For generations of biology students, the fundamental model of the cell membrane was defined by the fluid mosaic model: a fluid and dynamic phospholipid bilayer studded with various integral and peripheral proteins. While elegant, this well-known picture of the cell membrane omits a crucial component: a dense, carbohydrate-rich coating that covers most eukaryotic cells. This structure, known as the glycocalyx, forms the true outer interface between cells and their environment. The glycocalyx is not simply a decorative sugar coating. It participates in cell signaling, mechanical protection, immune recognition, host-pathogen interactions, and the regulation of vascular function. The glycocalyx is present on all cells in our body, but its thickness and composition vary enormously amongst cell types. On many cells, the glycocalyx is only tens of nanometers deep, whereas on specialized surfaces, it can be much more substantial. For instance, the glycocalyx of the vascular endothelium can reach hundreds of nanometers to several micrometers in thickness, forming a dynamic molecular forest that projects outward from the membrane.

Despite the growing prevalence of post-traumatic osteoarthritis (OA), early and effective treatment options to delay surgical intervention remain limited. Lubricin is a naturally occurring glycoprotein produced by articular chondrocytes and synovial fibroblasts that functions as the principal boundary lubricant in joints. Recently, a recombinant lubricin-like glycoprotein (rhLub) with codon optimized mucin domain was reported to have a sustained residence time (> 6 weeks) following injection into the knee joint of healthy rats. To evaluate outcomes of rhLub injection, skeletally mature Sprague-Dawley rats of both sexes (n = 18) underwent bilateral anterior cruciate ligament transection (ACLT) to initiate post-traumatic OA, followed by three intra-articular injections of 25 µL of rhLub (1.3 mg/mL) or phosphate-buffered saline (PBS) at weekly intervals beginning 1 week post-operatively. Pain sensitization was evaluated weekly throughout the 12-week study with weightbearing and pressure application measurement (PAM). Frontal plane histologic sections of the medial femorotibial joint compartment were scored using Osteoarthritis Research Society International criteria. Injections of rhLub were well-tolerated. rhLub injections protected against cartilage degeneration and improved histologic scores in male rats. Mechanical hyperalgesia and cartilage degeneration scores were greater in female rats compared to males, irrespective of treatment. This study suggests that intra-articular rhLub therapy may be most efficacious in mild-to-moderate disease.

Altered glucose metabolism is an auspicious feature of solid tumors, but is it an intrinsic property of tumor cells or a metabolic adaptation to the tumor microenvironment? Using normal epithelial cells cultured in a physiological culture medium under conditions that mimic the physical properties of healthy or cancerous tissues, we establish multiomics relationships between the biochemical and physical properties of the microenvironment and its impact on biosynthetic outputs of altered glucose metabolism. We find that microenvironmental properties, such as hyperglycemia, can affect the composition and thickness of the epithelial glycocalyx, in part through the activity of mechanosensitive stress responses associated with Heat Shock Factor 1 (HSF1). Because glycocalyx thickness alters immune surveillance of epithelial-origin tumor cells, we examined the relationship between the HSF1-hyperglycemia axis in human breast tumors and validate it as a druggable vulnerability to license natural killer cell lethality against cancer cells.

The cancer glycocalyx is characterized by the overexpression of large glycoconjugates, including mucins, proteoglycans, and polysialic acid, which collectively increase glycocalyx thickness and stiffness to promote tumor survival, metastasis, and progression. Previous work demonstrated that enzymatic degradation of cancer cell-surface mucins reduced tumor burden and metastasis in mouse models of breast cancer, thus validating glycocalyx remodeling as a therapeutic strategy. However, this work relied on an engineered bacterial mucin-selective protease, or mucinase, which does not degrade other bulky glycoconjugates and raises immunogenicity concerns because of its bacterial origin. A human enzyme that can degrade cell-surface mucins and other bulky glycoconjugates would address both limitations. We screened all 15 human cathepsins for their ability to degrade purified and cell-surface mucins, since multiple cathepsins have previously been shown to degrade mucins within regions of dense glycosylation. We found that cathepsin K (CTSK) uniquely degrades cell-surface mucins, proteoglycans, and polysialylated glycoproteins, and we demonstrated that CTSK reduces total glycocalyx thickness. These findings establish CTSK as a promising starting point for the development of a glycocalyx-debulking enzyme for cancer therapeutics.

Biological cells are often damaged by interactions with fluid interfaces, yet the cell-scale processes that underlie their destruction have not been fully elucidated. Here we investigate whether interactions of cells at aqueous interfaces of thermotropic liquid crystals (LCs) drive changes in LC ordering, thereby generating spatiotemporal optical signatures that yield insights into the pathways by which single cells are damaged at fluid interfaces and how molecular adsorbates influence those pathways. MCF10A breast epithelial cells were sedimented onto micrometer-thick films of nematic LCs and imaged using optical microscopy. We found that the cells exhibited a lag phase on the interface (lasting seconds to an hour during which no optical response of the LC was evident) that was followed by rapid lateral displacement of the cells and the generation of complex spatiotemporal patterns in the LC. Fingering instabilities evident in the patterned optical response revealed deconstruction of cells and redistribution of cellular components driven by interfacial tension gradients. Pretreatment of the LC interface with amphiphilic adsorbates altered spreading dynamics and domain shapes, suggesting a role for Marangoni stresses in the disassembly pathway and revealing angular spatial patterns consistent with a crossover to capillary fracturing driven by interfacial elasticity. Fluorescence imaging along with control experiments with synthetic vesicles and red blood cells enabled spatiotemporal features of the LC response to epithelial cells to be associated with specific intracellular structures (nucleus, plasma membrane, and cytosol). The duration of the lag phase was also found to be influenced by cell-surface expression levels of the mucin MUC1. Overall, our findings reveal key physical processes that occur when cells interact with fluid interfaces and highlight the potential of LC interfaces to form the basis of single cell-level analyses of biophysical properties.

ABSTRACT Collagen type II (Col‐II) and collagen type I (Col‐I) are major components of articular cartilage present at different ratios at its surface. Understanding how each of these components mediates the assembly of molecular films derived from synovial fluid (SF), the lubricant of synovial joints, is critical to explain the loss of mechanical performance in pathological conditions, guide the design of biomaterial implants meant to be in contact with SF, and develop molecular therapies to restore SF properties. This work demonstrates that Col‐II articular surface model assists in scaffolding SF‐derived films, while Col‐I model lacks SF film scaffolding capabilities. However, when Col‐II and Col‐I are exposed to recombinant equine lubricin (rEqLub), an analog of the major boundary lubricant in SF, both adsorbed and retained similar amounts. These insights, deduced from quartz crystal microbalance with dissipation, diffuse reflectance circular dichroism, and atomic force microscopy, reveal possible mechanisms underlying the loss of mechanical performance of synovial joints in pathology, where Col‐I becomes the major collagenous component of the articular cartilage surface, as well as considerations for designing functional biomaterial implants. Furthermore, this work reinforces the idea of rEqLub as an intra‐articular osteoarthritis therapy with the ability to bind to Col‐II and Col‐I, irrespectively.

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 40x variation in MUC1 density between GPMVs from a single preparation and a strong correlation between GPMV size and MUC1 density. By dispersing GPMVs in aqueous liquid crystals (LCs), we show that the elasticity of the LC can be used to strain individual GPMVs into spindle-like shapes, consistent with the straining of fluid-like membranes. To quantify the influence of MUC1 on membrane mechanical properties, we analyze the shapes of strained GPMVs within a theoretical framework that integrates the effects of MUC1 density and GPMV size on strain. We measure the spontaneous curvature of GPMV membranes to be 2-10 mu m-1 and weakly influenced by the 40x variation in MUC1 density, a conclusion we validate by performing independent experiments in which MUC1 is enzymatically removed from GPMVs. Overall, our study advances the understanding of heterogeneity in size and MUC1 density in GPMVs, and establishes single-vesicle-level methods for characterization of mechanical properties within a heterogeneous population of GPMVs. Furthermore, our measurements highlight differences between membrane properties of GPMVs and their parent cells.

Collagen is a key extracellular matrix protein found in connective tissues. The structure and organization of collagen fibers play a crucial role in determining tissue function and how tissues respond to mechanical loads. Small leucine-rich proteoglycans (SLRPs) are well-known facilitators of collagen fibrillogenesis in connective tissues. While the role of SLRPs has been extensively documented in tissues such as tendon and skin, their functions are primarily inferred from changes observed in knockout models. Additionally, their specific roles and influences of their addition to a system, particularly in collagen gel-based materials, remain underexplored. Previous in vitro studies of SLRPs have been partly limited by the challenges associated with obtaining pure SLRPs in sufficient quantities and with appropriate glycosylation. Therefore, novel methods to reliably produce SLRPs at the required quality and scale are needed. In this study, we first evaluated the feasibility of producing recombinant decorin, biglycan, and fibromodulin using HEK293-F cells. Subsequently, we investigated the effect of SLRP supplementation on high-density collagen gels using scanning electron microscopy and assessed the impact on tensile properties. Our findings demonstrated that each SLRP uniquely influenced collagen structure at both the fibril and fiber levels, consequently modifying the tissues' mechanical response to load. Decorin, in particular, exhibited significant differences in tensile properties compared to biglycan and fibromodulin, underscoring its distinct role in promoting a structurally and mechanically robust response under tensile load.

Osteochondral lesions (OCL) are common among young patients and often require surgical interventions since cartilage has a poor capacity for self-repair. Bone marrow stimulation (BMS) has been used clinically for decades to treat OCLs, however a persisting challenge with BMS and other cartilage repair strategies is the inferior quality of the resulting fibrocartilaginous repair tissue. Lubrication-based therapies have the potential to improve the quality of cartilage repair tissue as joint lubrication is linked to local cartilage tissue strains and subsequent cellular responses including death and apoptosis. Recently, a full length recombinant human lubricin (rhLubricin) was developed and has been shown to lower friction in cartilage. This study investigated the effect of a single delayed injection of rhLubricin on cartilage repair in an in vivo rabbit OCL model using gross macroscopic evaluation, surface profilometry, histology, and tribology. Moderate improvement in macroscopic scores for cartilage repair were observed. Notably, quantitative analysis of Safranin-O histology showed that rhLubricin treated joints had significantly higher glycosaminoglycan content compared to saline treated joints, and there were no differences in repair integration between groups. Furthermore, rhLubricin treated joints had significantly lower friction coefficients tested across three sliding speeds compared to saline treated joints (rhLubricin: 0.15 ± 0.03 at 0.1 mm/s to 0.12 ± 0.03 at 10 mm/s, Saline: 0.22 ± 0.06 at 0.1 mm/s to 0.19 ± 0.05 at 10 mm/s). Overall, a single delayed injection of rhLubricin improved the quality and lubricating ability of the repair cartilage tissue without inhibiting repair tissue integration.

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. Recently, a novel method using codon scrambling was developed to produce a stable, full-length bioengineered equine lubricin (eLub) in large reproducible quantities. While preliminary frictional analysis of eLub and other recombinantly produced forms revealed they can lubricate cartilage, a complete tribological characterization is lacking, with previous studies evaluating the friction coefficient only at a single dose or a single speed. The objective of this study was to analyze the dose-dependent tribological properties of eLub using the Stribeck framework of tribological analysis. Recombinantly produced eLub at doses greater than 1.5 mg/mL exhibits friction coefficients on par with healthy bovine SF, and a maximal 5 mg/mL dose exhibits a nearly 50% lower friction coefficient than healthy SF. eLub also modulates the shift in lubrication mode of the cartilage from the high friction boundary mode to the low friction minimum mode at high concentrations.

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. Here, a combination of synthetic bone matrix models with controlled mineral content, nanoscale optical imaging, and flow cytometry are utilized to evaluate how collagen type I mineralization affects the biochemical and biophysical properties of the tumor cell glycocalyx, a dense layer of glycosylated proteins and lipids decorating their cell surface. These results suggest that collagen mineralization upregulates mucin-type O-glycosylation and sialylation by tumor cells, which increases their glycocalyx thickness while enhancing resistance to attack by natural killer (NK) cells. These changes are functionally linked as treatment with a sialylation inhibitor decreased mineralization-dependent glycocalyx thickness and made tumor cells more susceptible to NK cell attack. Together, these results suggest that interference with glycocalyx sialylation may represent a therapeutic strategy to enhance cancer immunotherapies targeting bone-metastatic breast cancer.

Lubricin, a lubricating glycoprotein abundant in synovial fluid, forms a low-friction brush polymer interface in tissues exposed to sliding motion including joints, tendon sheaths, and the surface of the eye. Despite its therapeutic potential in diseases such as osteoarthritis and dry eye disease, there are few sources available. Through rational design, we developed a series of recombinant lubricin analogs that utilize the species-specific tissue-binding domains at the N- and C-termini to increase biocompatibility while replacing the central mucin domain with an engineered variant that retains the lubricating properties of native lubricin. In this study, we demonstrate the tissue binding capacity of our engineered lubricin product and its retention in the joint space of rats. Next, we present a new bioprocess chain that utilizes a human-derived cell line to produce O-glycosylation consistent with that of native lubricin and a purification strategy that capitalizes on the positively charged, hydrophobic N- and C-terminal domains. The bioprocess chain is demonstrated at 10 L scale in industry-standard equipment utilizing commonly available ion exchange, hydrophobic interaction and size exclusion chromatography resins. Finally, we confirmed the purity and lubricating properties of the recombinant biolubricant. The biomolecular engineering and bioprocessing strategies presented here are an effective means of lubricin production and could have broad applications to the study of mucins in general.

The glycocalyx is a densely crowded matrix of glycoproteins and glycolipids on the cell surface. Macromolecular crowding can strongly influence many biochemical and biophysical processes, including biomolecular transport and assembly. However, understanding the potential significance of macromolecular crowding forces in signaling processes that initiate at the cell surface is hampered by a lack of tools to parameterize crowding. To address this gap, we have designed and developed a fluorescence resonance energy transfer (FRET)-based sensor and a particle exclusion-based sensor for measurement of colloidal osmotic pressures in glycocalyx. The sensor modules were fused with genetically encoded nanobodies or affibody domains to target the glycocalyx. The configuration of the FRET-base sensor changed in response to colloidal osmotic pressure in the glycocalyx, resulting in concomitant changes in the FRET signal. The particle exclusion-based sensor consisted of a binding motif, a fluorescent reporter, and a globular domain with various particle sizes. Due to the particle exclusion effect, the apparent association constant of the sensor to the target on the cell surface was dependent on the size of the globular domain and the colloidal osmotic pressure in the glycocalyx. By measuring the FRET efficiency of the FRET-base sensor and the apparent association constant of the particle exclusion-based sensor, we quantified the colloidal osmotic pressure in the glycocalyx. By exploiting our sensors, we demonstrated that the expression of cancer-associated glycopolymers, such as mucin 1 (Muc1), significantly increase crowding pressures in the glycocalyx.

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 C. elegans, we discovered that the Inversin complex is activated by dimerization. Genome engineering of an RFP tag onto the nematode homologues of INVS (MLT-4) and NEK8 (NEKL-2) induced a gain-of-function, cyst-like phenotype that was suppressed by monomerization of the fluorescent tag. Stimulated dimerization of MLT-4 or NEKL-2 using optogenetics was sufficient to recapitulate the phenotype of a constitutively active Inversin complex. Further, dimerization of NEKL-2 bypassed a lethal MLT-4 mutant, demonstrating that the dimeric form is required for function. We propose that dynamic switching between at least two functionally distinct states - an active dimer and an inactive monomer - gates the output of the Inversin complex.

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. Here, we demonstrate the application of a novel mucinase (StcE) to selectively degrade lubricin's mucin domain in SF to measure its impact on joint lubrication and friction. Notably, StcE effectively degraded the lubricating ability of SF in a dose-dependent manner starting at nanogram concentrations (1-3.2 ng/mL). Further, the highest StcE doses effectively degraded lubrication to levels on par with trypsin, suggesting that cleavage at the mucin domain of lubricin is sufficient to completely inhibit the lubrication mechanism of the collective protein component in SF. These findings demonstrate the value of mucin-specific experimental approaches to characterize the lubricating properties of SF and reveal key trends in joint lubrication that help us better understand cartilage function in lubrication-deficient joints.

Cancer cell glycocalyx is a major line of defence against immune surveillance. However, how specific physical properties of the glycocalyx are regulated on a molecular level, contribute to immune evasion and may be overcome through immunoengineering must be resolved. Here we report how cancer-associated mucins and their glycosylation contribute to the nanoscale material thickness of the glycocalyx and consequently modulate the functional interactions with cytotoxic immune cells. Natural-killer-cell-mediated cytotoxicity is inversely correlated with the glycocalyx thickness of the target cells. Changes in glycocalyx thickness of approximately 10 nm can alter the susceptibility to immune cell attack. Enhanced stimulation of natural killer and T cells through equipment with chimeric antigen receptors can improve the cytotoxicity against mucin-bearing target cells. Alternatively, cytotoxicity can be enhanced through engineering effector cells to display glycocalyx-editing enzymes, including mucinases and sialidases. Together, our results motivate the development of immunoengineering strategies that overcome the glycocalyx armour of cancer cells. A nanoscale polymer layer formed by mucins at the surface of tumour cells protects them against immune cell attack. This shield can be circumvented through immune cell engineering, using chimeric antigen receptors to stimulate natural killer and T cells or by tethering glycocalyx-editing enzymes to immune cells.

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. Using this platform, we evaluated the effects of glycosylation inhibitors and targeted enzymatic degraders of the glycocalyx, with particular focus on strategies for cholangiocarcinoma (CCA), a highly lethal malignancy with limited therapeutic options. We found that CCA had the highest mean expression of the cancer-associated mucin, MUC1, across all cancers represented in the cancer cell line encyclopedia. Pharmacological inhibitors of mucin-type O-glycosylation and mucin-specific proteases, such as StcE, could dramatically reduce the glycocalyx layer in the YSCCC model of intrahepatic CCA. Motivated by these findings, we engineered Natural Killer (NK) cells tethered with StcE to enhance NK cell-mediated cytotoxicity against CCA. In a CCA xenograft model, these engineered NK cells demonstrated superior anti-tumor efficacy compared to wild-type NK cells, with no observable adverse effects. Our findings not only provide a reliable imaging-based screening platform for evaluating glycocalyx-targeting pharmacological interventions but also offer mechanistic insights into how CCA may avoid immune elimination through fortification of the glycocalyx layer with mucins. Additionally, this work presents a novel therapeutic strategy for mucin-overexpressing cancers, potentially improving immunotherapy efficacy across various cancer types.

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. The protocol includes the preparation of engineered cell lines with homozygous knockout (KO) of glycosyltransferases using CRISPR/Cas9 and homology-directed repair (HDR) templates designed for efficient screening of clones. Strategies are provided for the stable introduction of mucin expression cassettes into the HEK293-F genome and the subsequent isolation of high-expressing cell populations. The high-titer production of recombinant mucins in conventional shaker flasks is described as an example production strategy using these cell lines.

Complex carbohydrates called glycans play crucial roles in regulating cell and tissue physiology, but how they map to nanoscale anatomical features must still be resolved. Here, we present the first nanoscale map of mucin-type O-glycans throughout the entirety of the Caenorhabditis elegans model organism. We constructed a library of multifunctional linkers to probe and anchor metabolically labeled glycans in expansion microscopy (ExM). A flexible strategy was demonstrated for the chemical synthesis of linkers with a broad inventory of bio-orthogonal functional groups, fluorophores, anchorage chemistries, and linker arms. Employing C. elegans as a test bed, metabolically labeled O-glycans were resolved on the gut microvilli and other nanoscale anatomical features. Transmission electron microscopy images of C. elegans nanoanatomy validated the fidelity and isotropy of gel expansion. Whole organism maps of C. elegans O-glycosylation in the first larval stage revealed O-glycan "hotspots" in unexpected anatomical locations, including the body wall furrows. Beyond C. elegans, we validated ExM protocols for nanoscale imaging of metabolically labeled glycans on cultured mammalian cells. Together, our results suggest the broad applicability of the multifunctional reagents for imaging glycans and other metabolically labeled biomolecules at enhanced resolutions with ExM.

Cancer is often associated with aberrant expression of cell-surface mucins, resulting in a thick cellular glycocalyx that coats the cancer cell membrane and governs its interactions with surveilling immune cells. Densely packed mucin biopolymers on the cell surface can assemble into a nanoscale barrier that protects cells from immune cell-mediated lysis. However, the precise mechanisms by which the mucin barrier acts to disrupt the many dynamic events leading up to cell-mediated cytotoxicity are not known.