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.
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
Robust spatiotemporal organization of cytoskeletal networks is crucial, enabling cellular processes such as cell migration and division. α-Actinin and fascin are two actin crosslinking proteins localized to distinct regions of eukaryotes to form actin bundles with optimized spacing for cell contractile machinery and sensory projections, respectively. In vitro reconstitution assays and coarse-grained simulations have shown that these actin bundling proteins segregate into distinct domains with a bundler size-dependent competition-based mechanism, driven by the minimization of F-actin bending energy. However, it is not known how physical confinement imposed by the cell membrane contributes to sorting of actin bundling proteins and the concomitant reorganization of actin networks in intracellular environment. Here, by encapsulating actin, α-actinin, and fascin in giant unilamellar vesicles (GUVs), we show that the size of such a spherical boundary determines equilibrated structure of actin networks among three typical structures: single rings, astral structures, and star-like structures. We show that α-actinin bundling activity and its tendency for clustering actin is central to the formation of these structures. By analyzing physical features of crosslinked actin networks, we show that spontaneous sorting and domain formation of α-actinin and fascin are intimately linked to the resulting structures. We propose that the observed boundary-imposed effect on sorting and structure formation is a general mechanism by which cells can select between different structural dynamical steady states.
For example, the intermediates of the synthesis of dopamine and serotonin are L-DOPA (L-dihydroxy-phenylalanine) and 5-HTP (5-hydroxytryptophan), respectively, and have found use as administered precursors that are capable of crossing the blood-brain barrier. Although several studies have focused on the characterization of the separate enzymes involved in the biosynthesis of serotonin and dopamine, [3-5] surprisingly little effort has been put into developing enzymatic methods for the production of the final neurotransmitters starting from their proteinogenic amino acids precursors, i.e., L-tryptophan (Trp) and L-tyrosine (Tyr). If developed, such a system may open up new opportunities to build nanofactories and artificial cells for the treatment of neurological disorders. [6-8] To produce the monoamine neurotransmitters serotonin and dopamine in vitro, hydroxylases specific to the amino acid substrate were exploited. The catalytic domain of human tryptophan hydroxylase isoform 2 (TPH) was chosen for the synthesis of 5-HTP from Trp, [9] because this enzyme was previously shown to not be inhibited by high concentrations of the substrate. [10] Two versions of the catalytic domain of rat tyrosine hydroxylase were tested for the production of L-DOPA, including a recombinant, wild type version (rTH) and a truncated construct (ΔTH) that was not inhibited by substrate. [11] Additionally, a hydroxylase from the bacterium Chlamydia pneumoniae, Cpn1046, was tested. Cpn1046 has broader substrate specificity compared to TH and is homologous to eukaryotic aromatic amino acid hydroxylases. [12] Since the aromatic amino acid decarboxylase from Drosophila melanogaster (AADC) is active on both 5-HTP and L-DOPA, [13] a single enzyme was used for the decarboxylation step for the synthesis of both serotonin and dopamine. We found that TPH and ΔTH completely hydroxylated Trp and Tyr to produce the intermediates 5-HTP and L-DOPA, respectively. When TPH and ΔTH were coupled with AADC, serotonin and dopamine were efficiently produced in vitro. Each enzyme (TPH, rTH, ΔΤΗ, AADC, and Cpn1046) expressed well when fused to maltose binding protein (MBP), and each protein was purified in a single step with amylose resin (Figure S1, Supporting Information). However, multiple bands were observed on a SDS-PAGE of Cpn1046. Typically, 50 mg of purified protein was obtained per liter of bacterial culture expressing each construct. Conversely, the use of 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, respectiv...
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