Inferring a spatial code of cell-cell interactions across a whole animal body.

Cell-cell interactions shape cellular function and ultimately organismal phenotype. Interacting cells can sense their mutual distance using combinations of ligand-receptor pairs, suggesting the existence of a spatial code, i.e., signals encoding spatial properties of cellular organization. However, this code driving and sustaining the spatial organization of cells remains to be elucidated. Here we present a computational framework to infer the spatial code underlying cell-cell interactions from the transcriptomes of the cell types across the whole body of a multicellular organism. As core of this framework, we introduce our tool cell2cell, which uses the coexpression of ligand-receptor pairs to compute the potential for intercellular interactions, and we test it across the Caenorhabditis elegans' body. Leveraging a 3D atlas of C. elegans' cells, we also implement a genetic algorithm to identify the ligand-receptor pairs most informative of the spatial organization of cells across the whole body. Validating the spatial code extracted with this strategy, the resulting intercellular distances are negatively correlated with the inferred cell-cell interactions. Furthermore, for selected cell-cell and ligand-receptor pairs, we experimentally confirm the communicatory behavior inferred with cell2cell and the genetic algorithm. Thus, our framework helps identify a code that predicts the spatial organization of cells across a whole-animal body.

The Escherichia coli DnaK chaperone stimulates the α-complementation of ß-galactosidase.

pUC18 and pUC19 are well-known high copy-number plasmid vectors routinely used for DNA cloning purposes. We show here that, in Escherichia coli transformed by native pUC18, the α-complementation of ß-galactosidase (i.e., mediated by the peptide LacZα18) is intrinsically weak and slow, but is greatly stimulated by the DnaK/DnaJ/GrpE chaperone system. In contrast, the α-complementation mediated by the peptide LacZα19 (in E. coli transformed by the native pUC19) is much more efficient and therefore does not require the assistance of the DnaK chaperone machinery. The marked difference between these two LacZα peptides is reproduced in a cell-free protein expression system coupled with α-complementation. We conclude that: (i) α-complementation of ß-galactosidase is DnaK-mediated depending upon the LacZα peptide donor; (ii) DnaK, sensu stricto, is not necessary for α-complementation, but can enhance it to a great extent; (iii) this observation could be used to establish an easy and inexpensive method for screening small molecules libraries in search of DnaK inhibitors and also for deciphering the DnaK-mediated protein quality control mechanism.

Sculpting conducting nanopore size and shape through de novo protein design.

Transmembrane ß-barrels have considerable potential for a broad range of sensing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels, which provide suboptimal starting points. By contrast, de novo protein design can in principle create an unlimited number of new nanopores with any desired properties. Here we describe a general approach to designing transmembrane ß-barrel pores with different diameters and pore geometries. Nuclear magnetic resonance and crystallographic characterization show that the designs are stably folded with structures resembling those of the design models. The designs have distinct conductances that correlate with their pore diameter, ranging from 110 picosiemens (~0.5 nanometer pore diameter) to 430 picosiemens (~1.1 nanometer pore diameter). Our approach opens the door to the custom design of transmembrane nanopores for sensing and sequencing applications.

Increased alcohol dehydrogenase 1 activity promotes longevity.

Several molecules can extend healthspan and lifespan across organisms. However, most are upstream signaling hubs or transcription factors orchestrating complex anti-aging programs. Therefore, these molecules point to but do not reveal the fundamental mechanisms driving longevity. Instead, downstream effectors that are necessary and sufficient to promote longevity across conditions or organisms may reveal the fundamental anti-aging drivers. Toward this goal, we searched for effectors acting downstream of the transcription factor EB (TFEB), known as HLH-30 in C. elegans, because TFEB/HLH-30 is necessary across anti-aging interventions and its overexpression is sufficient to extend C. elegans lifespan and reduce biomarkers of aging in mammals including humans. As a result, we present an alcohol-dehydrogenase-mediated anti-aging response (AMAR) that is essential for C. elegans longevity driven by HLH-30 overexpression, caloric restriction, mTOR inhibition, and insulin-signaling deficiency. The sole overexpression of ADH-1 is sufficient to activate AMAR, which extends healthspan and lifespan by reducing the levels of glycerol-an age-associated and aging-promoting alcohol. Adh1 overexpression is also sufficient to promote longevity in yeast, and adh-1 orthologs are induced in calorically restricted mice and humans, hinting at ADH-1 acting as an anti-aging effector across phyla.

Sculpting conducting nanopore size and shape through de novo protein design.

Transmembrane ß-barrels (TMBs) are widely used for single molecule DNA and RNA sequencing and have considerable potential for a broad range of sensing and sequencing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels such as CsgG, which have evolved to carry out functions very different from sensing, and hence provide sub-optimal starting points. In contrast, de novo protein design can in principle create an unlimited number of new nanopores with any desired properties. Here we describe a general approach to the design of transmembrane ß-barrel pores with different diameter and pore geometry. NMR and crystallographic characterization shows that the designs are stably folded with structures close to the design models. We report the first examples of de novo designed TMBs with 10, 12 and 14 stranded ß-barrels. The designs have distinct conductances that correlate with their pore diameter, ranging from 110 pS (~0.5 nm pore diameter) to 430 pS (~1.1 nm pore diameter), and can be converted into sensitive small-molecule sensors with high signal to noise ratio. The capability to generate on demand ß-barrel pores of defined geometry opens up fundamentally new opportunities for custom engineering of sequencing and sensing technologies.

Phospholipid synthesis inside phospholipid membrane vesicles.

Construction of living artificial cells from genes and molecules can expand our understanding of life system and establish a new aspect of bioengineering. However, growth and division of cell membrane that are basis of cell proliferation are still difficult to reconstruct because a high-yielding phospholipid synthesis system has not been established. Here, we developed a cell-free phospholipid synthesis system that combines fatty acid synthesis and cell-free gene expression system synthesizing acyltransferases. The synthesized fatty acids were sequentially converted into phosphatidic acids by the cell-free synthesized acyltransferases. Because the system can avoid the accumulation of intermediates inhibiting lipid synthesis, sub-millimolar phospholipids could be synthesized within a single reaction mixture. We also performed phospholipid synthesis inside phospholipid membrane vesicles, which encapsulated all the components, and showed the phospholipids localized onto the mother membrane. Our approach would be a platform for the construction of self-reproducing artificial cells since the membrane can grow sustainably.

A Bifunctional Polyphosphate Kinase Driving the Regeneration of Nucleoside Triphosphate and Reconstituted Cell-Free Protein Synthesis.

Reconstituted cell-free protein synthesis systems (e.g., the PURE system) allow the expression of toxic proteins, hetero-oligomeric protein subunits, and proteins with noncanonical amino acids with high levels of homogeneity. In these systems, an artificial ATP/GTP regeneration system is required to drive protein synthesis, which is accomplished using three kinases and phosphocreatine. Here, we demonstrate the replacement of these three kinases with one bifunctional Cytophaga hutchinsonii polyphosphate kinase that phosphorylates nucleosides in an exchange reaction from polyphosphate. The optimized single-kinase system produced a final sfGFP concentration (∼530 µg/mL) beyond that of the three-kinase system (∼400 µg/mL), with a 5-fold faster mRNA translation rate in the first 90 min. The single-kinase system is also compatible with the expression of heat-sensitive firefly luciferase at 37 °C. Potentially, the single-kinase nucleoside triphosphate regeneration approach developed herein could expand future applications of cell-free protein synthesis systems and could be used to drive other biochemical processes in synthetic biology which require both ATP and GTP.

Artificial photosynthetic cell producing energy for protein synthesis.

Attempts to construct an artificial cell have widened our understanding of living organisms. Many intracellular systems have been reconstructed by assembling molecules, however the mechanism to synthesize its own constituents by self-sufficient energy has to the best of our knowledge not been developed. Here, we combine a cell-free protein synthesis system and small proteoliposomes, which consist of purified ATP synthase and bacteriorhodopsin, inside a giant unilamellar vesicle to synthesize protein by the production of ATP by light. The photo-synthesized ATP is consumed as a substrate for transcription and as an energy for translation, eventually driving the synthesis of bacteriorhodopsin or constituent proteins of ATP synthase, the original essential components of the proteoliposome. The de novo photosynthesized bacteriorhodopsin and the parts of ATP synthase integrate into the artificial photosynthetic organelle and enhance its ATP photosynthetic activity through the positive feedback of the products. Our artificial photosynthetic cell system paves the way to construct an energetically independent artificial cell.