In Vitro Reconstitution Platforms of Mammalian Cell-Free Expressed Membrane Proteins.

Membrane proteins are essential components in cell membranes and enable cells to communicate with their outside environment and to carry out intracellular signaling. Functional reconstitution of complex membrane proteins using cell-free expression (CFE) systems has been proved to be challenging mainly due to the lack of necessary machinery for proper folding and translocation of nascent membrane proteins and their delivery to the supplied synthetic bilayers. Here, we provide protocols for detergent-free, cell-free reconstitution of functional membrane proteins using HeLa-based CFE system and outline assays for studying their membrane insertion, topology, and their orientation upon incorporation into the supported lipid bilayers or bilayers of giant unilamellar vesicles as well as methods to isolate functional translocated cell-free produced membrane proteins.

Actin crosslinker competition and sorting drive emergent GUV size-dependent actin network architecture.

The proteins that make up the actin cytoskeleton can self-assemble into a variety of structures. In vitro experiments and coarse-grained simulations have shown that the actin crosslinking proteins α-actinin and fascin segregate into distinct domains in single actin bundles with a molecular size-dependent competition-based mechanism. Here, by encapsulating actin, α-actinin, and fascin in giant unilamellar vesicles (GUVs), we show that physical confinement can cause these proteins to form much more complex structures, including rings and asters at GUV peripheries and centers; the prevalence of different structures depends on GUV size. Strikingly, we found that α-actinin and fascin self-sort into separate domains in the aster structures with actin bundles whose apparent stiffness depends on the ratio of the relative concentrations of α-actinin and fascin. The observed boundary-imposed effect on protein sorting may be a general mechanism for creating emergent structures in biopolymer networks with multiple crosslinkers.

Engineering spatiotemporal organization and dynamics in synthetic cells.

Constructing synthetic cells has recently become an appealing area of research. Decades of research in biochemistry and cell biology have amassed detailed part lists of components involved in various cellular processes. Nevertheless, recreating any cellular process in vitro in cell-sized compartments remains ambitious and challenging. Two broad features or principles are key to the development of synthetic cells-compartmentalization and self-organization/spatiotemporal dynamics. In this review article, we discuss the current state of the art and research trends in the engineering of synthetic cell membranes, development of internal compartmentalization, reconstitution of self-organizing dynamics, and integration of activities across scales of space and time. We also identify some research areas that could play a major role in advancing the impact and utility of engineered synthetic cells. This article is categorized under: Biology-Inspired Nanomaterials > Lipid-Based Structures Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.

Cell-Free Synthesis of Dopamine and Serotonin in Two Steps with Purified Enzymes.

The synthesis of serotonin and dopamine with purified enzymes is described. Both pathways start from an amino acid substrate and synthesize the monoamine neurotransmitter in two enzymatic steps. The enzymes human tryptophan hydroxylase isoform 2, Rattus norvegicus tyrosine hydroxylase, Chlamydia pneumoniae Cpn1046, and aromatic amino acid decarboxylase from Drosophila melanogaster are recombinantly expressed, purified, and shown to be functional in vitro. The hydroxylases efficiently convert L-DOPA (L-dihydroxy-phenylalanine) and 5-HTP (5-hydroxytryptophan) from L-tyrosine and L-tryptophan, respectively. A single aromatic amino acid decarboxylase is capable of converting both hydroxylated intermediates into the final neurotransmitter. The platform described here may facilitate future efforts to generate medically useful artificial cells and nanofactories.