Genetically-encoded phase separation sensors for intracellular probing of biomolecular condensates.

Biomolecular condensates are dynamic membraneless compartments with enigmatic roles across intracellular phenomena. Intrinsically-disordered proteins (IDPs) often function as condensate scaffolds, fueled by their liquid-liquid phase separation (LLPS) dynamics. Intracellular probing of these condensates relies on live-cell imaging of IDP-scaffolds tagged with fluorescent proteins.

Programmability and biomedical utility of intrinsically-disordered protein polymers.

Intrinsically disordered proteins (IDPs) exhibit molecular-level conformational dynamics that are functionally harnessed across a wide range of fascinating biological phenomena. The low sequence complexity of IDPs has led to the design and development of intrinsically-disordered protein polymers (IDPPs), a class of engineered repeat IDPs with stimuli-responsive properties. The perfect repetitive architecture of IDPPs allows for repeat-level encoding of tunable protein functionality.

Cracking the Skin Barrier: Liquid-Liquid Phase Separation Shines under the Skin.

Central to forming and sustaining the skin's barrier, epidermal keratinocytes (KCs) fluxing to the skin surface undergo a rapid and enigmatic transformation into flat, enucleated squames. At the crux of this transformation are intracellular keratohyalin granules (KGs) that suddenly disappear as terminally differentiating KCs transition to the cornified skin surface. Defects in KGs have long been linked to skin barrier disorders.

Nanoscopic Dynamics Dictate the Phase Separation Behavior of Intrinsically Disordered Proteins.

Many intrinsically disordered proteins (IDPs) in nature may undergo liquid-liquid phase separation to assemble membraneless organelles with varied liquid-like properties and stability/dynamics. While solubility changes underlie these properties, little is known about hydration dynamics in phase-separating IDPs. Here, by studying IDP polymers of similar composition but distinct liquid-like dynamics and stability upon separation, namely, thermal hysteresis, we probe at a nanoscopic level hydration/dehydration dynamics in IDPs as they reversibly switch between phase separation states.

Publisher Correction: Mechanics of a multilayer epithelium instruct tumour architecture and function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Liquid-liquid phase separation drives skin barrier formation.

At the body surface, skin's stratified squamous epithelium is challenged by environmental extremes. The surface of the skin is composed of enucleated, flattened surface squames. They derive from underlying, transcriptionally active keratinocytes that display filaggrin-containing keratohyalin granules (KGs) whose function is unclear.

Intrinsically disordered proteins access a range of hysteretic phase separation behaviors.

The phase separation behavior of intrinsically disordered proteins (IDPs) is thought of as analogous to that of polymers that undergo equilibrium lower or upper critical solution temperature (LCST and UCST, respectively) phase transition. This view, however, ignores possible nonequilibrium properties of protein assemblies. Here, by studying IDP polymers (IDPPs) composed of repeat motifs that encode LCST or UCST phase behavior, we discovered that IDPs can access a wide spectrum of nonequilibrium, hysteretic phase behaviors.

Sequence Directionality Dramatically Affects LCST Behavior of Elastin-Like Polypeptides.

Elastin-like polypeptides (ELP) exhibit an inverse temperature transition or lower critical solution temperature (LCST) transition phase behavior in aqueous solutions. In this paper, the thermal responsive properties of the canonical ELP, poly(VPGVG), and its reverse sequence poly(VGPVG) were investigated by turbidity measurements of the cloud point behavior, circular dichroism (CD) measurements, and all-atom molecular dynamics (MD) simulations to gain a molecular understanding of mechanism that controls hysteretic phase behavior. It was shown experimentally that both poly(VPGVG) and poly(VGPVG) undergo a transition from soluble to insoluble in aqueous solution upon heating above the transition temperature ( T).

Micellar Self-Assembly of Recombinant Resilin-/Elastin-Like Block Copolypeptides.

Reported here is the synthesis of perfectly sequence defined, monodisperse diblock copolypeptides of hydrophilic elastin-like and hydrophobic resilin-like polypeptide blocks and characterization of their self-assembly as a function of structural parameters by light scattering, cryo-TEM, and small-angle neutron scattering. A subset of these diblock copolypeptides exhibit lower critical solution temperature and upper critical solution temperature phase behavior and self-assemble into spherical or cylindrical micelles. Their morphologies are dictated by their chain length, degree of hydrophilicity, and hydrophilic weight fraction of the ELP block.

Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers.

Proteins and synthetic polymers that undergo aqueous phase transitions mediate self-assembly in nature and in man-made material systems. Yet little is known about how the phase behaviour of a protein is encoded in its amino acid sequence. Here, by synthesizing intrinsically disordered, repeat proteins to test motifs that we hypothesized would encode phase behaviour, we show that the proteins can be designed to exhibit tunable lower or upper critical solution temperature (LCST and UCST, respectively) transitions in physiological solutions.

Molecular description of the LCST behavior of an elastin-like polypeptide.

Elastin-like polypeptides (ELPs) with the repeat sequence of VPGVG are widely used as a model system for investigation of lower critical solution temperature (LCST) transition behavior. In this paper, the effect of temperature on the structure, dynamics and association of (VPGVG)18 in aqueous solution is investigated using atomistic molecular dynamics simulations. Our simulations show that as the temperature increases the ELP backbones undergo gradual conformational changes, which are attributed to the formation of more ordered secondary structures such as β-strands.

Brachytherapy using injectable seeds that are self-assembled from genetically encoded polypeptides in situ.

Brachytherapy is a common clinical technique involving implantation of sealed radioactive "seeds" within a tumor to selectively irradiate the tumor mass while minimizing systemic toxicity. To mitigate the disadvantages associated with complex surgical implantation and subsequent device removal procedures, we have developed an alternative approach using a genetically encoded peptide polymer solution composed of a thermally responsive elastin-like polypeptide (ELP) radiolabeled with (131)I that self-assembles into radionuclide seeds upon intratumoral injection. The formation of these nontoxic and biodegradable polymer seeds led to prolonged intratumoral retention (~85% ID/tumor 7 days postinjection) of the radionuclide, elicited a tumor growth delay in 100% of the tumors in two human xenografts (FaDu and PC-3), and cured more than 67% of tumor-bearing animals after a single administration of labeled ELP.

Protein polymer hydrogels by in situ, rapid and reversible self-gelation.

Protein-based biomaterials are an important class of materials for applications in biotechnology and medicine. The exquisite control of their composition, stereochemistry, and chain length offers unique opportunities to engineer biofunctionality, biocompatibility, and biodegradability into these materials. Here, we report the synthesis of a thermally responsive peptide polymer-based hydrogel composed of a recombinant elastin-like polypeptide (ELP) that rapidly forms a reversibly cross-linked hydrogel by the formation of intermolecular disulfide cross-links.

Reinforced Portland cement porous scaffolds for load-bearing bone tissue engineering applications.

Modified Portland cement porous scaffolds with suitable characteristics for load-bearing bone tissue engineering applications were manufactured by combining the particulate leaching and foaming methods. Non-crosslinked polydimethylsiloxane was evaluated as a potential reinforcing material. The scaffolds presented average porosities between 70 and 80% with mean pore sizes ranging from 300 μm up to 5.

Portland cement for bone tissue engineering: Effects of processing and metakaolin blends.

The need for a suitable scaffolding material for load bearing bone tissue engineering still has yet to be met satisfactorily. In this study, Portland cement and Portland cement/metakaolin (MK) blends were processed to render them biologically and mechanically suitable for such application. Portland cement was mixed with MK at different ratios.

A highly parallel method for synthesizing DNA repeats enables the discovery of 'smart' protein polymers.

Robust high-throughput synthesis methods are needed to expand the repertoire of repetitive protein-polymers for different applications. To address this need, we developed a new method, overlap extension rolling circle amplification (OERCA), for the highly parallel synthesis of genes encoding repetitive protein-polymers. OERCA involves a single PCR-type reaction for the rolling circle amplification of a circular DNA template and simultaneous overlap extension by thermal cycling.

Housekeeping gene stability influences the quantification of osteogenic markers during stem cell differentiation to the osteogenic lineage.

Real-time reverse transcription PCR (RT-qPCR) relies on a housekeeping or normalizer gene whose expression remains constant throughout the experiment. RT-qPCR is commonly used for characterization of human bone marrow mesenchymal stem cells (hBMSCs). However, to the best of our knowledge, there are no studies validating the expression stability of the genes used as normalizers during hBMSCs differentiation.

Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes.

This paper reports a new strategy, recursive directional ligation by plasmid reconstruction (PRe-RDL), to rapidly clone highly repetitive polypeptides of any sequence and specified length over a large range of molecular weights. In a single cycle of PRe-RDL, two halves of a parent plasmid, each containing a copy of an oligomer, are ligated together, thereby dimerizing the oligomer and reconstituting a functional plasmid. This process is carried out recursively to assemble an oligomeric gene with the desired number of repeats.

Amyloid-beta-induced ion flux in artificial lipid bilayers and neuronal cells: resolving a controversy.

Understanding the pathogenicity of amyloid-beta (Abeta) peptides constitutes a major goal in research on Alzheimer's disease (AD). One hypothesis entails that Abeta peptides induce uncontrolled, neurotoxic ion flux through cellular membranes. The exact biophysical mechanism of this ion flux is, however, a subject of an ongoing controversy which has attenuated progress toward understanding the importance of Abeta-induced ion flux in AD.