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.

Bioengineered lubricin alters the lubrication modes of cartilage in a dose-dependent manner.

The low friction nature of articular cartilage has been attributed to the synergistic interaction between lubricin and hyaluronic acid in the synovial fluid (SF). Lubricin is a mucinous glycoprotein that lowers the boundary mode coefficient of friction of articular cartilage in a dose-dependent manner. While there have been multiple attempts to produce recombinant lubricin and lubricin mimetic cartilage lubricants over the last two decades, these materials have not found clinical use due to challenges associated with large scale production, manufacturing, and purification.

Specific Degradation of the Mucin Domain of Lubricin in Synovial Fluid Impairs Cartilage Lubrication.

Progressive cartilage degradation, synovial inflammation, and joint lubrication dysfunction are key markers of osteoarthritis. The composition of synovial fluid (SF) is altered in OA, with changes to both hyaluronic acid and lubricin, the primary lubricating molecules in SF. Lubricin's distinct bottlebrush mucin domain has been speculated to contribute to its lubricating ability, but the relationship between its structure and mechanical function in SF is not well understood.

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.

Recombinant Production of Glycoengineered Mucins in HEK293-F Cells.

Recombinant mucins are attractive polymeric building blocks for new biomaterials, biolubricants, and therapeutics. Advances in glycoengineered host cell systems now enable the recombinant production of mucins with tailored O-glycan side chains, offering new opportunities to tune the functionality of mucins and investigate the biology of specific O-glycan structures. Here, we provide a protocol for the scalable production of glycoengineered mucins and mucin-like glycoproteins in suspension-adapted HEK293-F cells.

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.

Bone-matrix mineralization dampens integrin-mediated mechanosignalling and metastatic progression in breast cancer.

In patients with breast cancer, lower bone mineral density increases the risk of bone metastasis. Although the relationship between bone-matrix mineralization and tumour-cell phenotype in breast cancer is not well understood, mineralization-induced rigidity is thought to drive metastatic progression via increased cell-adhesion forces. Here, by using collagen-based matrices with adjustable intrafibrillar mineralization, we show that, unexpectedly, matrix mineralization dampens integrin-mediated mechanosignalling and induces a less proliferative stem-cell-like phenotype in breast cancer cells.

The lamin A/C Ig-fold undergoes cell density-dependent changes that alter epitope binding.

Lamins A/C are nuclear intermediate filament proteins that are involved in diverse cellular mechanical and biochemical functions. Here, we report that recognition of Lamins A/C by a commonly used antibody (JOL-2) that binds the Lamin A/C Ig-fold and other antibodies targeting similar epitopes is highly dependent on cell density, even though Lamin A/Clevels do not change. We propose that the effect is caused by partial unfolding or masking of the C'E and/or EF loops of the Ig-fold in response to cell spreading.

Convergent Approaches to Delineate the Metabolic Regulation of Tumor Invasion by Hyaluronic Acid Biosynthesis.

Metastasis is the leading cause of breast cancer-related deaths and is often driven by invasion and cancer-stem like cells (CSCs). Both the CSC phenotype and invasion are associated with increased hyaluronic acid (HA) production. How these independent observations are connected, and which role metabolism plays in this process, remains unclear due to the lack of convergent approaches integrating engineered model systems, computational tools, and cancer biology.

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.

Azimuthal Beam Scanning Microscope Design and Implementation for Axial Localization with Scanning Angle Interference Microscopy.

Azimuthal beam scanning, also referred to as circle scanning, is an effective way of eliminating coherence artifacts with laser illumination in widefield microscopy. With a static excitation spot, dirt on the optics and internal reflections can produce an uneven excitation field due to interference fringes. These artifacts become more pronounced in TIRF microscopy, where the excitation is confined to an evanescent field that extends a few hundred nanometers above the coverslip.

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.

Investigation of synovial fluid lubricants and inflammatory cytokines in the horse: a comparison of recombinant equine interleukin 1 beta-induced synovitis and joint lavage models.

Background: Lameness is a debilitating condition in equine athletes that leads to more performance limitation and loss of use than any other medical condition. There are a limited number of non-terminal experimental models that can be used to study early inflammatory and synovial fluid biophysical changes that occur in the equine joint. Here, we compare the well-established carpal IL-1β-induced synovitis model to a tarsal intra-articular lavage model, focusing on serial changes in synovial fluid inflammatory cytokines/chemokines and the synovial fluid lubricating molecules lubricin/proteoglycan 4 and hyaluronic acid.

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.