Myosin II isoforms play distinct roles in junction biogenesis.

Adherens junction (AJ) assembly under force is essential for many biological processes like epithelial monolayer bending, collective cell migration, cell extrusion and wound healing. The acto-myosin cytoskeleton acts as a major force-generator during the de novo formation and remodeling of AJ. Here, we investigated the role of non-muscle myosin II isoforms (NMIIA and NMIIB) in epithelial junction assembly.

Cell-free microcompartmentalised transcription-translation for the prototyping of synthetic communication networks.

Recent efforts in synthetic biology have shown the possibility of engineering distributed functions in populations of living cells, which requires the development of highly orthogonal, genetically encoded communication pathways. Cell-free transcription-translation (TXTL) reactions encapsulated in microcompartments enable prototyping of molecular communication channels and their integration into engineered genetic circuits by mimicking critical cell features, such as gene expression, cell size, and cell individuality within a community. In this review, we discuss the uses of cell-free transcription-translation reactions for the development of synthetic genetic circuits, with a special focus on the use of microcompartments supporting this reaction.

Self-Assembling VHH-Elastin-Like Peptides for Photodynamic Nanomedicine.

Recombinant llama heavy-chain antibody fragments (VHHs) are promising tools in the field of targeted nanomedicine. 7D12, a VHH against the epidermal growth factor receptor (EGFR) that is overexpressed in various cancers, has been evaluated as an effective cancer-targeting VHH in multiple studies. The small size of VHHs (15-20 kDa) results in a low circulation half-life, which can be disadvantageous for certain applications.

Continuous fabrication of polymeric vesicles and nanotubes with fluidic channels.

Fluidic channels were employed to induce the self-assembly of poly(ethylene glycol)-b-polystyrene into polymeric vesicles and nanotubes. The laminar flow in the device enables controlled diffusion of two miscible liquids at the phase boundary, leading to the formation of homogeneous polymeric structures of different shapes. These structures could be easily loaded with small molecule cargoes and functionalized with nanometer sized catalytic platinum nanoparticles.

Legomedicine-A Versatile Chemo-Enzymatic Approach for the Preparation of Targeted Dual-Labeled Llama Antibody-Nanoparticle Conjugates.

Conjugation of llama single domain antibody fragments (Variable Heavy chain domains of Heavy chain antibodies, VHHs) to diagnostic or therapeutic nanoparticles, peptides, proteins, or drugs offers many opportunities for optimized targeted cancer treatment. Currently, mostly nonspecific conjugation strategies or genetic fusions are used that may compromise VHH functionality. In this paper we present a versatile modular approach for bioorthogonal VHH modification and conjugation.

A Compartmentalized Out-of-Equilibrium Enzymatic Reaction Network for Sustained Autonomous Movement.

Every living cell is a compartmentalized out-of-equilibrium system exquisitely able to convert chemical energy into function. In order to maintain homeostasis, the flux of metabolites is tightly controlled by regulatory enzymatic networks. A crucial prerequisite for the development of lifelike materials is the construction of synthetic systems with compartmentalized reaction networks that maintain out-of-equilibrium function.

Supramolecular Adaptive Nanomotors with Magnetotaxis Behavior.

With a convenient bottom-up approach, magnetic metallic nickel is grown in situ of a supramolecular nanomotor using the catalytic activities of preloaded platinum nanoparticles. After introducing magnetic segments, simultaneous guidance and steering of catalytically powered motors with additional magnetic fields are achieved. Guided motion in a tissue model is demonstrated.

Coiled-Coil-Mediated Activation of Oligoarginine Cell-Penetrating Peptides.

A supramolecular approach was undertaken to create functionally activatable cell-penetrating peptides. Two tetra-arginines were assembled into an active cell-penetrating peptide by heterodimerizing leucine zippers. Three different leucine-zipper pairs were evaluated: activation was found to depend on the association constant of the coiled-coil peptides.

Click-MS: Tagless Protein Enrichment Using Bioorthogonal Chemistry for Quantitative Proteomics.

Epitope-tagging is an effective tool to facilitate protein enrichment from crude cell extracts. Traditionally, N- or C-terminal fused tags are employed, which, however, can perturb protein function. Unnatural amino acids (UAAs) harboring small reactive handles can be site-specifically incorporated into proteins, thus serving as a potential alternative for conventional protein tags.

Highly efficient enzyme encapsulation in a protein nanocage: towards enzyme catalysis in a cellular nanocompartment mimic.

The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved.

Formation of Well-Defined, Functional Nanotubes via Osmotically Induced Shape Transformation of Biodegradable Polymersomes.

Polymersomes are robust, versatile nanostructures that can be tailored by varying the chemical structure of copolymeric building blocks, giving control over their size, shape, surface chemistry, and membrane permeability. In particular, the generation of nonspherical nanostructures has attracted much attention recently, as it has been demonstrated that shape affects function in a biomedical context. Until now, nonspherical polymersomes have only been constructed from nondegradable building blocks, hampering a detailed investigation of shape effects in nanomedicine for this category of nanostructures.

An integrated, peptide-based approach to site-specific protein immobilization for detection of biomolecular interactions.

We have developed an integrated solution for the site-specific immobilization of proteins on a biosensor surface, which may be widely applicable for high throughput analytical purposes. The gold surface of a biosensor was coated with an anti-fouling layer of zwitterionic peptide molecules from which leucine zipper peptides protrude. Proteins of interest, the autoantigenic proteins La and U1A, were immobilized via a simple incubation procedure by using the complementary leucine zipper sequence as a genetically fused binding tag.

Metal Ion-Induced Self-Assembly of a Multi-Responsive Block Copolypeptide into Well-Defined Nanocapsules.

Protein cages are an interesting class of biomaterials with potential applications in bionanotechnology. Therefore, substantial effort is spent on the development of capsule-forming designer polypeptides with a tailor-made assembly profile. The expanded assembly profile of a triblock copolypeptide consisting of a metal ion chelating hexahistidine-tag, a stimulus-responsive elastin-like polypeptide block, and a pH-responsive morphology-controlling viral capsid protein is presented.

Tuning Ice Nucleation with Supercharged Polypeptides.

Supercharged unfolded polypeptides (SUPs) are exploited for controlling ice nucleation via tuning the nature of charge and charge density of SUPs. The results show that positively charged SUPs facilitate ice nucleation, while negatively charged ones suppress it. Moreover, the charge density of the SUP backbone is another parameter to control it.

A covalent and cleavable antibody-DNA conjugation strategy for sensitive protein detection via immuno-PCR.

Immuno-PCR combines specific antibody-based protein detection with the sensitivity of PCR-based quantification through the use of antibody-DNA conjugates. The production of such conjugates depends on the availability of quick and efficient conjugation strategies for the two biomolecules. Here, we present an approach to produce cleavable antibody-DNA conjugates, employing the fast kinetics of the inverse electron-demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO).

Dynamic Loading and Unloading of Proteins in Polymeric Stomatocytes: Formation of an Enzyme-Loaded Supramolecular Nanomotor.

Self-powered artificial nanomotors are currently attracting increased interest as mimics of biological motors but also as potential components of nanomachinery, robotics, and sensing devices. We have recently described the controlled shape transformation of polymersomes into bowl-shaped stomatocytes and the assembly of platinum-driven nanomotors. However, the platinum encapsulation inside the structures was low; only 50% of the structures contained the catalyst and required both high fuel concentrations for the propelling of the nanomotors and harsh conditions for the shape transformation.

Compartmentalization Approaches in Soft Matter Science: From Nanoreactor Development to Organelle Mimics.

Compartmentalization is an essential feature found in living cells to ensure that biological processes occur without being affected by undesired external influences. Over the years many scientists have designed self-assembled soft matter structures that mimic these natural catalytic compartments. The rationale behind this research is threefold.

Sortase A-Mediated N-Terminal Modification of Cowpea Chlorotic Mottle Virus for Highly Efficient Cargo Loading.

A new strategy is described for the modification of CCMV for loading of cargoes inside the viral capsid. Sortase A, an enzyme which is present in Gram-positive bacteria, was used to attach cargo to the glycine-tagged N-termini of several CCMV variants. We show that small molecules and proteins bearing a C-terminal LPETG-motif can be attached in this way.

Self-Guided Supramolecular Cargo-Loaded Nanomotors with Chemotactic Behavior towards Cells.

Delivery vehicles that are able to actively seek and precisely locate targeted tissues using concentration gradients of signaling molecules have hardly been explored. The directed movement toward specific cell types of cargo-loaded polymeric nanomotors along a hydrogen peroxide concentration gradient (chemotaxis) is reported. Through self-assembly, bowl-shaped poly(ethylene glycol)-b-polystyrene nanomotors, or stomatocytes, were formed with platinum nanoparticles entrapped in the cavity while a model drug was encapsulated in the inner compartment.

Soft PEG-Hydrogels with Independently Tunable Stiffness and RGDS-Content for Cell Adhesion Studies.

Poly(ethylene)glycol (PEG)-based hydrogels are often used as matrix material for cell culturing. An efficient method to prepare soft PEG gels is by cross-linking via copper-free strain-promoted azide-alkyne cycloaddition (SPAAC). Here, the effect of polymer density and RGDS-content on hydrogel formation and cell adhesion was studied, by varying the total polymer content (10, 20 and 30 mg · mL(-1) ) and the amount of RGDS moieties (0-100%) independently of each other.

Reversibly Triggered Protein-Ligand Assemblies in Giant Vesicles.

External small-molecule triggers were used to reversibly control dynamic protein-ligand interactions in giant vesicles. An alcohol dehydrogenase was employed to increase or decrease the interior pH upon conversion of two different small-molecule substrates, thereby modulating the pH-sensitive interaction between a Ni-NTA ligand on the vesicle membrane and an oligohistidine-tagged protein in the lumen. By alternating the small-molecule substrates the interaction could be reversed.

Enzyme-Activatable Cell-Penetrating Peptides through a Minimal Side Chain Modification.

Activatable cell-penetrating peptides are of great interest in drug delivery because of their enhanced selectivity which can be controlled by the external stimuli that trigger their activation. The use of a specific enzymatic reaction to trigger uptake of an inert peptide offers a relevant targeting strategy because the activation process takes place in a short time and only in areas where the specific cell surface enzyme is present. To this aim, the lysine side chain of Tat peptides was modified with an enzyme-cleavable domain of minimal size.

The influence of amino acid sequence on structure and morphology of polydiacetylene containing peptide fibres.

A systematic study was performed on the influence of charge and steric hindrance on the assembly into fibres of a series of pentameric peptides based on the well-known β-sheet forming sequence Gly-Ala-Gly-Ala-Gly, which were N-terminally acylated with pentacosadiynoic acid. To investigate the effect of steric hindrance and charge repulsion on the fibre structure, either the N-terminal or the C-terminal amino acid in the sequence was replaced by a glutamic acid or lysine residue. Furthermore, peptide amphiphiles (PAs) with an amide or a free acid group at the C-terminus were compared.

A fast and activatable cross-linking strategy for hydrogel formation.

Strain-promoted oxidation-controlled cyclo-octyne-1,2-quinone cycloaddition (SPOCQ) is a fast and activatable cross-linking strategy for hydrogel formation. Gelation is induced by oxidation, which is performed both chemically using sodium periodate and enzymatically using mushroom tyrosinase. Due to the fast reaction kinetics, SPOCQ-formed hydrogels can be functionalized in one-pot with an azido-containing moiety using SPAAC cross-linking.

Strain-promoted oxidation-controlled cyclooctyne-1,2-quinone cycloaddition (SPOCQ) for fast and activatable protein conjugation.

A main challenge in the area of bioconjugation is to devise reactions that are both activatable and fast. Here, we introduce a temporally controlled reaction between cyclooctynes and 1,2-quinones, induced by facile oxidation of 1,2-catechols. This so-called strain-promoted oxidation-controlled cyclooctyne-1,2-quinone cycloaddition (SPOCQ) shows a remarkably high reaction rate when performed with bicyclononyne (BCN), outcompeting the well-known cycloaddition of azides and BCN by 3 orders of magnitude, thereby allowing a new level of orthogonality in protein conjugation.