Reengineering of an Artificial Protein Cage for Efficient Packaging of Active Enzymes.

Protein cages that readily encapsulate active enzymes of interest present useful nanotools for delivery and catalysis, wherein those with programmable disassembly characteristics serve as particularly attractive platforms. Here, a general guest packaging system based on an artificial protein cage, TRAP-cage, the disassembly of which can be induced by the addition of reducing agents, is established. In this system, TRAP-cage with SpyCatcher moieties in the lumen is prepared using genetic modification of the protein building block and assembled into a cage structure with either monovalent gold ions or molecular crosslinkers. The resulting protein cage can efficiently capture guest proteins equipped with a SpyTag by simply mixing them in an aqueous solution. This post-assembly loading system, which circumvents the exposure of guests to thiol-reactive crosslinkers, enables the packaging of enzymes possessing a catalytic cysteine or a metal cofactor while retaining their catalytic activity.

Synergistic effects of plant genotype and soil microbiome on growth in Lotus japonicus.

The biological interactions between plants and their root microbiomes are essential for plant growth, and even though plant genotype (G), soil microbiome (M), and growth conditions (environment; E) are the core factors shaping root microbiome, their relationships remain unclear. In this study, we investigated the effects of G, M, and E and their interactions on the Lotus root microbiome and plant growth using an in vitro cross-inoculation approach, which reconstructed the interactions between nine Lotus accessions and four soil microbiomes under two different environmental conditions. Results suggested that a large proportion of the root microbiome composition is determined by M and E, while G-related (G, G × M, and G × E) effects were significant but small. In contrast, the interaction between G and M had a more pronounced effect on plant shoot growth than M alone. Our findings also indicated that most microbiome variations controlled by M have little effect on plant phenotypes, whereas G × M interactions have more significant effects. Plant genotype-dependent interactions with soil microbes warrant more attention to optimize crop yield and resilience.

Complementary charge-driven encapsulation of functional protein by engineered protein cages in cellulo.

Charge-driven inclusion complex formation in live cells was examined using a degradation-prone fluorescent protein and a series of protein cages. The results show that sufficiently strong host-guest ionic interaction and an intact shell-like structure are crucial for the protective guest encapsulation.

Systematic analysis of cell morphodynamics in C. elegans early embryogenesis.

The invariant cell lineage of Caenorhabditis elegans allows unambiguous assignment of the identity for each cell, which offers a unique opportunity to study developmental dynamics such as the timing of cell division, dynamics of gene expression, and cell fate decisions at single-cell resolution. However, little is known about cell morphodynamics, including the extent to which they are variable between individuals, mainly due to the lack of sufficient amount and quality of quantified data. In this study, we systematically quantified the cell morphodynamics in 52 C. elegans embryos from the two-cell stage to mid-gastrulation at the high spatiotemporal resolution, 0.5 µm thickness of optical sections, and 30-second intervals of recordings. Our data allowed systematic analyses of the morphological features. We analyzed sphericity dynamics and found a significant increase at the end of metaphase in every cell, indicating the universality of the mitotic cell rounding. Concomitant with the rounding, the volume also increased in most but not all cells, suggesting less universality of the mitotic swelling. Combining all features showed that cell morphodynamics was unique for each cell type. The cells before the onset of gastrulation could be distinguished from all the other cell types. Quantification of reproducibility in cell-cell contact revealed that variability in division timings and cell arrangements produced variability in contacts between the embryos. However, the area of such contacts occupied less than 5% of the total area, suggesting the high reproducibility of spatial occupancies and adjacency relationships of the cells. By comparing the morphodynamics of identical cells between the embryos, we observed diversity in the variability between cells and found it was determined by multiple factors, including cell lineage, cell generation, and cell-cell contact. We compared the variabilities of cell morphodynamics and cell-cell contacts with those in ascidian Phallusia mammillata embryos. The variabilities were larger in C. elegans, despite smaller differences in embryo size and number of cells at each developmental stage.

Chemically induced protein cage assembly with programmable opening and cargo release.

Engineered protein cages are promising tools that can be customized for applications in medicine and nanotechnology. A major challenge is developing a straightforward strategy for endowing cages with bespoke, inducible disassembly. Such cages would allow release of encapsulated cargoes at desired timing and location. Here, we achieve such programmable disassembly using protein cages, in which the subunits are held together by different molecular cross-linkers. This modular system enables cage disassembly to be controlled in a condition-dependent manner. Structural details of the resulting cages were determined using cryo­electron microscopy, which allowed observation of bridging cross-linkers at intended positions. Triggered disassembly was demonstrated by high-speed atomic force microscopy and subsequent cargo release using an encapsulated Förster resonance energy transfer pair whose signal depends on the quaternary structure of the cage.

Connectability of protein cages.

Regular, hollow proteinaceous nanoparticles are widespread in nature. The well-defined structures as well as diverse functions of naturally existing protein cages have inspired the development of new nanoarchitectures with desired capabilities. In such approaches, a key functionality is "connectability". Engineering of interfaces between cage building blocks to modulate intra-cage connectability leads to protein cages with new morphologies and assembly-disassembly properties. Modification of protein cage surfaces to control inter-cage connectability enables their arrangement into lattice-like nanomaterials. Here, we review the current progress in control of intra- and inter-cage connectability for protein cage-based nanotechnology development.

Cytoplasmic glycoengineering enables biosynthesis of nanoscale glycoprotein assemblies.

Glycosylation of proteins profoundly impacts their physical and biological properties. Yet our ability to engineer novel glycoprotein structures remains limited. Established bacterial glycoengineering platforms require secretion of the acceptor protein to the periplasmic space and preassembly of the oligosaccharide substrate as a lipid-linked precursor, limiting access to protein and glycan substrates respectively. Here, we circumvent these bottlenecks by developing a facile glycoengineering platform that operates in the bacterial cytoplasm. The Glycoli platform leverages a recently discovered site-specific polypeptide glycosyltransferase together with variable glycosyltransferase modules to synthesize defined glycans, of bacterial or mammalian origin, directly onto recombinant proteins in the E. coli cytoplasm. We exploit the cytoplasmic localization of this glycoengineering platform to generate a variety of multivalent glycostructures, including self-assembling nanomaterials bearing hundreds of copies of the glycan epitope. This work establishes cytoplasmic glycoengineering as a powerful platform for producing glycoprotein structures with diverse future biomedical applications.

Dipicolylamine/Metal Complexes that Promote Direct Cell-Membrane Penetration of Octaarginine.

Marked promotion of membrane permeation of a cell-penetrating peptide, octaarginine (R8), was attained by attachment to a single 2,2'-dipicolylamine moiety (DPA-R8) that forms 1:1 complexes with metal ions. Studies using giant unilamellar vesicles demonstrated that DPA targets phospholipids and enhances R8 binding to the membranes in the presence of metal ions. While DPA/Zn(II) complex has been most frequently employed for chelate formation with phosphates, Ni(II) had the most prominent effect on the membrane binding and penetration of DPA-R8. Facile cytosolic distribution of DPA-R8 was also attained in a few minutes in the presence of Ni(II). Analysis of the cellular uptake methods of DPA-R8/Ni(II) suggested the involvement of direct permeation through cell membrane without the use of endocytosis. The applicability of this system to the intracellular delivery of bioactive compounds was exemplified using a peptidomimetic farnesyltransferase inhibitor, FTI277.

Modular Redesign of a Cationic Lytic Peptide To Promote the Endosomal Escape of Biomacromolecules.

Endocytosis is an important route for the intracellular delivery of biomacromolecules, wherein their inefficient endosomal escape into the cytosol remains a major barrier. Based on the understanding that endosomal membranes are negatively charged, we focused on the potential of cationic lytic peptides for developing endosomolysis agents to release such entrapped molecules. As such, a venom peptide, Mastoparan X, was employed and redesigned to serve as a delivery tool. Appending a tri-glutamate unit to the N-terminus attenuates the cytotoxicity of Mastoparan X by about 40 fold, while introduction of a NiII -dipicolylamine complex enhances cellular uptake of the peptide by about 17 fold. Using the optimized peptide, various fluorescently labeled macromolecules were successfully delivered to the cytosol, enabling live-cell imaging of acetylated histones.

Enzyme Encapsulation in an Engineered Lumazine Synthase Protein Cage.

The packaging of active enzymes in protein cages is a powerful strategy to control catalytic activity. Using a positively supercharged variant of green fluorescent protein, GFP(+36), as a genetically programmable tag, enzymes can be rapidly and quantitatively loaded into an engineered variant of the Aquifex aeolicus cage-forming protein lumazine synthase (AaLS-13) that possesses a negatively charged lumen. The cargo is spontaneously localized within AaLS-13 cages by simply mixing the components in aqueous solution. This chapter describes a detailed protocol for the preparation of AaLS-13 cages and GFP(+36)-enzyme fusions, as well as characterization of the inclusion complexes. Suitable conditions for encapsulation and enzyme kinetic assays are also discussed.

Laboratory evolution of virus-like nucleocapsids from nonviral protein cages.

Viruses are remarkable nanomachines that efficiently hijack cellular functions to replicate and self-assemble their components within a complex biological environment. As all steps of the viral life cycle depend on formation of a protective proteinaceous shell that packages the DNA or RNA genome, bottom-up construction of virus-like nucleocapsids from nonviral materials could provide valuable insights into virion assembly and evolution. Such constructs could also serve as safe alternatives to natural viruses for diverse nano- and biotechnological applications. Here we show that artificial virus-like nucleocapsids can be generated-rapidly and surprisingly easily-by engineering and laboratory evolution of a nonviral protein cage formed by Aquifex aeolicus lumazine synthase (AaLS) and its encoding mRNA. Cationic peptides were appended to the engineered capsid proteins to enable specific recognition of packaging signals on cognate mRNAs, and subsequent evolutionary optimization afforded nucleocapsids with expanded spherical structures that encapsulate their own full-length RNA genome in vivo and protect the cargo molecules from nucleases. These findings provide strong experimental support for the hypothesis that subcellular protein-bounded compartments may have facilitated the emergence of ancient viruses.

Tailoring lumazine synthase assemblies for bionanotechnology.

Nanoscale compartments formed by hierarchical protein self-assembly are valuable platforms for nanotechnology development. The well-defined structure and broad chemical functionality of protein cages, as well as their amenability to genetic and chemical modification, have enabled their repurposing for diverse applications. In this review, we summarize progress in the engineering of the cage-forming enzyme lumazine synthase. This bacterial nanocompartment has proven to be a malleable scaffold. The natural protein has been diversified to afford a family of unique proteinaceous capsules that have been modified, evolved and assembled with other components to produce nanoreactors, artificial organelles, delivery vehicles and virus mimics.

Substrate Sorting by a Supercharged Nanoreactor.

Compartmentalization of proteases enables spatially and temporally controlled protein degradation in cells. Here we show that an engineered lumazine synthase protein cage, which possesses a negatively supercharged lumen, can exploit electrostatic effects to sort substrates for an encapsulated protease. This proteasome-like nanoreactor preferentially cleaves positively charged polypeptides over both anionic and zwitterionic substrates, inverting the inherent substrate specificity of the guest enzyme approximately 480 fold. Our results suggest that supercharged nanochambers could provide a simple and potentially general means of conferring substrate specificity to diverse encapsulated catalysts.

Modular Protein Cages for Size-Selective RNA Packaging in Vivo.

Protein cages have recently emerged as an important platform for nanotechnology development. Of the naturally existing protein cages, viruses are among the most efficient nanomachines, overcoming various barriers to achieve component replication and efficient self-assembly in complex biological milieu. We have designed an artificial system that can carry out the most basic steps of viral particle assembly in vivo. Our strategy is based on patchwork capsids formed from Aquifex aeolicus lumazine synthase and a circularly permuted variant with appended cationic peptides. These two-component protein containers self-assemble in vivo, capturing endogenous RNA molecules in a size-selective manner. By varying the number and design of the RNA-binding peptides displayed on the lumenal surface, the length of guest RNA can be further controlled. Using a fluorescent aptamer, we also show that short-lived RNA species are captured by the protein cage. This platform has potential as a model system for investigating virus assembly, as well as developing RNA regulation or sampling tools to augment biotechnology.

Diversification of Protein Cage Structure Using Circularly Permuted Subunits.

Self-assembling protein cages are useful as nanoscale molecular containers for diverse applications in biotechnology and medicine. To expand the utility of such systems, there is considerable interest in customizing the structures of natural cage-forming proteins and designing new ones. Here we report that a circularly permuted variant of lumazine synthase, a cage-forming enzyme from Aquifex aeolicus (AaLS) affords versatile building blocks for the construction of nanocompartments that can be easily produced, tailored, and diversified. The topologically altered protein, cpAaLS, self-assembles into spherical and tubular cage structures with morphologies that can be controlled by the length of the linker connecting the native termini. Moreover, cpAaLS proteins integrate into wild-type and other engineered AaLS assemblies by coproduction in Escherichia coli to form patchwork cages. This coassembly strategy enables encapsulation of guest proteins in the lumen, modification of the exterior through genetic fusion, and tuning of the size and electrostatics of the compartments. This addition to the family of AaLS cages broadens the scope of this system for further applications and highlights the utility of circular permutation as a potentially general strategy for tailoring the properties of cage-forming proteins.

Biologically constrained optimization based cell membrane segmentation in C. elegans embryos.

BACKGROUND: Recent advances in bioimaging and automated analysis methods have enabled the large-scale systematic analysis of cellular dynamics during the embryonic development of Caenorhabditis elegans. Most of these analyses have focused on cell lineage tracing rather than cell shape dynamics. Cell shape analysis requires cell membrane segmentation, which is challenging because of insufficient resolution and image quality. This problem is currently solved by complicated segmentation methods requiring laborious and time consuming parameter adjustments. RESULTS: Our new framework BCOMS (Biologically Constrained Optimization based cell Membrane Segmentation) automates the extraction of the cell shape of C. elegans embryos. Both the segmentation and evaluation processes are automated. To automate the evaluation, we solve an optimization problem under biological constraints. The performance of BCOMS was validated against a manually created ground truth of the 24-cell stage embryo. The average deviation of 25 cell shape features was 5.6%. The deviation was mainly caused by membranes parallel to the focal planes, which either contact the surfaces of adjacent cells or make no contact with other cells. Because segmentation of these membranes was difficult even by manual inspection, the automated segmentation was sufficiently accurate for cell shape analysis. As the number of manually created ground truths is necessarily limited, we compared the segmentation results between two adjacent time points. Across all cells and all cell cycles, the average deviation of the 25 cell shape features was 4.3%, smaller than that between the automated segmentation result and ground truth. CONCLUSIONS: BCOMS automated the accurate extraction of cell shapes in developing C. elegans embryos. By replacing image processing parameters with easily adjustable biological constraints, BCOMS provides a user-friendly framework. The framework is also applicable to other model organisms. Creating the biological constraints is a critical step requiring collaboration between an experimentalist and a software developer.

The C-terminal peptide of Aquifex aeolicus riboflavin synthase directs encapsulation of native and foreign guests by a cage-forming lumazine synthase.

Encapsulation of specific enzymes in self-assembling protein cages is a hallmark of bacterial compartments that function as counterparts to eukaryotic organelles. The cage-forming enzyme lumazine synthase (LS) from Bacillus subtilis (BsLS), for example, encapsulates riboflavin synthase (BsRS), enabling channeling of lumazine from the site of its generation to the site of its conversion to vitamin B2 Elucidating the molecular mechanisms underlying the assembly of these supramolecular complexes could help inform new approaches for metabolic engineering, nanotechnology, and drug delivery. To that end, we investigated a thermostable LS from Aquifex aeolicus (AaLS) and found that it also forms cage complexes with the cognate riboflavin synthase (AaRS) when both proteins are co-produced in the cytosol of Escherichia coli A 12-amino acid-long peptide at the C terminus of AaRS serves as a specific localization sequence responsible for targeting the guest to the protein compartment. Sequence comparisons suggested that analogous peptide segments likely direct RS complexation by LS cages in other bacterial species. Covalent fusion of this peptide tag to heterologous guest molecules led to their internalization into AaLS assemblies both in vivo and in vitro, providing a firm foundation for creating tailored biomimetic nanocompartments for medical and biotechnological applications.

Quantitative Packaging of Active Enzymes into a Protein Cage.

Genetic fusion of cargo proteins to a positively supercharged variant of green fluorescent protein enables their quantitative encapsulation by engineered lumazine synthase capsids possessing a negatively charged lumenal surface. This simple tagging system provides a robust and versatile means of creating hierarchically ordered protein assemblies for use as nanoreactors. The generality of the encapsulation strategy and its effect on enzyme function were investigated with eight structurally and mechanistically distinct catalysts.

Controlling leucine-zipper partner recognition in cells through modification of a-g interactions.

By focusing on the a-g interactions, successful design and selection were accomplished to obtain a leucine-zipper segment that discriminates the appropriate partner over another that provides very similar patterns of electrostatic interactions.

Evaluation of the effectiveness of simple nuclei-segmentation methods on Caenorhabditis elegans embryogenesis images.

BACKGROUND: For the analysis of spatio-temporal dynamics, various automated processing methods have been developed for nuclei segmentation. These methods tend to be complex for segmentation of images with crowded nuclei, preventing the simple reapplication of the methods to other problems. Thus, it is useful to evaluate the ability of simple methods to segment images with various degrees of crowded nuclei. RESULTS: Here, we selected six simple methods from various watershed based and local maxima detection based methods that are frequently used for nuclei segmentation, and evaluated their segmentation accuracy for each developmental stage of the Caenorhabditis elegans. We included a 4D noise filter, in addition to 2D and 3D noise filters, as a pre-processing step to evaluate the potential of simple methods as widely as possible. By applying the methods to image data between the 50- to 500-cell developmental stages at 50-cell intervals, the error rate for nuclei detection could be reduced to ≤ 2.1% at every stage until the 350-cell stage. The fractions of total errors throughout the stages could be reduced to ≤ 2.4%. The error rates improved at most of the stages and the total errors improved when a 4D noise filter was used. The methods with the least errors were two watershed-based methods with 4D noise filters. For all the other methods, the error rate and the fraction of errors could be reduced to ≤ 4.2% and ≤ 4.1%, respectively. The minimum error rate for each stage between the 400- to 500-cell stages ranged from 6.0% to 8.4%. However, similarities between the computational and manual segmentations measured by volume overlap and Hausdorff distance were not good. The methods were also applied to Drosophila and zebrafish embryos and found to be effective. CONCLUSIONS: The simple segmentation methods were found to be useful for detecting nuclei until the 350-cell stage, but not very useful after the 400-cell stage. The incorporation of a 4D noise filter to the simple methods could improve their performances. Error types and the temporal biases of errors were dependent on the methods used. Combining multiple simple methods could also give good segmentations.