Nanoscale Membrane Research Articles

Extracting nanoscale membrane morphology from single-molecule localizations

Membrane surface reconstruction at the nanometer scale is required for understanding mechanisms of subcellular shape change. This historically has been the domain of electron microscopy, but extraction of surfaces from specific labels is a difficult task in this imaging modality. Existing methods for extracting surfaces from fluorescence microscopy have poor resolution or require high-quality super-resolution data that are manually cleaned and curated. Here, we present NanoWrap, a new method for extracting surfaces from generalized single-molecule localization microscopy data. This makes it possible to study the shape of specifically labeled membranous structures inside cells. We validate NanoWrap using simulations and demonstrate its reconstruction capabilities on single-molecule localization microscopy data of the endoplasmic reticulum and mitochondria. NanoWrap is implemented in the open-source Python Microscopy Environment.

Advances in Therapeutic Applications of Extracellular Vesicles.

Extracellular vesicles (EVs) are nanoscale bilayer phospholipid membrane vesicles released by cells. Contained large molecules such as nucleic acid, protein, and lipid, EVs are an integral part of cell communication. The contents of EVs vary based on the cell source and play an important role in both pathological and physiological conditions. EVs can be used as drugs or targets in disease treatment, and changes in the contents of EVs can indicate the progression of diseases. In recent years, with the continuous exploration of the structure, characteristics, and functions of EVs, the potential of engineered EVs for drug delivery and therapy being constantly explored. This review provides a brief overview of the structure, characteristics and functions of EVs, summarizes the advanced application of EVs and outlook on the prospect of it. It is our hope that this review will increase understanding of the current development of medical applications of EVs and help us overcome future challenges.

Elucidating a fresh perspective on the interplay between exosomes and rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by chronic synovitis and the destruction of bones and joints. Exosomes are nanoscale lipid membrane vesicles originating from multivesicular bodies and are used as a vital means of intercellular communication. Both exosomes and the microbial community are essential in RA pathogenesis. Multiple types of exosomes from different origins have been demonstrated to have effects on various immune cells through distinct mechanisms in RA, which depend on the specific cargo carried by the exosomes. Tens of thousands of microorganisms exist in the human intestinal system. Microorganisms exert various physiological and pathological effects on the host directly or through their metabolites. Gut microbe-derived exosomes are being studied in the field of liver disease; however, information on their role in the context of RA is still limited. Gut microbe-derived exosomes may enhance autoimmunity by altering intestinal permeability and transporting cargo to the extraintestinal system. Therefore, we performed a comprehensive literature review on the latest progress on exosomes in RA and provided an outlook on the potential role of microbe-derived exosomes as emerging players in clinical and translational research on RA. This review aimed to provide a theoretical basis for developing new clinical targets for RA therapy.

Revealing the static and dynamic nanomechanical properties of diatom frustules—Nature's glass lace

Diatoms are single cell microalgae enclosed in silica exoskeletons (frustules) that provide inspiration for advanced hybrid nanostructure designs mimicking multi-scale porosity to achieve outstanding mechanical and optical properties. Interrogating the structure and properties of diatoms down to nanometer scale leads to breakthrough advances reported here in the nanomechanical characterization of Coscinodiscus oculus-iridis diatom pure silica frustules, as well as of air-dried and wet cells with organic content. Static and dynamic mode Atomic Force Microscopy (AFM) and in-SEM nanoindentation revealed the peculiarities of diatom response with separate contributions from material nanoscale behavior and membrane deformation of the entire valve. Significant differences in the nanomechanical properties of the different frustule layers were observed. Furthermore, the deformation response depends strongly on silica hydration and on the support from the internal organic content. The cyclic loading revealed that the average compliance of the silica frustule is 0.019 m/N and increases with increasing number of cycles. The structure–mechanical properties relationship has a direct impact on the vibrational properties of the frustule as a complex micrometer-sized mechanical system. Lessons from Nature’s nanostructuring of diatoms open up pathways to new generations of nano- and microdevices for electronic, electromechanical, photonic, liquid, energy storage, and other applications.

A Simple Radioassay to Detect Nanoscale Membrane Disruption.

Understanding the mechanisms and kinetics of membrane damage is of interest to researchers in several overlapping fields of biology. In this study, we describe the development and validation of a simple 32PO43- release radioassay used to track nanometer-scale damage to the bacterial cell membrane. Nanoscale membrane damage will result in the release of small cytoplasmic molecules, such as amino acids, sugars, and osmolytes. Our radioassay tracks the release of these molecules using the release of cytoplasmic 32PO43- as a proxy. Our assay can both detect 32PO43- release and track release kinetics in the order of minutes. We demonstrate the use of our radioassay using A. baumannii treated with colistin and Ω76: two agents known to cause membrane damage. Our assay tracks greater membrane damage in A. baumannii treated with both these agents, compared to an untreated control. Our assay fills a niche that is not covered by traditional 51Cr release radioassays and fluorescent staining techniques. Furthermore, our assay can potentially be used to track membrane damage in other membrane systems such as lipid vesicles, animal cells, and organelles.

Conformation-dependent diffusion properties of a transmembrane protein.

Fundamental cell physiology originates from conformational changes and diffusion dynamics of membrane proteins. However, studying the interplay between nanoscale protein conformations and membrane mechanics is inherently challenging in cell-based experiments. In this work, we investigate the diffusion dynamics of multidrug resistance ABC transporter BmrA as the function of protein conformations: apo (open) or post-hydrolytic (closed) upon in-vitro membrane reconstitution. We perform single-particle tracking measurements on BmrA reconstituted in giant unilamellar vesicles with controlled and varying membrane tensions. We observe a distinct conformation-dependent diffusion profile of BmrA which is inhibited upon increase in membrane tension. Further, experiments involving saturating levels of ATP indicate confinement of proteins to specific “hubs” indicating a new mechanism of protein clustering that competes with membrane tension, and protein enzymatic reactions. Our work gives insights into the intrinsic diffusion dynamics of an active membrane transporter, its interconnection with membrane mechanics, and conformational cycles; all possibly influencing the cell function.

Nanoscale membrane dynamics in chain asymmetric lipid bilayers.

Lipids that have two tails of different lengths are found throughout biomembranes in nature, yet the effects of this asymmetry on the membrane properties are not well understood, especially the membrane dynamics. Accommodating such a difference in tail length could significantly impact the membrane dynamics and elastic properties depending on how the longer tail is situated within the bilayer. To probe these effects, here we study the nanoscale bending fluctuations in model chain-asymmetric 14:0-18:0 PC (MSPC) and 18:0-14:0 PC (SMPC) lipid bilayers using neutron spin echo (NSE) spectroscopy. We find that despite the partial interdigitation that is known to persist in the fluid phase of these membranes, the collective fluctuations are enhanced on timescales of tens of nanoseconds, and the chain-asymmetric lipid bilayers are softer than an analogous chain-symmetric lipid with the same average number of carbons in the acyl tails, di-16:0 PC (DPPC). Quantitative comparison of the NSE results suggests that the enhanced bending fluctuations at the nanosecond timescales are consistent with experimental and computational studies that showed the compressibility moduli of chain-asymmetric lipid membranes are 20 % to 40% lower than chain-symmetric lipid membranes. These studies add to growing evidence that the partial interdigitation in chain-asymmetric lipid membranes in the fluid phase is highly dynamic and influences membrane dynamic processes from the molecular to mesoscopic length scales without significantly changing the bilayer thickness or area per lipid.

From Conventional to Microfluidic: Progress in Extracellular Vesicle Separation and Individual Characterization.

Extracellular vesicles (EVs) are nanoscale membrane vesicles, which contain a wide variety of cargo such as proteins, miRNAs, and lipids. A growing body of evidence suggests that EVs are promising biomarkers for disease diagnosis and therapeutic strategies. Although the excellent clinical value, their use in personalized healthcare practice is not yet feasible due to their highly heterogeneous nature. Taking the difficulty of isolation and the small size of EVs into account, the characterization of EVs at a single-particle level is both imperative and challenging. In a bid to address this critical point, more research has been directed into a microfluidic platform because of its inherent advantages in sensitivity, specificity, and throughput. This review discusses the biogenesis and heterogeneity of EVs and takes a broad view of state-of-the-art advances in microfluidics-based EV research, including not only EV separation, but also the single EV characterization of biophysical detection and biochemical analysis. To highlight the advantages of microfluidic techniques, conventional technologies are included for comparison. The current status of artificial intelligence (AI) for single EV characterization is then presented. Furthermore, the challenges and prospects of microfluidics and its combination with AI applications in single EV characterization are also discussed. In the foreseeable future, recent breakthroughs in microfluidic platforms are expected to pave the way for single EV analysis and improve applications for precision medicine.

Brominated lipid probes expose structural asymmetries in constricted membranes

Lipids in biological membranes are thought to be functionally organized, but few experimental tools can probe nanoscale membrane structure. Using brominated lipids as contrast probes for cryo-EM and a model ESCRT-III membrane-remodeling system composed of human CHMP1B and IST1, we observed leaflet-level and protein-localized structural lipid patterns within highly constricted and thinned membrane nanotubes. These nanotubes differed markedly from protein-free, flat bilayers in leaflet thickness, lipid diffusion rates and lipid compositional and conformational asymmetries. Simulations and cryo-EM imaging of brominated stearoyl-docosahexanenoyl-phosphocholine showed how a pair of phenylalanine residues scored the outer leaflet with a helical hydrophobic defect where polyunsaturated docosahexaenoyl tails accumulated at the bilayer surface. Combining cryo-EM of halogenated lipids with molecular dynamics thus enables new characterizations of the composition and structure of membranes on molecular length scales.

Nanoscale membrane curvature sorts lipid phases and alters lipid diffusion

The precise spatiotemporal control of nanoscale membrane shape and composition is the result of a complex interplay of individual and collective molecular behaviors. Here, we employed single-molecule localization microscopy and computational simulations to observe single-lipid diffusion and sorting in model membranes with varying compositions, phases, temperatures, and curvatures. Supported lipid bilayers were created over 50-nm-radius nanoparticles to mimic the size of naturally occurring membrane buds, such as endocytic pits and the formation of viral envelopes. The curved membranes recruited liquid-disordered lipid phases while altering the diffusion and sorting of tracer lipids. Disorder-preferring fluorescent lipids sorted to and experienced faster diffusion on the nanoscale curvature only when embedded in a membrane capable of sustaining lipid phase separation at low temperatures. The curvature-induced sorting and faster diffusion even occurred when the sample temperature was above the miscibility temperature of the planar membrane, implying that the nanoscale curvature could induce phase separation in otherwise homogeneous membranes. Further confirmation and understanding of these results are provided by continuum and coarse-grained molecular dynamics simulations with explicit and spontaneous curvature-phase coupling, respectively. The curvature-induced membrane compositional heterogeneity and altered dynamics were achieved only with a coupling of the curvature with a lipid phase separation. These cross-validating results demonstrate the complex interplay of lipid phases, molecular diffusion, and nanoscale membrane curvature that are critical for membrane functionality.

Clinical usage of dental stem cells and their derived extracellular vesicles.

Stem cell-based therapies remain at the forefront of tissue engineering and regenerative medicine because stem cells are a unique cell source with enormous potential to treat incurable diseases and even extend lifespans. The search for the best stem cell candidates continues to evolve and in recent years, dental stem cells have received significant attention due to their easy accessibility, high plasticity, and multipotential properties. Dental stem cells have been the subject of extensive research in both animal models and human clinical trials over the past two decades, and have demonstrated significant potential in ocular therapy, bone tissue engineering, and, of course, therapeutic applications in dentistry such as regenerative endodontics and periodontal tissue regeneration. These new sources of cells may be advantageous for cellular therapy and the advancement of regenerative medicine strategies, such as allogeneic transplantation or therapy with extracellular vesicles (EVs), which are functional nanoscale membrane vesicles produced by cells. This chapter discusses the accumulating research findings on cell-based regenerative therapy utilizing dental stem cells and their derived EVs, which could be a viable tool for the treatment of a variety of diseases and hence extremely valuable to mankind in the long run.

How clathrin-coated pits control nanoparticle avidity for cells.

The paramount relevance of clathrin-coated pits (CCPs) to receptor-mediated endocytosis of nanoparticles, extracellular vesicles, and viruses has made them the focus of many studies; however, the role of CCP geometry in the ligand-receptor interactions between multivalent nanoparticles and cells has not been investigated. We hypothesized the general dependence of nanoparticle binding energy on local membrane curvature to be expandable to the specific case of ligand-functionalized nanoparticles binding cell membranes, in the sense that membrane structures whose curvature matches that of the particle (e.g., CCPs) signficantly contribute to binding avidity. We investigated this hypothesis with nanoparticles that bind multivalently to angiotensin II receptor type 1, which is subject to clathrin-mediated endocytosis. When we used cholesterol extraction to prevent the action of CCPs, we found a 67 to 100-fold loss in avidity. We created a theoretical model that predicts this decrease based on the loss of ligand-receptor interactions when CCPs, which perfectly match nanoparticle geometry, are absent. Our findings shed new light on how cells "see" nanoparticles. The presence or absence of CPPs is so influential on how cells interact with nanoparticles that the number of particles required to be visible to cells changes by two orders of magnitude depending on CCP presence.

Abstract IA020: Early cancer detection through the analyses of extracellular vesicles

Abstract Breakthroughs in deep molecular profiling of non-invasive clinical samples (“liquid biopsy”) promise to revolutionize how cancer is detected and monitored. Analyzing circulating biomarkers in patient blood has shown their diagnostic potential in advanced disease states. Early detection, however, remains challenging – most biomarkers either (i) arise in advanced disease and at low abundance or (ii) often suffer from poor specificity, thus creating false positives that are costly and stressful for patients. We have been exploring extracellular vesicles (EVs) as a new biomarker that may overcome these challenges. EVs are nanoscale membrane particles secreted by cells. Most types of cancer shed large numbers of EVs that carry molecular cargo from the parent tumor. Analyzing EVs thus can represent a new real-time window to detect, diagnose, and serially monitor tumor molecular status. This presentation will describe our recent advances that leverage EVs for early cancer detection. Specifically, we will discuss the single EV analysis (SEA) platform that characterizes individual EVs for multiple protein markers. This method helps us unveil EV heterogeneity, which maximizes biological information content (specificity and sensitivity) for tumor-derived vesicles. We will review the SEA’s development and pilot application to diagnose early cancers. Citation Format: Hakho Lee, Cesar M. Castro. Early cancer detection through the analyses of extracellular vesicles. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr IA020.

Plant-derived exosomal nanoparticles: potential therapeutic for inflammatory bowel disease.

Inflammatory bowel disease (IBD), encompassing Crohn's disease and ulcerative colitis, is a chronic autoimmune disorder characterized by inflammation. However, currently available disease-modifying anti-IBD drugs exhibit limited efficacy in IBD therapy. Furthermore, existing therapeutic approaches provide only partial relief from IBD symptoms and are associated with certain side effects. In recent years, a novel category of nanoscale membrane vesicles, known as plant-derived exosome-like nanoparticles (PDENs), has been identified in edible plants. These PDENs are abundant in bioactive lipids, proteins, microRNAs, and other pharmacologically active compounds. Notably, PDENs possess immunomodulatory, antitumor, regenerative, and anti-inflammatory properties, making them particularly promising for the treatment of intestinal diseases. Moreover, PDENs can be engineered as targeted delivery systems for the efficient transport of chemical or nucleic acid drugs to the site of intestinal inflammation. In the present study, we provided an overview of PDENs, including their biogenesis, extraction, purification, and construction strategies, and elucidated their physiological functions and therapeutic effects on IBD. Additionally, we summarized the applications and potential of PDENs in IBD treatment while highlighting the future directions and challenges in the field of emerging nanotherapeutics for IBD therapy.

Surface morphology live-cell imaging reveals how macropinocytosis inhibitors affect membrane dynamics

Macropinocytosis is a unique endocytic pathway that involves dynamic membrane ruffling and is important in various cellular functions beyond nutrient uptake. Due to its impact, macropinocytosis has been the subject of various studies and macropinocytosis inhibitors have been widely used. However, inhibitors exhibit offtarget effects and could be less specific for macropinocytosis, thereby affecting various cellular mechanisms with effects on nanoscale membrane dynamics that remain elusive. In this study, we revealed how five different inhibitors affect membrane dynamics before and after treatment using scanning ion conductance microscopic imaging on living cells. Our results indicated that cytochalasins and sodium azide exhibited similar slowing effects on horizontal ruffle movements, even though their inhibitory mechanisms are different. In contrast, ruffle deformation and elongation were less strongly inhibited compared to the movements along cell membrane direction. These results suggested the involvement of mechanisms beyond actin polymerization. The ruffles disappeared and the cell surface smoothened upon the 5-(N-ethyl-N-isopropyl)-Amiloride treatment. In the presence of wortmannin, we observed active ruffles but no cup-like structures. These findings and scanning ion conductance microscopy live-cell imaging would potentially contribute to a better understanding of inhibitor effects.

Control of phosphatidylinositol-3-kinase signaling by nanoscale membrane compartmentalization.

Phosphatidylinositol-3-kinases (PI3Ks) are lipid kinases that produce 3-phosphorylated derivatives of phosphatidylinositol upon activation by various cues. These 3-phosphorylated lipids bind to various protein effectors to control many cellular functions. Lipid phosphatases such as phosphatase and tensin homolog (PTEN) terminate PI3K-derived signals and are critical to ensure appropriate signaling outcomes. Many lines of evidence indicate that PI3Ks and PTEN, as well as some specific lipid effectors are highly compartmentalized, either in plasma membrane nanodomains or in endosomal compartments. We examine the evidence for specific recruitment of PI3Ks, PTEN, and other related enzymes to membrane nanodomains and endocytic compartments. We then examine the hypothesis that scaffolding of the sources (PI3Ks), terminators (PTEN), and effectors of these lipid signals with a common plasma membrane nanodomain may achieve highly localized lipid signaling and ensure selective activation of specific effectors. This highlights the importance of spatial regulation of PI3K signaling in various physiological and disease contexts.

Membrane curvature governs the distribution of Piezo1 in live cells

Piezo1 is a bona fide mechanosensitive ion channel ubiquitously expressed in mammalian cells. The distribution of Piezo1 within a cell is essential for various biological processes including cytokinesis, cell migration, and wound healing. However, the underlying principles that guide the subcellular distribution of Piezo1 remain largely unexplored. Here, we demonstrate that membrane curvature serves as a key regulator of the spatial distribution of Piezo1 in the plasma membrane of living cells. Piezo1 depletes from highly curved membrane protrusions such as filopodia and enriches to nanoscale membrane invaginations. Quantification of the curvature-dependent sorting of Piezo1 directly reveals the in situ nano-geometry of the Piezo1-membrane complex. Piezo1 density on filopodia increases upon activation, independent of calcium, suggesting flattening of the channel upon opening. Consequently, the expression of Piezo1 inhibits filopodia formation, an effect that diminishes with channel activation.

Dihydroquercetin composite nanofibrous membrane prevents UVA radiation-mediated inflammation, apoptosis and oxidative stress by modulating MAPKs/Nrf2 signaling in human epidermal keratinocytes

Exposure to ultraviolet (UV) radiation is a key cause of skin inflammation and photodamage in the environment. Dihydroquercetin composite nanofiber membrane (CPD) is a nano-scale membrane cloth prepared by electrospinning technology. The results in this study showed that CPD could enhance the activities of endogenous antioxidant enzymes such as SOD and GSH-Px induced by UVA radiation, and reduce the overexpression of ROS. MAPKs/Nrf2 signaling is associated with inflammation, apoptosis and oxidative stress. Compared with control HaCaT cells, we found that CPD pretreatment prevents MAPK (p-ERK, p-JNK, and p-P38)/Nrf2-induced inflammation, apoptosis, and oxidative stress signaling during UVA exposure pathway overexpression. Immunofluorescence experiments also showed that CPD could reduce the fluorescence intensity of Caspase-3 and TNF-α. These results suggest that CPD may be a successful healing agent that provides reinforcement against UVA-induced oxidative and irritating skin compensation.

Correlation analyses reveal differential diffusion behavior of eisosomal proteins between mother and daughter cells

Eisosomes are nanoscale plasma membrane domains shaped as furrow-like invaginations. In Saccharomyces cerevisiae these relatively immobile and uniform structures are mainly composed of two cytoplasmic proteins Pil1 and Lsp1. The present work uses fluctuation of fluorescence signals and analytical methods to determine Pil1 and Lsp1 dynamics at different subcellular locations. Using scanning techniques and autocorrelation analysis we determine that the cytoplasmic pools of Pil1 and Lsp1 behave mainly by passive diffusion. Single-point FCS experiments performed at several subcellular locations reveal that Pil1 mobility is faster in daughter cells. Furthermore, pair correlation function analysis indicates a rapid dynamic of Pil1 near the plasma membrane of growing yeast buds, where the membrane is expected to be actively assembling eisosomes.

Fast and specific enrichment and quantification of cancer-related exosomes by DNA-nanoweight-assisted centrifugation.

Exosomes are nanoscale membrane vesicles actively released by cells and play an important role in the diagnosis of cancer-related diseases. However, it is challenging to efficiently enrich exosomes from extracellular fluids. In this work, we used DNA nanostructures as "nanoweights" during centrifugation to facilitate the enrichment of cancerous exosomes in human serum. Two different DNA tetrahedral nanostructures (DTNs), each carrying a specific aptamer for exosome biomarker recognition, were incubated with clinical samples simultaneously. One DTN triggered the cross-linking of multiple target exosomes and, therefore, enabled low-speed and fast centrifugation for enrichment. The other DTN further narrowed down the target exosome subtype and initiated a hybridization chain reaction (HCR) for sensitive signal amplification. The method enabled the detection of 1.8 × 102 MCF-7-derived exosomes per microliter and 5.6 × 102 HepG2-derived exosomes per microliter, with 1000-fold higher sensitivity than conventional ELISA and 10-fold higher sensitivity than some recently reported fluorescence assays. Besides, the dual-aptamer system simultaneously recognized multiple surface proteins, eliminating the interference risk from free proteins. Thus, this easy-to-operate method can enrich exosomes with excellent specificity and sensitivity and therefore will be appealing in biomedical research and clinical diagnosis.