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Microglia are resident central nervous system macrophages and the first responders to neural injury. Until recently, microglia have been studied only in animal models with exogenous or transgenic labeling. While these studies provided a wealth of information on the delicate balance between neuroprotection and neurotoxicity within which these cells operate, extrapolation to human immune function has remained an open question. Here we examine key characteristics of retinal macrophage cells in live human eyes, both healthy and diseased, with the unique capabilities of our adaptive optics-optical coherence tomography approach and owing to their propitious location above the inner limiting membrane (ILM), allowing direct visualization of cells. Our findings indicate that human ILM macrophage cells may be distributed distinctly, age differently, and have different dynamic characteristics than microglia in other animals. For example, we observed a macular pattern that was sparse centrally and peaked peripherally in healthy human eyes. Moreover, human ILM macrophage density decreased with age (~2% of cells per year). Our results in glaucomatous eyes also indicate that ILM macrophage cells appear to play an early and regionally specific role of nerve fiber layer phagocytosis in areas of active disease. While we investigate ILM macrophage cells distinct from the larger sample of overall retinal microglia, the ability to visualize macrophage cells without fluorescent labeling in the live human eye represents an important advance for both ophthalmology and neuroscience, which may lead to novel disease biomarkers and new avenues of exploration in disease progression. [ABSTRACT FROM AUTHOR]

The selectin family of leukocyte adhesion receptors is principally recognized for mediating transient rolling interactions during the inflammatory response. Recent studies using ultrasensitive force probes to characterize the force-lifetime relationship between P- and L-selectin and their endogenous ligands have underscored the ability of increasing levels of force to initially extend the lifetime of these complexes before disrupting bond integrity. This so-called "catch-slip" transition has provided an appealing explanation for shear threshold phenomena in which increasing levels of shear stress stabilize leukocyte rolling under flow. We recently incorporated catch-slip kinetics into a mechanical model for cell adhesion and corroborated this hypothesis for neutrophils adhering via L-selectin. Here, using adhesive dynamics simulations, we demonstrate that biomembrane force probe measurements of various P- and L-selectin catch bonds faithfully predict differences in cell adhesion patterns that have been described extensively in vitro. Using phenomenological parameters to characterize the dominant features of molecular force spectra, we construct a generalized phase map that reveals that robust shear-threshold behavior is possible only when an applied force very efficiently stabilizes the bound receptor complex. This criteria explains why only a subset of selectin catch bonds exhibit a shear threshold and leads to a quantitative relationship that may be used to predict the magnitude of the shear threshold for families of catch-slip bonds directly from their force spectra. Collectively, our results extend the conceptual framework of adhesive dynamics as a means to translate complex single-molecule biophysics to macroscopic cell behavior. [ABSTRACT FROM AUTHOR]

Investigates the ability of hydrodynamic collisions between cells to induce capture of free-stream leukocytes to a selection-bearing surface under shear flow. Use of cell-free assay in the study; Attachment of carbohydrate-coated spherical beads to the adhesive wall; Occurrence of hydrodynamic recruitment in the absence of buoyancy effects.

Using recombinant amphlphlllc proteins to self-assemble suprastructures would allow precise control over surfactant chemistry and the facile incorporation of biological functionality. We used cryo-TEWl to confirm self-assembled structures from recombinantly produced mutants of the naturally occurring sunflower protein, oleosin. We studied the phase behavior of protein self-assembly as a function of solution ionic strength and protein hydrophilic fraction, observing nanometric fibers, sheets, and vesicles. Vesicle membrane thickness correlated with increasing hydrophilic fraction for a fixed hydrophobic domain length. The existence of a bilayer membrane was corroborated in giant vesicles through the localized encapsulation of hydrophobic Nile red and hydrophilic calcein. Circular dichroism revealed that changes in nanostructural morphology in this family of mutants was unrelated to changes in secondary structure. Ultimately, we envision the use of recombinant techniques to introduce novel functionality into these materials for biological applications. [ABSTRACT FROM AUTHOR]

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction.We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation. [ABSTRACT FROM AUTHOR]

Precise translation of glycan-encoded information into cellular activity depends critically on highly specific functional pairing between glycans and their human lectin counter receptors. Sulfoglycolipids, such as sulfatides, are important glycolipid components of the biological membranes found in the nervous and immune systems. The optimal molecular and spatial design aspects of sulfated and nonsulfated glycans with high specificity for lectin-mediated bridging are unknown. To elucidate how different molecular and spatial aspects combine to ensure the high specificity of lectin-mediated bridging, a bottom-up toolbox is devised. To this end, negatively surface-charged glycodendrimersomes (GDSs), of different nanoscale dimensions, containing sulfo-lactose groups are self-assembled in buffer from a synthetic sulfatide mimic: Janus glycodendrimer (JGD) containing a 3'-O-sulfo-lactose headgroup. Also prepared for comparative analysis are GDSs with nonsulfated lactose, a common epitope of human membranes. These selfassembled GDSs are employed in aggregation assays with 15 galectins, comprising disease-related human galectins, and other natural and engineered variants from four families, having homodimeric, heterodimeric, and chimera architectures. There are pronounced differences in aggregation capacity between human homodimeric and heterodimeric galectins, and also with respect to their responsiveness to the charge of carbohydrate-derived ligand. Assays reveal strong differential impact of ligand surface charge and density, as well as lectin concentration and structure, on the extent of surface cross-linking. These findings demonstrate how synthetic JGD-headgroup tailoring teamed with protein engineering and network assays can help explain how molecular matchmaking operates in the cellular context of glycan and lectin complexity. [ABSTRACT FROM AUTHOR]

A three-component system of Janus dendrimers (JDs) including hydrogenated, fluorinated, and hybrid hydrogenated–fluorinated JDs are reported to coassemble by film hydration at specific ratios into an unprecedented class of supramolecular Janus particles (JPs) denoted Janus dendrimersomes (JDSs). They consist of a dumbbell-shaped structure composed of an onion-like hydrogenated vesicle and an onion-like fluorinated vesicle tethered together. The synthesis of dye-tagged analogs of each JD component enabled characterization of JDS architectures with confocal fluorescence microscopy. Additionally, a simple injection method was used to prepare submicron JDSs, which were imaged with cryogenic transmission electron microscopy (cryo-TEM). As reported previously, different ratios of the same three-component system yielded a variety of structures including homogenous onion-like vesicles, core-shell structures, and completely self-sorted hydrogenated and fluorinated vesicles. Taken together with the JDSs reported herein, a self-sorting pathway is revealed as a function of the relative concentration of the hybrid JD, which may serve to stabilize the interface between hydrogenated and fluorinated bilayers. The fission-like pathway suggests the possibility of fusion and fission processes in biological systems that do not require the assistance of proteins but instead may result from alterations in the ratios of membrane composition. [ABSTRACT FROM AUTHOR]

A library of amphiphilic Janus dendrimers including two that are fluorescent and one glycodendrimer presenting lactose were used to construct giant dendrimersomes and glycodendrimersomes. Coassembly with the components of bacterial membrane vesicles by a dehydration-rehydration process generated giant cell-like hybrid vesicles, whereas the injection of their ethanol solution into PBS produced monodisperse nanometer size assemblies. These hybrid vesicles contain transmembrane proteins including a small membrane protein, MgrB, tagged with a red fluorescent protein, lipopolysaccharides, and glycoproteins from the bacterium Escherichia coli. Incorporation of two colored fluorescent probes in each of the components allowed fluorescence microscopy to visualize and demonstrate coassembly and the incorporation of functional membrane channels. Importantly, the hybrid vesicles bind a human galectin, consistent with the display of sugar moieties from lipopolysaccharides or possibly glycosylated membrane proteins. The present coassembly method is likely to create cell-like hybrids from any biological membrane including human cells and thus may enable practical application in nanomedicine. [ABSTRACT FROM AUTHOR]

A library of eight amphiphilic Janus glycodendrimers (GDs) with D-mannose (Man) headgroups, a known routing signal for lectinmediated transport processes, was constructed via an iterative modular methodology. Sequence-defined variations of the Janus GD modulate the surface density and sequence of Man after selfassembly into multilamellar glycodendrimersomes (GDSs). The spatial mode of Man presentation is decisive for formation of either unilamellar or onion-like GDS vesicles. Man presentation and Janus GD concentration determine GDS size and number of bilayers. Beyond vesicle architecture, Man topological display affects kinetics and plateau level of GDS aggregation by a tetravalent model lectin: the leguminous agglutinin Con A, which is structurally related to endogenous cargo transporters. The agglutination process was rapid, efficient, and readily reversible for onion-like GDSs, demonstrating their value as versatile tools to explore the nature of physiologically relevant glycan/lectin pairing. [ABSTRACT FROM AUTHOR]

Probes embedded within a structure can enable prediction of material behavior or failure. Carefully assembled composites that respond intelligently to physical changes within a material could be useful as intrinsic sensors. Molecular rotors are one such tool that can respond optically to physical environmental changes. Here, we propose to use molecular rotors within a polymersome membrane to report membrane stress. Using supermolecular porphyrin-based fluorophores as rotors, we characterize changes in the optical emission of these near-infrared (NIR) emissive probes embedded within the hydrophobic core of the polymersome membrane. The configuration of entrapped fluorophore depends on the available space within the membrane; in response to increased volume, emission is blue shifted. We used this feature to study how shifts in fluorescence correlate to membrane integrity, imparted by membrane stress. We monitored changes in emission of these porphyrin-based fluorophores resulting from membrane stress produced through a range of physical and chemical perturbations, including surfactant-induced lysis, hydrolytic lysis, thermal degradation, and applied stress by micropipette aspiration. This paper comprehensively illustrates the potential for supermolecular porphyrin-based fluorophores to detect intrinsic physical changes in a wide variety of environments, and suggests how molecular rotors may be used in soft materials science and biology as sensors. [ABSTRACT FROM AUTHOR]

We demonstrate that synthetic soft materials can extend the utility of natural vesicles, from predominantly hydrophilic reservoirs to functional colloidal carriers that facilitate the biomedical application of large aqueous-insoluble compounds. Near-infrared (NIR)- emissive polymersomes (50-nm- to 50-μm-diameter polymer yes-ides) were generated through cooperative self assembly of amphiphilic diblock copolymers and conjugated multi(porphyrin)- based NIR fluorophores (NIRFs). When compared with natural vesicles comprised of phospholipids, polymersomes were uniquely capable of incorporating and uniformly distributing numerous large hydrophobic NIRFs exclusively in their lamellar membranes. Within these sequestered compartments, long polymer chains regulate the mean fluorophore-fluorophore interspatial separation as well as the fluorophore-localized electronic environment. Porphyrin-based NIRFs manifest photophysical properties within the polymersomal matrix akin to those established for these high-emission dipole strength fluorophores in organic solvents, thereby yielding uniquely emissive vesicles. Furthermore, the total fluorescence emanating from the assemblies gives rise to a localized optical signal of sufficient intensity to penetrate through the dense tumor tissue of a live animal. Robust NIR-emissive polymersomes thus define a soft matter platform with exceptional potential to facilitate deep-tissue fluorescence-based imaging for in vivo diagnostic and drug-delivery applications [ABSTRACT FROM AUTHOR]