Allosteric interactions prime androgen receptor dimerization and activation.

The androgen receptor (AR) is a nuclear receptor that governs gene expression programs required for prostate development and male phenotype maintenance. Advanced prostate cancers display AR hyperactivation and transcriptome expansion, in part, through AR amplification and interaction with oncoprotein cofactors. Despite its biological importance, how AR domains and cofactors cooperate to bind DNA has remained elusive. Using single-particle cryo-electron microscopy, we isolated three conformations of AR bound to DNA, showing that AR forms a non-obligate dimer, with the buried dimer interface utilized by ancestral steroid receptors repurposed to facilitate cooperative DNA binding. We identify novel allosteric surfaces which are compromised in androgen insensitivity syndrome and reinforced by AR's oncoprotein cofactor, ERG, and by DNA-binding motifs. Finally, we present evidence that this plastic dimer interface may have been adopted for transactivation at the expense of DNA binding. Our work highlights how fine-tuning AR's cooperative interactions translate to consequences in development and disease.

Connected Peptide Modules Enable Controlled Co-Existence of Self-Assembled Fibers Inside Liquid Condensates.

Supramolecular self-assembly of fibrous components and liquid-liquid phase separation are at the extremes of the order-to-disorder spectrum. They collectively play key roles in cellular organization. It is still a major challenge to design systems where both highly ordered nanostructures and liquid-liquid phase-separated domains can coexist. We present a three-component assembly approach that generates fibrous domains that exclusively form inside globally disordered, liquid condensates. This is achieved by creating amphiphilic peptides that combine the features of fibrillar assembly (the amyloid domain LVFFA) and complex coacervation (oligo-arginine and adenosine triphosphate (ATP)) in one peptide, namely, LVFFAR9. When this hybrid peptide is mixed in different ratios with R9 and ATP, we find that conditions can be created where fibrous assembly is exclusively observed inside liquid coacervates. Through fluorescence and atomic force microscopy characterization, we investigate the dynamic evolution of ordered and disordered features over time. It was observed that the fibers nucleate and mature inside the droplets and that these fiber-containing liquid droplets can also undergo fusion, showing that the droplets remain liquid-like. Our work thus generates opportunities for the design of ordered structures within the confined environment of biomolecular condensates, which may be useful to create supramolecular materials in defined compartments and as model systems that can enhance understanding of ordering principles in biology.

Atomic Force Microscopy-Based Measurements of Retinal Microvessel Stiffness in Mice with Endothelial-Specific Deletion of CCN1.

Vascular stiffness is an independent predictor of human vascular diseases and is linked to ischemia, diabetes, high blood pressure, hyperlipidemia, and/or aging. Blood vessel stiffening increases owing to changes in the microscale architecture and/or content of extracellular, cytoskeletal, and nuclear matrix proteins. These alterations, while best appreciated in large blood vessels, also gradually occur in the microvasculature and play an important role in the initiation and progression of numerous microangiopathies including diabetic retinopathy. Although macroscopic measurements of arterial stiffness by pulse wave velocity are often used for clinical diagnosis, stiffness changes of intact microvessels and their causative factors have not been characterized. Herein, we describe the use of atomic force microscopy (AFM) to determine stiffness of mouse retinal capillaries and assess its regulation by the cellular communication network (CCN) 1, a stiffness-sensitive gene-encoded matricellular protein. AFM yields reproducible measurements of retinal capillary stiffness in lightly fixed freshly isolated retinal flat mounts. AFM measurements also show significant changes in compliance properties of the retinal microvasculature of mice with endothelial-specific deletion of CCN1, indicating that CCN1 expression, or lack thereof, affects the mechanical properties of microvascular cells in vivo. Thus, AFM has the force sensitivity and the spatial resolution necessary to measure the local modulus of retinal capillaries in situ and eventually to investigate microvascular compliance heterogeneities as key components of disease pathogenesis.

Rear traction forces drive adherent tissue migration in vivo.

During animal embryogenesis, homeostasis and disease, tissues push and pull on their surroundings to move forward. Although the force-generating machinery is known, it is unknown how tissues exert physical stresses on their substrate to generate motion in vivo. Here, we identify the force transmission machinery, the substrate and the stresses that a tissue, the zebrafish posterior lateral line primordium, generates during its migration. We find that the primordium couples actin flow through integrins to the basement membrane for forward movement. Talin- and integrin-mediated coupling is required for efficient migration, and its loss is partially compensated for by increased actin flow. Using Embryogram, an approach to measure stresses in vivo, we show that the rear of the primordium exerts higher stresses than the front, which suggests that this tissue pushes itself forward with its back. This unexpected strategy probably also underlies the motion of other tissues in animals.

Peptide density targets and impedes triple negative breast cancer metastasis.

The C-X-C chemokine receptor type 4 (CXCR4, CD184) pathway is a key regulator of cancer metastasis. Existing therapeutics that block CXCR4 signaling are dependent on single molecule-receptor interactions or silencing CXCR4 expression. CXCR4 localizes in lipid rafts and forms dimers therefore CXCR4 targeting and signaling may depend on ligand density. Herein, we report liposomes presenting a CXCR4 binding peptide (DV1) as a three-dimensional molecular array, ranging from 9k to 74k molecules µm-2, target triple negative breast cancer (TNBC). TNBC cells exhibit a maxima in binding and uptake of DV1-functionalized liposomes (L-DV1) in vitro at a specific density, which yields a significant reduction in cell migration. This density inhibits metastasis from a primary tumor for 27 days, resulting from peptide density dependent gene regulation. We show that complementing cell membrane receptor expression may be a strategy for targeting cells and regulating signaling.

Nanoparticle elasticity directs tumor uptake.

To date, the role of elasticity in drug delivery remains elusive due to the inability to measure microscale mechanics and alter rheology without affecting chemistry. Herein, we describe the in vitro cellular uptake and in vivo tumor uptake of nanolipogels (NLGs). NLGs are composed of identical lipid bilayers encapsulating an alginate core, with tunable elasticity. The elasticity of NLGs was evaluated by atomic force microscopy, which demonstrated that they exhibit Young's moduli ranging from 45 ± 9 to 19,000 ± 5 kPa. Neoplastic and non-neoplastic cells exhibited significantly greater uptake of soft NLGs (Young's modulus <1.6 MPa) relative to their elastic counterparts (Young's modulus >13.8 MPa). In an orthotopic breast tumor model, soft NLGs accumulated significantly more in tumors, whereas elastic NLGs preferentially accumulated in the liver. Our findings demonstrate that particle elasticity directs tumor accumulation, suggesting that it may be a design parameter to enhance tumor delivery efficiency.

Exceptional thermoelectric properties of flexible organic-inorganic hybrids with monodispersed and periodic nanophase.

Flexible organic-inorganic hybrids are promising thermoelectric materials to recycle waste heat in versatile formats. However, current organic/inorganic hybrids suffer from inferior thermoelectric properties due to aggregate nanostructures. Here we demonstrate flexible organic-inorganic hybrids where size-tunable Bi2Te3 nanoparticles are discontinuously monodispersed in the continuous conductive polymer phase, completely distinct from traditional bi-continuous hybrids. Periodic nanofillers significantly scatter phonons while continuous conducting polymer phase provides favored electronic transport, resulting in ultrahigh power factor of ~1350 µW m-1 K-2 and ultralow in-plane thermal conductivity of ~0.7 W m-1 K-1. Consequently, figure-of-merit (ZT) of 0.58 is obtained at room temperature, outperforming all reported organic materials and organic-inorganic hybrids. Thermoelectric properties of as-fabricated hybrids show negligible change for bending 100 cycles, indicating superior mechanical flexibility. These findings provide significant scientific foundation for shaping flexible thermoelectric functionality via synergistic integration of organic and inorganic components.

Minimizing antibody surface density on liposomes while sustaining cytokine-activated EC targeting.

Liposomes may be engineered to target inflamed endothelium by mimicking ligand-receptor interactions between leukocytes and cytokine-activated endothelial cells (ECs). The upregulation and assembly of vascular cell adhesion molecule-1 (VCAM1) and E-selectin on the cell membrane upon exposure to cytokines have shown potential for drug delivery vehicles to target sites of chronic endothelial inflammation, such as atherosclerosis and cancer. Herein, we characterized EC surfaces by measuring the E-selectin and VCAM1 surface densities and adhesion forces of aVCAM1 and aE-selectin to ECs. We quantified the antibody density, ratio, and diffusivity of liposomes to achieve significant binding and internalization. At 1 h, the 1:1 ratio of VCAM1:E-selectin antibodies was significantly higher than 1:0 and 0:1. Significant binding and uptake was achieved at aE-selectin densities as low as 400 molecules/µm(2). The highest levels of binding and uptake were achieved when using a 1:1 ratio of VCAM1:E-selectin antibodies at a density of 1000 molecules/µm(2); this density is 85% lower than previous reports. The binding and uptake of functionalized liposomes were reduced to levels comparable to IgG functionalized liposomes upon a 10-fold reduction in liposome membrane diffusivity. We conclude with a liposomal design that discriminates between healthy and inflamed endothelium while reducing antibody surface presentation.

Mapping the CXCR4 receptor on breast cancer cells.

The CXCR4 receptor triggers cell migration and, in breast cancer, promotes metastasis. To date, the dynamic assembly of CXCR4 on the cell surface as a mediator of receptor binding is not well characterized. The objective of this work is to quantify the density, spatial organization, and magnitude of binding of the CXCR4 receptor on live metastatic breast cancer (MBC) cells. We measured the Young's modulus, the CXCR4 surface density, and CXCR4 unbinding force on MBC cells by atomic force microscopy. We conclude that the CXCR4 density, spatial organization, and matrix stiffness are paramount to achieve strong binding.

A general approach for the microrheology of cancer cells by atomic force microscopy.

The determination of the viscoelastic properties of cells by atomic force microscopy (AFM) is mainly realized by looking at the relaxation of the force when a constant position of the AFM head is maintained or at the evolution of the indentation when a constant force is maintained. In both cases the analysis rests on the hypothesis that the motion of the probe before the relaxation step is realized in a time which is much smaller than the characteristic relaxation time of the material. In this paper we carry out a more general analysis of the probe motion which contains both the indentation and relaxation steps, allowing a better determination of the rheological parameters. This analysis contains a correction of the Hertz model for large indentation and also the correction due to the finite thickness of the biological material; it can be applied to determine the parameters representing any kind of linear viscoelastic model. This approach is then used to model the rheological behavior of one kind of cancer cell called Hep-G2. For this kind of cell, a power law model does not well describe the low and high frequency modulus contrary to a generalized Maxwell model.