatment. Red blood cell membrane was peeled and coated onto the surface of the Au/polydopamine nanoparticle-containing aptamer. The composite nanomaterials based on Au/polydopamine exhibit highest photothermal conversion capability. Moreover, these assembled nanoparticles can quickly enter the body's circular system with a specific capability to recognise bacteria. In vivo experiments demonstrated that the composites could kill bacteria from infected blood while significantly reducing the level of bacteria in various organs. Such assemblies offer a paradigm for the treatment of bacterial infections caused by the side effects of antibiotics.

Lipid scrambling is a general feature of protein insertases.

Glycerophospholipids are synthesized primarily in the cytosolic leaflet of the endoplasmic reticulum (ER) membrane and must be equilibrated between bilayer leaflets to allow the ER and membranes derived from it to grow. Lipid equilibration is facilitated by integral membrane proteins called "scramblases." These proteins feature a hydrophilic groove allowing the polar heads of lipids to traverse the hydrophobic membrane interior, similar to a credit card moving through a reader. Nevertheless, despite their fundamental role in membrane expansion and dynamics, the identity of most scramblases has remained elusive. Here, combining biochemical reconstitution and molecular dynamics simulations, we show that lipid scrambling is a general feature of protein insertases, integral membrane proteins which insert polypeptide chains into membranes of the ER and organelles disconnected from vesicle trafficking. Our data indicate that lipid scrambling occurs in the same hydrophilic channel through which protein insertion takes place and that scrambling is abolished in the presence of nascent polypeptide chains. We propose that protein insertases could have a so-far-overlooked role in membrane dynamics as scramblases.

The role of the AP-1 adaptor complex in outgoing and incoming membrane traffic.

The AP-1 adaptor complex is found in all eukaryotes, but it has been implicated in different pathways in different organisms. To look directly at AP-1 function, we generated stably transduced HeLa cells coexpressing tagged AP-1 and various tagged membrane proteins. Live cell imaging showed that AP-1 is recruited onto tubular carriers trafficking from the Golgi apparatus to the plasma membrane, as well as onto transferrin-containing early/recycling endosomes. Analysis of single AP-1 vesicles showed that they are a heterogeneous population, which starts to sequester cargo 30 min after exit from the ER. Vesicle capture showed that AP-1 vesicles contain transmembrane proteins found at the TGN and early/recycling endosomes, as well as lysosomal hydrolases, but very little of the anterograde adaptor GGA2. Together, our results support a model in which AP-1 retrieves proteins from post-Golgi compartments back to the TGN, analogous to COPI's role in the early secretory pathway. We propose that this is the function of AP-1 in all eukaryotes.

Molecular Dynamics Simulation of Permeation Through Connexin Channels.

Molecular dynamics (MD) simulations are a collection of computational tools that can be used to trace intermolecular interactions at the sub-nanometer level. They offer possibilities that are often unavailable to experimental methods, making MD an ideal complementary technique for the understanding a plethora of biological processes. Thanks to significant efforts by many groups of developers around the world, setting up and running MD simulations has become progressively simpler. However, simulating ionic permeation through membrane channels still presents significant caveats.MD simulations of connexin (Cx) hemichannels (HCs) are particularly problematic because HCs create wide pores in the plasma membrane, and the lateral sizes of the extracellular and intracellular regions are quite different. In this chapter, we provide a detailed instruction to perform MD simulations aimed at computationally modeling the permeation of inorganic ions and larger molecules through Cx HCs.

Purification, Reconstitution, and Functional Analysis of Connexin Hemichannels.

Connexins are the proteins that form the gap junction channels that are essential for cell-to-cell communication. These channels are formed by head-to-head docking of hemichannels (each from one of two adjacent cells). Free "undocked" hemichannels at the plasma membrane are mostly closed, although they are still important under physiological conditions. However, abnormal and sustained increase in hemichannel activity due to connexin mutations or acquired conditions can produce or contribute to cell damage. For example, mutations of Cx26, a connexin isoform, can increase hemichannel activity and cause deafness. Studies using purified isolated systems under well-controlled conditions are essential for a full understanding of molecular mechanisms of hemichannel function under normal conditions and in disease, and here, we present methodology for the expression, purification, and functional analysis of hemichannels formed by Cx26.

Evaluation of Connexin Hemichannel Activity In Vivo.

Connexin hemichannels (Cx HCs) are hexameric structures at the cell plasma membrane, whose function as membrane transport proteins allows for the passive flow of small hydrophilic molecules and ions (≤1 kDa) between the cytosol and the extracellular environment. Activation of Cx HCs is highly dependent on pathological conditions. HC activity provokes changes in the microenvironment, inducing the dissemination of signaling molecules in both an autocrine and paracrine manner. Given the elicitation of a variety of signaling pathways, and assortment of Cx species and dispersion throughout the body, Cx HCs have been implicated in a range of processes such as cell proliferation, differentiation, cell death, and tissue modeling and remodeling. While studying the expression and localization of Cx HCs can be done using traditional laboratory techniques, such as immunoblot analysis, measuring the functionality/activity of the HCs requires a more explicit methodology and is essential for determining Cx-mediated physiological changes. The study of Cx HC function/activity has focused mainly on in vitro measurements through electrophysiological characterization or, more commonly, using HC-permeable dye uptake studies. Here, we describe the use of dye uptake to measure Cx HC activity in vivo using mechanically stimulated osteocytic Cx43 HCs with Evans blue dye as our model.

Measuring Connexin Hemichannel Opening in Response to an InsP3-Mediated Cytosolic Ca2+ Increase.

The opening of connexin hemichannels (HCs) expressed at the plasma membrane of mammalian cells is regulated by a number of physiological parameters, including extracellular and intracellular Ca2+ ions. Submicromolar variations of the cytosolic Ca2+ concentration ([Ca2+]c) are per se sufficient to trigger extracellular bursts of messenger molecules through connexin HCs, thus mediating paracrine signaling. In this chapter, we present a quantitative method to measure the opening dynamics of connexin HCs expressed in a single HeLa cell upon stimulation by a canonical InsP3-mediated [Ca2+]c transient. The protocol relies on a combination of Ca2+ imaging and patch-clamp techniques. The insights gained from our method are expected to make a significant contribution to understanding the structure-function relationship of connexin HCs. The protocol is also suitable to screen candidate therapeutic compounds to treat connexin-related diseases linked to HC dysfunction.

Quantitative measurement of cell-surface displayed proteins based on split-GFP assembly.

BACKGROUND: Microbial cell surface display technology allows immobilizing proteins on the cell surface by fusing them to anchoring motifs, thereby endowing the cells with diverse functionalities. However, the assessment of successful protein display and the quantification of displayed proteins remain challenging. The green fluorescent protein (GFP) can be split into two non-fluorescent fragments, while they spontaneously assemble and emit fluorescence when brought together through complementation. Based on split-GFP assembly, we aim to: (1) confirm the success display of passenger proteins, (2) quantify the number of passenger proteins displayed on individual cells. RESULTS: In this study, we propose two innovative methods based on split-green fluorescent protein (split-GFP), named GFP1-10/GFP11 and GFP1-9/GFP10-11 assembly, for the purpose of confirming successful display and quantifying the number of proteins displayed on individual cells. We evaluated the display efficiency of SUMO and ubiquitin using different anchor proteins to demonstrate the feasibility of the two split-GFP assembly systems. To measure the display efficiency of functional proteins, laccase expression was measured using the split-GFP assembly system by co-displaying GFP11 or GFP10-11 tags, respectively. CONCLUSIONS: Our study provides two split-GFP based methods that enable qualitative and quantitative analyses of individual cell display efficiency with a simple workflow, thus facilitating further comprehensive investigations into microbial cell surface display technology. Both split-GFP assembly systems offer a one-step procedure with minimal cost, simplifying the fluorescence analysis of surface-displaying cells.

Dynamic Mode Decomposition of Multiphoton and Stimulated Emission Depletion Microscopy Data for Analysis of Fluorescent Probes in Cellular Membranes.

An analysis of the membrane organization and intracellular trafficking of lipids often relies on multiphoton (MP) and super-resolution microscopy of fluorescent lipid probes. A disadvantage of particularly intrinsically fluorescent lipid probes, such as the cholesterol and ergosterol analogue, dehydroergosterol (DHE), is their low MP absorption cross-section, resulting in a low signal-to-noise ratio (SNR) in live-cell imaging. Stimulated emission depletion (STED) microscopy of membrane probes like Nile Red enables one to resolve membrane features beyond the diffraction limit but exposes the sample to a lot of excitation light and suffers from a low SNR and photobleaching. Here, dynamic mode decomposition (DMD) and its variant, higher-order DMD (HoDMD), are applied to efficiently reconstruct and denoise the MP and STED microscopy data of lipid probes, allowing for an improved visualization of the membranes in cells. HoDMD also allows us to decompose and reconstruct two-photon polarimetry images of TopFluor-cholesterol in model and cellular membranes. Finally, DMD is shown to not only reconstruct and denoise 3D-STED image stacks of Nile Red-labeled cells but also to predict unseen image frames, thereby allowing for interpolation images along the optical axis. This important feature of DMD can be used to reduce the number of image acquisitions, thereby minimizing the light exposure of biological samples without compromising image quality. Thus, DMD as a computational tool enables gentler live-cell imaging of fluorescent probes in cellular membranes by MP and STED microscopy.

Impact of TRP Channels on Extracellular Matrix Remodeling: Focus on TRPV4 and Collagen.

Disturbed remodeling of the extracellular matrix (ECM) is frequently observed in several high-prevalence pathologies that include fibrotic diseases of organs such as the heart, lung, periodontium, liver, and the stiffening of the ECM surrounding invasive cancers. In many of these lesions, matrix remodeling mediated by fibroblasts is dysregulated, in part by alterations to the regulatory and effector systems that synthesize and degrade collagen, and by alterations to the functions of the integrin-based adhesions that normally mediate mechanical remodeling of collagen fibrils. Cell-matrix adhesions containing collagen-binding integrins are enriched with regulatory and effector systems that initiate localized remodeling of pericellular collagen fibrils to maintain ECM homeostasis. A large cadre of regulatory molecules is enriched in cell-matrix adhesions that affect ECM remodeling through synthesis, degradation, and contraction of collagen fibrils. One of these regulatory molecules is Transient Receptor Potential Vanilloid-type 4 (TRPV4), a mechanically sensitive, Ca2+-permeable plasma membrane channel that regulates collagen remodeling. The gating of Ca2+ across the plasma membrane by TRPV4 and the consequent generation of intracellular Ca2+ signals affect several processes that determine the structural and mechanical properties of collagen-rich ECM. These processes include the synthesis of new collagen fibrils, tractional remodeling by contractile forces, and collagenolysis. While the specific mechanisms by which TRPV4 contributes to matrix remodeling are not well-defined, it is known that TRPV4 is activated by mechanical forces transmitted through collagen adhesion receptors. Here, we consider how TRPV4 expression and function contribute to physiological and pathological collagen remodeling and are associated with collagen adhesions. Over the long-term, an improved understanding of how TRPV4 regulates collagen remodeling could pave the way for new approaches to manage fibrotic lesions.

Generating Concentration Gradients across Membranes for Molecular Dynamics Simulations of Periodic Systems.

The plasma membrane forms the boundary between a living entity and its environment and acts as a barrier to permeation and flow of substances. Several computational means of calculating permeability have been implemented for molecular dynamics (MD) simulations-based approaches. Except for double bilayer systems, most permeability studies have been performed under equilibrium conditions, in large part due to the challenges associated with creating concentration gradients in simulations utilizing periodic boundary conditions. To enhance the scientific understanding of permeation and complement the existing computational means of characterizing membrane permeability, we developed a non-equilibrium method that enables the generation and maintenance of steady-state gradients in MD simulations. We utilize PBCs advantageously by imposing a directional bias to the motion of permeants so that their crossing of the boundary replenishes the gradient, like a previous study on ions. Under these conditions, a net flow of permeants across membranes may be observed to determine bulk permeability by a direct application of J=PΔc. In the present study, we explore the results of its application to an exemplary O2 and POPC bilayer system, demonstrating accurate and precise permeability measurements. In addition, we illustrate the impact of permeant concentration and the choice of thermostat on the permeability. Moreover, we demonstrate that energetics of permeation can be closely examined by the dissipation of the gradient across the membrane to gain nuanced insights into the thermodynamics of permeability.

Identification of MATE Family and Characterization of GmMATE13 and GmMATE75 in Soybean's Response to Aluminum Stress.

The multidrug and toxic compound extrusion (MATE) proteins are coding by a secondary transporter gene family, and have been identified to participate in the modulation of organic acid exudation for aluminum (Al) resistance. The soybean variety Glycine max "Tamba" (TBS) exhibits high Al tolerance. The expression patterns of MATE genes in response to Al stress in TBS and their specific functions in the context of Al stress remain elusive. In this study, 124 MATE genes were identified from the soybean genome. The RNA-Seq results revealed significant upregulation of GmMATE13 and GmMATE75 in TBS upon exposure to high-dose Al3+ treatment and both genes demonstrated sequence homology to citrate transporters of other plants. Subcellular localization showed that both proteins were located in the cell membrane. Transgenic complementation experiments of Arabidopsis mutants, atmate, with GmMATE13 or GmMATE75 genes enhanced the Al tolerance of the plant due to citrate secretion. Taken together, this study identified GmMATE13 and GmMATE75 as citrate transporter genes in TBS, which could improve citrate secretion and enhance Al tolerance. Our findings provide genetic resources for the development of plant varieties that are resistant to Al toxicity.