Influence of the glycocalyx on the size and mechanical properties of plasma membrane-derived vesicles.

Recent studies have reported that the overexpression of MUC1 glycoproteins on cell surfaces changes the morphology of cell plasma membranes and increases the blebbing of vesicles from them, supporting the hypothesis that entropic forces exerted by MUC1 change the spontaneous curvature of cell membranes. However, how MUC1 is incorporated into and influences the size and biophysical properties of plasma-membrane-blebbed vesicles is not understood. Here we report single-vesicle-level characterization of giant plasma membrane vesicles (GPMVs) derived from cells overexpressing MUC1, revealing a 40× variation in MUC1 density between GPMVs from a single preparation and a strong correlation between GPMV size and MUC1 density.

Leucine zipper-based SAIM imaging identifies therapeutic agents to disrupt the cancer cell glycocalyx for enhanced immunotherapy.

The abnormally thick glycocalyx of cancer cells can provide a physical barrier to immune cell recognition and effective immunotherapy. Here, we demonstrate an optical method based on Scanning Angle Interference Microscopy (SAIM) for the screening of therapeutic agents that can disrupt the glycocalyx layer as a strategy to improve anti-cancer immune responses. We developed a new membrane labeling strategy utilizing leucine zipper pairs to fluorescently mark the glycocalyx layer boundary for precise and robust measurement of glycocalyx thickness with SAIM.

Dimerization activates the Inversin complex in .

Genetic, colocalization, and biochemical studies suggest that the ankyrin repeat-containing proteins Inversin (INVS) and ANKS6 function with the NEK8 kinase to control tissue patterning and maintain organ physiology. It is unknown whether these three proteins assemble into a static "Inversin complex" or one that adopts multiple bioactive forms. Through the characterization of hyperactive alleles in , we discovered that the Inversin complex is activated by dimerization.

Dimerization activates the Inversin complex in .

Genetic, colocalization, and biochemical studies suggest that the ankyrin repeat-containing proteins Inversin (INVS) and ANKS6 function with the NEK8 kinase to control tissue patterning and maintain organ physiology. It is unknown whether these three proteins assemble into a static "Inversin complex" or one that adopts multiple bioactive forms. Through characterization of hyperactive alleles in , we discovered that the Inversin complex is activated by dimerization.

Bio-orthogonal Glycan Imaging of Culture Cells and Whole Animal with Expansion Microscopy.

Complex carbohydrates called glycans play crucial roles in the regulation of cell and tissue physiology, but how glycans map to nanoscale anatomical features must still be resolved. Here, we present the first nanoscale map of mucin-type -glycans throughout the entirety of the model organism. We construct a library of multifunctional linkers to probe and anchor metabolically labelled glycans in expansion microscopy (ExM), an imaging modality that overcomes the diffraction limit of conventional optical microscopes through the physical expansion of samples embedded in a polyelectrolyte gel matrix.

COLLAGEN MINERALIZATION DECREASES NK CELL-MEDIATED CYTOTOXICITY OF BREAST CANCER CELLS VIA INCREASED GLYCOCALYX THICKNESS.

Skeletal metastasis is common in patients with advanced breast cancer, and often caused by immune evasion of disseminated tumor cells (DTCs). In the skeleton, tumor cells not only disseminate to the bone marrow, but also to osteogenic niches in which they interact with newly mineralizing bone extracellular matrix (ECM). However, it remains unclear how mineralization of collagen type I, the primary component of bone ECM, regulates tumor-immune cell interactions.

Collagen Mineralization Decreases NK Cell-Mediated Cytotoxicity of Breast Cancer Cells via Increased Glycocalyx Thickness.

Skeletal metastasis is common in patients with advanced breast cancer and often caused by immune evasion of disseminated tumor cells (DTCs). In the skeleton, tumor cells not only disseminate to the bone marrow but also to osteogenic niches in which they interact with newly mineralizing bone extracellular matrix (ECM). However, it remains unclear how mineralization of collagen type I, the primary component of bone ECM, regulates tumor-immune cell interactions.

The microenvironment dictates glycocalyx construction and immune surveillance.

Efforts to identify anti-cancer therapeutics and understand tumor-immune interactions are built with models that do not match the microenvironmental characteristics of human tissues. Using models which mimic the physical properties of healthy or cancerous tissues and a physiologically relevant culture medium, we demonstrate that the chemical and physical properties of the microenvironment regulate the composition and topology of the glycocalyx. Remarkably, we find that cancer and age-related changes in the physical properties of the microenvironment are sufficient to adjust immune surveillance via the topology of the glycocalyx, a previously unknown phenomenon observable only with a physiologically relevant culture medium.

Recombinant mucin biotechnology and engineering.

Mucins represent a largely untapped class of polymeric building block for biomaterials, therapeutics, and other biotechnology. Because the mucin polymer backbone is genetically encoded, sequence-specific mucins with defined physical and biochemical properties can be fabricated using recombinant technologies. The pendent O-glycans of mucins are increasingly implicated in immunomodulation, suppression of pathogen virulence, and other biochemical activities.

Glycocalyx Curving the Membrane: Forces Emerging from the Cell Exterior.

Morphological transitions are typically attributed to the actions of proteins and lipids. Largely overlooked in membrane shape regulation is the glycocalyx, a pericellular membrane coat that resides on all cells in the human body. Comprised of complex sugar polymers known as glycans as well as glycosylated lipids and proteins, the glycocalyx is ideally positioned to impart forces on the plasma membrane.

Hyaluronic acid synthesis, degradation, and crosslinking in equine osteoarthritis: TNF-α-TSG-6-mediated HC-HA formation.

Background: TNF-α-stimulated gene 6 (TSG-6) protein, a TNF-α-responsive hyaladherin, possesses enzymatic activity that can catalyze covalent crosslinks of the polysaccharide hyaluronic acid (HA) to another protein to form heavy chain-hyaluronic acid (HC-HA) complexes in pathological conditions such as osteoarthritis (OA). Here, we examined HA synthase and inflammatory gene expression; synovial fluid HA, TNF-α, and viscosity; and TSG-6-mediated HC-HA complex formation in an equine OA model. The objectives of this study were to (1) evaluate the TNF-α-TSG-6-HC-HA signaling pathway across multiple joint tissues, including synovial membrane, cartilage, and synovial fluid, and (2) determine the impact of OA on synovial fluid composition and biophysical properties.

Nuclear Deformation Causes DNA Damage by Increasing Replication Stress.

Cancer metastasis, i.e., the spreading of tumor cells from the primary tumor to distant organs, is responsible for the vast majority of cancer deaths.

Litmus-Body: A Molecularly Targeted Sensor for Cell-Surface pH Measurements.

Precise pH measurements in the immediate environment of receptors is essential for elucidating the mechanisms through which local pH changes associated with diseased phenotypes manifest into aberrant receptor function. However, current pH sensors lack the ability to localize and target specific receptor molecules required to make these measurements. Herein we present the Litmus-body, our recombinant protein-based pH sensor, which through fusion to an anti-IgG nanobody is capable of piggybacking on IgG antibodies for molecular targeting to specific proteins on the cell surface.

The surface stress of biomedical silicones is a stimulant of cellular response.

Silicones are commonly used for lubrication of syringes, encapsulation of medical devices, and fabrication of surgical implants. While silicones are generally viewed as relatively inert to the cellular milieu, they can mediate a variety of inflammatory responses and other deleterious effects, but the mechanisms underlying the bioactivity of silicones remain unresolved. Here, we report that silicone liquids and gels have high surface stresses that can strongly resist deformation at cellular length scales.

Direct comparison of optical and electron microscopy methods for structural characterization of extracellular vesicles.

As interest in the role of extracellular vesicles in cell-to-cell communication has increased, so has the use of microscopy and analytical techniques to assess their formation, release, and morphology. In this study, we evaluate scanning electron microscopy (SEM) and cryo-SEM for characterizing the formation and shedding of vesicles from human breast cell lines, parental and hyaluronan synthase 3-(HAS3)-overexpressing MCF10A cells, grown directly on transmission electron microscopy (TEM) grids. While cells imaged with conventional and cryo-SEM exhibit distinct morphologies due to the sample preparation process for each technique, tubular structures protruding from the cell surfaces were observed with both approaches.

Sequence-Specific Mucins for Glycocalyx Engineering.

Few approaches exist for the stable and controllable synthesis of customized mucin glycoproteins for glycocalyx editing in eukaryotic cells. Taking advantage of custom gene synthesis and a biology-by-parts approach to cDNA construction, we build a library of swappable DNA bricks for mucin leader tags, membrane anchors, cytoplasmic motifs, and optical reporters, as well as codon-optimized native mucin repeats and newly designed domains for synthetic mucins. We construct a library of over 50 mucins, each with unique chemical, structural, and optical properties and describe how additional permutations could readily be constructed.

High-speed device synchronization in optical microscopy with an open-source hardware control platform.

Azimuthal beam scanning eliminates the uneven excitation field arising from laser interference in through-objective total internal reflection fluorescence (TIRF) microscopy. The same principle can be applied to scanning angle interference microscopy (SAIM), where precision control of the scanned laser beam presents unique technical challenges for the builders of custom azimuthal scanning microscopes. Accurate synchronization between the instrument computer, beam scanning system and excitation source is required to collect high quality data and minimize sample damage in SAIM acquisitions.

Physical Principles of Membrane Shape Regulation by the Glycocalyx.

Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface glycocalyx. Mucin biopolymers and long-chain polysaccharides within the glycocalyx can generate entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface.

Antibody-Mediated Endocytosis of Polysialic Acid Enables Intracellular Delivery and Cytotoxicity of a Glycan-Directed Antibody-Drug Conjugate.

The specific targeting of differentially expressed glycans in malignant cells has emerged as an attractive anticancer strategy. One such target is the oncodevelopmental antigen polysialic acid (polySia), a polymer of α2,8-linked sialic acid residues that is largely absent during postnatal development but is re-expressed during progression of several malignant human tumors, including small-cell and non-small cell lung carcinomas, glioma, neuroblastoma, and pancreatic carcinoma. In these cancers, expression of polySia correlates with tumor progression and poor prognosis and appears to modulate cancer cell adhesion, invasiveness, and metastasis.

Equilibrium Modeling of the Mechanics and Structure of the Cancer Glycocalyx.

The glycocalyx is a thick coat of proteins and carbohydrates on the outer surface of all eukaryotic cells. Overproduction of large, flexible or rod-like biopolymers, including hyaluronic acid and mucins, in the glycocalyx strongly correlates with the aggression of many cancer types. However, theoretical frameworks to predict the effects of these changes on cancer cell adhesion and other biophysical processes remain limited.

Stable recombinant production of codon-scrambled lubricin and mucin in human cells.

Widespread therapeutic and commercial interest in recombinant mucin technology has emerged due to the unique ability of mucin glycoproteins to hydrate, protect, and lubricate biological surfaces. However, recombinant production of the large, highly repetitive domains that are characteristic of mucins remains a challenge in biomanufacturing likely due, at least in part, to the inherent instability of DNA repeats in the cellular genome. To overcome this challenge, we exploit codon redundancy to encode desired mucin polypeptides with minimal nucleotide repetition.

Mucin-coating technologies for protection and reduced aggregation of cellular production systems.

Optimization of host-cell production systems with improved yield and production reliability is desired to meet the increasing demand for biologics with complex posttranslational modifications. Aggregation of suspension-adapted mammalian cells remains a significant problem that can limit the cellular density and per volume yield of bioreactors. Here, we propose a genetically encoded technology that directs the synthesis of antiadhesive and protective coatings on the cellular surface.

Genetically Encoded Toolbox for Glycocalyx Engineering: Tunable Control of Cell Adhesion, Survival, and Cancer Cell Behaviors.

The glycocalyx is a coating of protein and sugar on the surface of all living cells. Dramatic perturbations to the composition and structure of the glycocalyx are frequently observed in aggressive cancers. However, tools to experimentally mimic and model the cancer-specific glycocalyx remain limited.