The Roles of Micro- and Nanoscale Materials in Cell-Engineering Systems.

Customizable manufacturing of ex vivo cell engineering is driven by the need for innovations in the biomedical field and holds substantial potential for addressing current therapeutic challenges; but it is still only in its infancy. Micro- and nanoscale-engineered materials are increasingly used to control core cell-level functions in cellular engineering. By reprogramming or redirecting targeted cells for extremely precise functions, these advanced materials offer new possibilities.

Transparent, Antibiofouling Window Obtained with Surface Nanostructuring.

Biofouling is one of the key factors which limits the long-term performance of seawater sensors. Common measures to hinder biofouling include toxic paints, mechanical cleaning and UV radiation. All of these measures have various limitations.

Membrane localization of actin filaments stabilizes giant unilamellar vesicles against external deforming forces.

Actin organization is crucial for establishing cell polarity, which influences processes such as directed cell motility and division. Despite its critical role in living organisms, achieving similar polarity in synthetic cells remains challenging. In this study, we employ a bottom-up approach to investigate how molecular crowders facilitate the formation of cortex-like actin networks and how these networks localize and organize based on membrane shape.

Evaluation of Acoustophoretic and Dielectrophoretic Forces for Droplet Injection in Droplet-Based Microfluidic Devices.

Acoustophoretic forces have been successfully implemented into droplet-based microfluidic devices to manipulate droplets. These acoustophoretic forces in droplet microfluidic devices are typically generated as in acoustofluidic devices through transducer actuation of a piezoelectric substrate such as lithium niobate (LiNbO), which is inherently accompanied by the emergence of electrical fields. Understanding acoustophoretic versus dielectrophoretic forces produced by electrodes and transducers within active microfluidic devices is important for the optimization of device performance during design iterations.

Designed Ankyrin Repeat Proteins as Actin Labels of Distinct Cytoskeletal Structures in Living Cells.

The orchestrated assembly of actin and actin-binding proteins into cytoskeletal structures coordinates cell morphology changes during migration, cytokinesis, and adaptation to external stimuli. The accurate and unbiased visualization of the diverse actin assemblies within cells is an ongoing challenge. We describe here the identification and use of designed ankyrin repeat proteins (DARPins) as synthetic actin binders.

An Efficient Method for the Production of High-Purity Bioinspired Large Unilamellar Vesicles.

In order to recapitulate complex eukaryotic compartmentalization, synthetic biology aims to recreate cellular membrane-lined compartments from the bottom-up. Many important cellular organelles and cell-produced extracellular vesicles are in the size range of several hundreds of nanometers. Although attaining a fundamental characterization and mimicry of their cellular functions is a compelling goal, the lack of methods for controlled vesicle formation in this size range has hindered full understanding.

Bottom-up Assembled Synthetic SARS-CoV-2 Miniviruses Reveal Lipid Membrane Affinity of Omicron Variant Spike Glycoprotein.

The ongoing COVID-19 pandemic has been brought on by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike glycoprotein (S), which decorates the viral envelope forming a corona, is responsible for the binding to the angiotensin-converting enzyme 2 (ACE2) receptor and initiating the infection. In comparison to previous variants, Omicron S presents additional binding sites as well as a more positive surface charge.

Elucidating the Morphology of the Endoplasmic Reticulum: Puzzles and Perspectives.

Artificial or synthetic organelles are a key challenge for bottom-up synthetic biology. So far, synthetic organelles have typically been based on spherical membrane compartments, used to spatially confine selected chemical reactions. In vivo, these compartments are often far from being spherical and can exhibit rather complex architectures.

Bottom-Up Assembly of Bioinspired, Fully Synthetic Extracellular Vesicles.

Extracellular vesicles (EVs) are lipid membrane-enclosed compartments released by cells for intercellular communication in homeostasis and disease. Studies have shown great therapeutic potential of EVs, including but not limited to regenerative and immunomodulatory therapies. Additionally, EVs are promising next-generation drug delivery systems due to their biocompatibility, low immunogenicity, and inherent target specificity.

Immune complex-induced haptokinesis in human non-classical monocytes.

Formation and deposition of immune complexes (ICs) are hallmarks of various autoimmune diseases. Detection of ICs by IC receptors on leukocytes induces downstream signaling and shapes the local immune response. In many cases the pathological relevance of ICs is not well understood.

Confined environments induce polarized paraspeckle condensates.

Cancer cells experience confinement as they navigate the tumour microenvironment during metastasis. Recent studies have revealed that the nucleus can function as a 'ruler' for measuring physical confinement via membrane tension, allowing for compression-sensitive changes in migration. Cell nuclei contain many nuclear bodies that form when their components phase separate and condense within permissive local regions within the nucleus.

Convenient site-selective protein coupling from bacterial raw lysates to coenzyme A-modified tobacco mosaic virus (TMV) by Bacillus subtilis Sfp phosphopantetheinyl transferase.

A facile enzyme-mediated strategy enables site-specific covalent one-step coupling of genetically tagged luciferase molecules to coenzyme A-modified tobacco mosaic virus (TMV-CoA) both in solution and on solid supports. Bacillus subtilis surfactin phosphopantetheinyl transferase Sfp produced in E. coli mediated the conjugation of firefly luciferase N-terminally extended by eleven amino acids forming a 'ybbR tag' as Sfp-selective substrate, which even worked in bacterial raw lysates.

Artificial Cytoskeleton Assembly for Synthetic Cell Motility.

Imitation of cellular processes in cell-like compartments is a current research focus in synthetic biology. Here, a method is introduced for assembling an artificial cytoskeleton in a synthetic cell model system based on a poly(N-isopropyl acrylamide) (PNIPAM) composite material. Toward this end, a PNIPAM-based composite material inside water-in-oil droplets that are stabilized with PNIPAM-functionalized and commercial fluorosurfactants is introduced.

Bottom-up assembly of viral replication cycles.

Bottom-up synthetic biology provides new means to understand living matter by constructing minimal life-like systems. This principle can also be applied to study infectious diseases. Here we summarize approaches and ethical considerations for the bottom-up assembly of viral replication cycles.

Microstructural Modeling and Simulation of a Carbon Black-Based Conductive Polymer-A Template for the Virtual Design of a Composite Material.

Carbon black is the most frequently applied conductive additive in rubber and polymer composites. In this work, we show how a carbon black microstructure in a polymer matrix can be conclusively modeled based on carbon black aggregation as well as an agglomeration mechanism using a state-of-the-art mathematical model. This novel and flexible microstructural modeling method enables us to virtually investigate the morphology of conductive additives within a polymer matrix and can be adapted to many conductive polymer combinations used for different applications.

Fibronectin anchoring to viscoelastic poly(dimethylsiloxane) elastomers controls fibroblast mechanosensing and directional motility.

The established link between deregulated tissue mechanics and various pathological states calls for the elucidation of the processes through which cells interrogate and interpret the mechanical properties of their microenvironment. In this work, we demonstrate that changes in the presentation of the extracellular matrix protein fibronectin on the surface of viscoelastic silicone elastomers have an overarching effect on cell mechanosensing, that is independent of bulk mechanics. Reduction of surface hydrophilicity resulted in altered fibronectin adsorption strength as monitored using atomic force microscopy imaging and pulling experiments.

Bottom-up assembly of target-specific cytotoxic synthetic cells.

Immune vigilance ensures body integrity by eliminating malignant cells through the complex but coordinated cooperation of highly diversified lymphocytes populations. The sheer complexity of the immune system has slowed development of immunotherapies based on top-down genetic engineering of lymphocytes. In contrast, bottom-up assembly of synthetic cell compartments has contributed novel engineering strategies to reverse engineer and understand cellular phenomena as molecularly defined systems.

Temperature-sensitive migration dynamics in neutrophil-differentiated HL-60 cells.

Cell migration plays an essential role in wound healing and inflammatory processes inside the human body. Peripheral blood neutrophils, a type of polymorphonuclear leukocyte (PMN), are the first cells to be activated during inflammation and subsequently migrate toward an injured tissue or infection site. This response is dependent on both biochemical signaling and the extracellular environment, one aspect of which includes increased temperature in the tissues surrounding the inflammation site.

Vesicle Induced Receptor Sequestration: Mechanisms behind Extracellular Vesicle-Based Protein Signaling.

Extracellular vesicles (EVs) are fundamental for proper physiological functioning of multicellular organisms. By shuttling nucleic acids and proteins between cells, EVs regulate a plethora of cellular processes, especially those involved in immune signalling. However, the mechanistic understanding concerning the biophysical principles underlying EV-based communication is still incomplete.

Synthetic virions reveal fatty acid-coupled adaptive immunogenicity of SARS-CoV-2 spike glycoprotein.

SARS-CoV-2 infection is a major global public health concern with incompletely understood pathogenesis. The SARS-CoV-2 spike (S) glycoprotein comprises a highly conserved free fatty acid binding pocket (FABP) with unknown function and evolutionary selection advantage. Deciphering FABP impact on COVID-19 progression is challenged by the heterogenous nature and large molecular variability of live virus.

Facile and Versatile Method for Micropatterning Poly(acrylamide) Hydrogels Using Photocleavable Comonomers.

We here present a micropatterning strategy to introduce small molecules and ligands on patterns of arbitrary shapes on the surface of poly(acrylamide)-based hydrogels. The main advantages of the presented approach are the ease of use, the lack of need to prefabricate photomasks, the use of mild UV light and biocompatible bioconjugation chemistries, and the capacity to pattern low-molecular-weight ligands, such as peptides, peptidomimetics, or DNA fragments. To achieve the above, a monomer containing a caged amine (NVOC group) was co-polymerized in the hydrogel network; upon UV light illumination using a commercially available setup, primary amines were locally deprotected and served as reactive groups for further functionalization.

Building a community to engineer synthetic cells and organelles from the bottom-up.

Employing concepts from physics, chemistry and bioengineering, 'learning-by-building' approaches are becoming increasingly popular in the life sciences, especially with researchers who are attempting to engineer cellular life from scratch. The SynCell2020/21 conference brought together researchers from different disciplines to highlight progress in this field, including areas where synthetic cells are having socioeconomic and technological impact. Conference participants also identified the challenges involved in designing, manipulating and creating synthetic cells with hierarchical organization and function.

pH-Triggered Assembly of Endomembrane Multicompartments in Synthetic Cells.

By using electrostatic interactions as driving force to assemble vesicles, the droplet-stabilized method was recently applied to reconstitute and encapsulate proteins, or compartments, inside giant unilamellar vesicles (GUVs) to act as minimal synthetic cells. However, the droplet-stabilized approach exhibits low production efficiency associated with the troublesome release of the GUVs from the stabilized droplets, corresponding to a major hurdle for the droplet-stabilized approach. Herein, we report the use of pH as a potential trigger to self-assemble droplet-stabilized GUVs (dsGUVs) by either bulk or droplet-based microfluidics.