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The incorporation of living cells into materials promises both significant challenges and new possibilities. Although recent years have seen important advances in this field, there is still much to be learned about engineering interfaces between cells and materials. Here, we present a new class of 3D-printable materials, based on poly(

Coinfection with two notorious opportunistic pathogens, the Gram-negativePseudomonas aeruginosaand Gram-positiveStaphylococcus aureus, dominates chronic pulmonary infections. While coinfection is associated with poor patient outcomes, the interspecies interactions responsible for such decline remain unknown. Here, we dissected molecular mechanisms of interspecies sensing betweenP. aeruginosaandS. aureus. We discovered thatP. aeruginosasensesS. aureussecreted peptides and, counterintuitively, moves towards these toxins.P. aeruginosatolerates such a strategy through “competition sensing”, whereby it preempts imminent danger/competition by arming cells with type six secretion (T6S) and iron acquisition systems. Intriguingly, while T6S is predominantly described as weaponry targeting Gram-negative and eukaryotic cells, we find that T6S is essential for fullP. aeruginosacompetition withS. aureus, a previously undescribed role for T6S. Importantly, competition sensing was activated during coinfection of bronchial epithelia, including T6S islands targeting human cells. This study reveals critical insight into both interspecies competition and how antagonism may cause collateral damage to the host environment.

Pancreatic ducts function to deliver digestive enzymes into the intestines. Upon injury, ducts can become proliferative and contribute to tissue regeneration; however, the identity of the ductal cells that contribute to these processes is unknown. We combined fluorescence-activated cell sorting, a methylcellulose-containing 3-dimensional culture, droplet RNA-sequencing, and a clonal lineage tracing tool to identify and isolate a distinct subpopulation of pancreatic ductal cells that exhibit progenitor cell properties. These ductal cells are unique in that they form tightly-bound clusters (termed FSCmid-high), with an average of 8 cells per cluster. FSCmid-high clusters comprise only about 0.1% of the total pancreas, are tri-potent for duct, acinar and endocrine lineages, and self-renew robustly in vitro. Transcriptomic analysis of FSCmid-high clusters reveals enrichment for genes involved in cell-cell interactions, organ development, and cancer pathways. FSCmid-high clusters express embryonic pancreatic progenitor markers Sox9, Pdx1, and Nkx6-1 at both transcription and protein levels. FSCmid-high clusters are resistant to enzymatic dissociation and survive severe in vivo acinar injury, which induces formation of ductal rosettes that become proliferative within 14 days. Thus, FSCmid-high clusters represent a small subset of ductal cells with progenitor cell properties. These rare progenitor-like duct cell clusters have implications in pancreas regeneration and tumor initiation/progression.

Engineered microbial communities show promise in a wide range of applications, including environmental remediation, microbiome engineering, and synthesis of fine chemicals. Here we present methods by which bacterial aggregates can be directed into several distinct architectures by inducible surface expression of hetero-associative protein domains (SpyTag/SpyCatcher and SynZip17/18). Programmed aggregation can be used to activate a quorum-sensing circuit, and aggregate size can be tuned via control of the amount of the associative protein displayed on the cell surface. We further demonstrate reversibility of SynZip-mediated assembly by addition of soluble competitor peptide. Genetically programmable bacterial assembly provides a starting point for the development of new applications of engineered microbial communities in environmental technology, agriculture, human health, and bioreactor design.

Proline residues are unique in the extent to which they constrain the conformational space available to the protein backbone. Because the conformational preferences of proline cannot be recapitulated by any of the other proteinogenic amino acids, standard mutagenesis approaches that seek to introduce new chemical functionality at proline positions unavoidably perturb backbone flexibility. Here, we detail the incorporation of proline analogs into recombinant proteins in Escherichia coli via a residue-specific mutagenesis strategy. This approach results in global replacement of proline residues with high yields of the recombinant protein of interest, minimal genetic manipulation, and maintenance of backbone conformational constraints.

Artificial protein hydrogels made from a triblock protein (designated AC10A, where A is an acidic zipper domain and C10 comprises 10 repeats of the nonapeptide sequence exhibit normalized plateau storage moduli (G′∞/nkT) less than 0.13 at all concentrations, pH values, and ionic strengths examined. These gels are surprisingly soft due to loop formation at the expense of bridges between physical junctions. Molecular-level evidence of loop formation is provided by strong fluorescence energy transfer (FRET) between distinct chromophores placed at the C- and N-termini of labelled chains diluted in an excess of unlabelled chains. The tendency to form loops originates from the compact size of the random coil midblock (mean RH(C10) ≈ 20 A, determined from quasi-elastic light scattering of C10), and is facilitated by the ability of the leucine zipper domains to form antiparallel aggregates. Although the aggregation number of the leucine zipper domains is small (tetrameric, determined from multi-angle static light scattering of AC10 diblock), the average center-to-center distance between aggregates is roughly 1.5 times the average end-to-end distance of the C10 domain in a 7% w/v network. To avoid stretching the C10 domain, the chains tend to form loops. Changes in pH or ionic strength that expand the polyelectrolyte midblock favor bridging, leading to greater G′∞ as long as leucine zipper endblocks do not dissociate. Understanding of the network structure provided successful design strategies to increase the rigidity of these hydrogels. In contrast to intuitive design concepts for rubber and gel materials, it was shown that increasing either the length or the charge density of the midblock increases rigidity, because fewer chains are wasted in loop formation.

Although advances in protein labeling methods have made it possible to measure the proteome of mixed cell populations, it has not been possible to isolate cell-type-specific proteomes in vivo. This is because the existing methods for metabolic protein labeling in vivo access all cell types. We report the development of a transgenic mouse line where Cre-recombinase-induced expression of a mutant methionyl-tRNA synthetase (L274G) enables the cell-type-specific labeling of nascent proteins with a non-canonical amino-acid and click chemistry. Using immunoblotting, imaging and mass spectrometry, we use our transgenic mouse to label and analyze proteins in excitatory principal neurons and Purkinje neurons in vitro (brain slices) and in vivo. We discover more than 200 proteins that are differentially regulated in hippocampal excitatory neurons by exposing mice to an environment with enriched sensory cues. Our approach can be used to isolate, analyze and quantitate cell-type-specific proteomes and their dynamics in healthy and diseased tissues.

Correlated cryo-fluorescence and cryo-electron microscopy (cryo-CLEM) has become an increasingly popular method for combining the resolving power of cryo-EM with the specificity of fluorescence. Although cryo-fluorescence microscopy suffers from optical limitations, it is a powerful way to target the resolving power of cryo-EM toward proteins of interest in heterogeneous cellular environments. Super-resolution microscopy at cryo temperatures has also been established using several different approaches. While fluorescence-derived localization is a key benefit of cryo-CLEM, fluorescence can also be used to bring orthogonal information into cryo-EM images. We recently developed a fusion assay compatible with cryo-CLEM equipment and conditions (Metskas and Briggs, Microscopy & Microanalysis 2019). This development employs auto-quenching by resonant energy transfer to specifically target a function rather than a protein in cryo-CLEM - in this case, adding information on lipid mixing to morphologies from micrographs of influenza virus fusion. However, further methods developments, particularly those involving FRET, are currently hampered by limited characterization of modern fluorophores at cryo-CLEM temperatures (77-100 K). Here, we present a study of commonly used synthetic fluorophores and fluorescent proteins, characterizing excitation and emission spectra, singlet state lifetime, and quantum yield at 77 K. We note that 10 nm shifts of the modes are common for both excitation and emission spectra, but are fluorophore specific in magnitude and even in direction. Vibronic coupling and spectral narrowing are visible in all cases characterized, and singlet state lifetimes increase or decrease in a fluorophore-specific manner. Taken together, these data suggest guidelines for choosing cryo-CLEM fluorophores and filter sets, and demonstrate promise for techniques such as FRET in carefully-adapted applications.

Methods that enable the super-resolution imaging of intracellular proteins in live bacterial cells provide powerful tools for the study of prokaryotic cell biology. Photoswitchable organic dyes exhibit many of the photophysical properties needed for super-resolution imaging, including high brightness, photostability, and photon output, but most such dyes require organisms to be fixed and permeabilized if intracellular targets are to be labeled. We recently reported a general strategy for the chemoenzymatic labeling of bacterial proteins with azide-bearing fatty acids in live cells using the eukaryotic enzyme N-myristoyltransferase. Here we demonstrate the labeling of proteins in live Escherichia coli using cell-permeant bicyclononyne-functionalized photoswitchable rhodamine spirolactams. Single-molecule fluorescence measurements on model rhodamine spirolactam salts show that these dyes emit hundreds of photons per switching event. Super-resolution imaging was performed on bacterial chemotaxis proteins Tar and CheA and cell division proteins FtsZ and FtsA. High-resolution imaging of Tar revealed a helical pattern; imaging of FtsZ yielded banded patterns dispersed throughout the cell. The precision of radial and axial localization in reconstructed images approaches 15 and 30 nm, respectively. The simplicity of the method, which does not require redox imaging buffers, should make this approach broadly useful for imaging intracellular bacterial proteins in live cells with nanometer resolution., We describe a chemoenzymatic protein labeling strategy that uses cell-permeant photoswitchable dyes to enable super-resolution imaging in live bacteria without the need for special imaging buffers.

Methods for selective protein imaging are critical for elucidating how cells orchestrate fundamental biological processes. We recently developed a chemoenzymatic method to modify bacterial proteins in situ for fluorescence imaging using N-myristoyl transferase (NMT). Target proteins outfitted with an N-terminal NMT recognition sequence are covalently modified with an azido fatty acid. Subsequent strain-promoted azide-alkyne cycloaddition allows for conjugation to cell-permeant fluorophores and imaging by fluorescence microscopy. Here we describe sample preparation and labeling protocols for imaging bacterial proteins in fixed and live cells.

Non‐canonical amino acid mutagenesis was used to examine the biophysical consequences of changing ring size and structure at the single proline site in insulin. Addition of a methylene spacer to the prolyl ring (by replacement of proline by pipecolic acid at position B28) led to an increase in stability and a decrease in the rate of hexamer dissociation. The results of this work illustrate the power of non‐canonical amino acid mutagenesis in the engineering of macromolecular aggregation and protein therapeutics.

Proteomic plasticity undergirds stress responses in plants, and understanding such responses requires accurate measurement of the extent to which proteins levels are adjusted to counter external stimuli. Here, we adapt bioorthogonal noncanonical amino acid tagging (BONCAT) to interrogate protein synthesis in vegetative Arabidopsis (Arabidopsis thaliana) seedlings. BONCAT relies on the translational incorporation of a noncanonical amino acid probe into cellular proteins. In this study, the probe is the Met surrogate azidohomoalanine (Aha), which carries a reactive azide moiety in its amino acid side chain. The azide handle in Aha can be selectively conjugated to dyes and functionalized beads to enable visualization and enrichment of newly synthesized proteins. We show that BONCAT is sensitive enough to detect Arabidopsis proteins synthesized within a 30-min interval defined by an Aha pulse and that the method can be used to detect proteins made under conditions of light stress, osmotic shock, salt stress, heat stress, and recovery from heat stress. We further establish that BONCAT can be coupled to tandem liquid chromatography-mass spectrometry to identify and quantify proteins synthesized during heat stress and recovery from heat stress. Our results are consistent with a model in which, upon the onset of heat stress, translation is rapidly reprogrammed to enhance the synthesis of stress mitigators and is again altered during recovery. All experiments were carried out with commercially available reagents, highlighting the accessibility of the BONCAT method to researchers interested in stress responses as well as translational and posttranslational regulation in plants.

Recombinant artificial proteins contain genetically encoded information that specifies their assembly into higher order structures by physical or chemical cross-linking as well as elastic behavior and biological or chemical function. This thesis describes the use of artificial proteins to construct molecular networks containing covalent cross-links involving the thiol side chain of cysteine residues and physical cross-links involving the association of helical domains as coiled coils. The goal of this work was to demonstrate how the viscoelastic properties of protein hydrogels could be encoded within an artificial protein sequence. Using genetic engineering methods, a telechelic protein denoted ERE was designed from elastin- and fibronectin-derived repeating units and expressed in Escherichia coli. ERE was end-linked by the reaction of terminal cysteine residues with tetrakis-vinyl sulfone-functionalized 4-arm star PEG to form hydrogel networks. The effects of varying the precursor concentration and cross-linker stoichiometry on the swelling ratio and mechanical properties of the hydrogels were studied in detail in Chapter 2. The capacity for ERE hydrogels to serve as an artificial extracellular matrix was also assessed by the encapsulation of mouse fibroblasts, which survived the cross-linking reaction and exhibited a spread morphology within the gel. Chapter 3 describes a set of recombinant artificial proteins that can be cross-linked by covalent bonds, by association of helical domains, or by both mechanisms. These proteins were used to construct chemical, physical, and chemical-physical hydrogel networks in which the mechanism of cross-linking determines whether the material response to mechanical deformation is elastic or viscoelastic. In viscoelastic networks, stress relaxation and energy dissipation could be tuned by controlling the ratio of physical cross-linking to chemical cross-linking, and the physical cross-links could be disrupted either by protein denaturation or by mutation of the primary sequence. Network dynamics control the viscoelasticity and erosion rate of materials and influence biological processes at multiple length scales.In Chapter 4, variation of the protein sequence was explored as a strategy to tune the characteristic relaxation timescale of protein networks. Single point mutations to coiled-coil physical cross-linking domains in chemical-physical hydrogels altered the characteristic relaxation time over five orders of magnitude. Using a pair of orthogonal coiled-coil physical cross-linking domains, networks with two distinct relaxation timescales were also engineered. The dynamic properties of protein hydrogels can also be controlled by interactions between protein domains and small molecule ligands. In Chapter 5, the viscoelastic behavior of chemical-physical protein gels was tuned by swelling the gels with small hydrophobic molecules including vitamin D3 and fatty acids. The proposed mechanism for this effect involves binding of the ligands within the hydrophobic pore or channel created by a coiled-coil physical cross-link. Exploiting natural and designed protein-ligand interactions represents a new approach to developing hydrogel “formulations” in which the viscoelastic properties of the material can be engineered to meet specific design criteria. In addition to exhibiting interesting dynamic properties, polymeric hydrogels containing permanent covalent cross-links and reversible physical cross-links often display enhanced toughness and extensibility. Protein hydrogels cross-linked by covalent thioether bonds and physical coiled coils could be extended further than control covalent hydrogels and exhibited a greater work of extension, which is considered a measure of material toughness. These results demonstrate progress toward engineering tougher, more extensible protein-based materials by the incorporation of coiled-coil physical cross-links within a covalent hydrogel network.

Methods for cell-selective analysis of proteome dynamics will facilitate studies of biological processes in multicellular organisms. Here we describe a mutant murine methionyl-tRNA synthetase (designated L274GMmMetRS) that charges the noncanonical amino acid azidonorleucine (Anl) to elongator tRNA^(Met) in hamster (CHO), monkey (COS7), and human (HeLa) cell lines. Proteins made in cells that express the synthetase can be labeled with Anl, tagged with dyes or affinity reagents, and enriched on affinity resin to facilitate identification by mass spectrometry. The method does not require expression of orthogonal tRNAs or depletion of canonical amino acids. Successful labeling of proteins with Anl in several mammalian cell lines demonstrates the utility of L274GMmMetRS as a tool for cell-selective analysis of mammalian protein synthesis.

Methods for selective protein imaging are critical for elucidating how cells orchestrate fundamental biological processes. We recently developed a chemoenzymatic method to modify bacterial proteins in situ for fluorescence imaging using N-myristoyl transferase (NMT). Target proteins outfitted with an N-terminal NMT recognition sequence are covalently modified with an azido fatty acid. Subsequent strain-promoted azide-alkyne cycloaddition allows for conjugation to cell-permeant fluorophores and imaging by fluorescence microscopy. Here we describe sample preparation and labeling protocols for imaging bacterial proteins in fixed and live cells.

We examined common models for atomic force microscope (AFM) nanoindentation of living cells and propose a new methodology for fitting the mechanical properties to the indentation data. By modeling the living cells as fluid-filled spherical membranes, the important parameters become the cell wall thickness, cell wall elastic modulus, and the cell’s turgor pressure. We modeled these parameters in addition to adhesion, plasticity, and viscoelasticity using finite element analysis to better describe the underlying physics and geometry of microbial indentation. This modeling approach applied different scaling laws from the classical Hertz model in order to better explain indentation data found in experiments on living E. coli bacterial cells. Work supported by DARPA Contract No. HR0011-17-2-0037.

Networks assembled by reversible association of telechelic polymers constitute a common class of soft materials. Various mechanisms of chain migration in associative networks have been proposed; yet there remains little quantitative experimental data to discriminate among them. Proposed mechanisms for chain migration include multichain aggregate diffusion as well as single-chain mechanisms such as “walking” and “hopping”, wherein diffusion is achieved by either partial (“walking”) or complete (“hopping”) disengagement of the associated chain segments. Here, we provide evidence that hopping can dominate the effective diffusion of chains in associative networks due to a strong entropic penalty for bridge formation imposed by local network structure; chains become conformationally restricted upon association with two or more spatially separated binding sites. This restriction decreases the effective binding strength of chains with multiple associative domains, thereby increasing the probability that a chain will hop. For telechelic chains this manifests as binding asymmetry, wherein the first association is effectively stronger than the second. We derive a simple thermodynamic model that predicts the fraction of chains that are free to hop as a function of tunable molecular and network properties. A large set of self-diffusivity measurements on a series of model associative polymers finds good agreement with this model.

Though bacteria in nature are often nutritionally limited and growing slowly, most of our understanding of core cellular processes such as transcription comes from studies in a handful of model organisms doubling rapidly under nutrient-replete conditions. We previously identified a small protein of unknown function, called SutA, in a global screen of proteins synthesized in Pseudomonas aeruginosa under growth arrest (Babin BM, et al. (2016) SutA is a bacterial transcription factor expressed during slow growth in Pseudomonas aeruginosa. PNAS 113(5):E597-605). SutA binds RNA polymerase (RNAP), causing widespread changes in gene expression, including upregulation of the ribosomal RNA (rRNA) genes. Here, using biochemical and structural methods, we examine how SutA interacts with RNAP and the functional consequences of these interactions. We show that SutA consists of a central α-helix with unstructured N- and C-terminal tails, and binds to the β1 domain of RNAP. It activates transcription from the P. aeruginosa rrn promoter by both the housekeeping sigma factor holoenzyme (Eσ70) and the general stress response sigma factor holoenzyme (EσS) in vitro, and its N-terminal tail is required for activation in both holoenzyme contexts. However, we find that the interaction between SutA and each holoenzyme is distinct, with the SutA C-terminal tail and an acidic loop unique to σ70 playing the determining roles in these differences. Our results add SutA to a growing list of transcription regulators that use their intrinsically disordered regions to remodel transcription complexes.SIGNIFICANCELittle is known about how bacteria regulate their activities during periods of dormancy, yet growth arrest dominates bacterial existence in most environments and is directly relevant to the problem of physiological antibiotic tolerance. Though much is known about transcription in the model organism, Escherichia coli, even there, our understanding of gene expression during dormancy is incomplete. Here we explore how transcription under growth arrest is modulated in Pseudomonas aeruginosa by the small acidic protein, SutA. We show that SutA binds to RNA polymerase and controls transcription by a mechanism that is distinct from other known regulators. Our work underscores the potential for fundamental, mechanistic discovery in this important and understudied realm of bacterial physiology.

Pluripotent stem cells may serve as an alternative source of beta-like cells for replacement therapy of type 1 diabetes; however, the beta-like cells generated in many differentiation protocols are immature. The maturation of endogenous beta cells involves an increase in insulin expression starting in late gestation and a gradual acquisition of the abilities to sense glucose and secrete insulin by week 2 after birth in mice; however, what molecules regulate these maturation processes are incompletely known. In this study, we aim to identify small molecules that affect immature beta cells. A cell-based assay, using pancreatic beta-like cells derived from murine embryonic stem (ES) cells harboring a transgene containing an insulin 1-promoter driven enhanced green fluorescent protein reporter, was used to screen a compound library (NIH Clinical Collection-003). Cortisone, a glucocorticoid, was among five positive hit compounds. Quantitative reverse transcription–polymerase chain reaction analysis revealed that glucocorticoids enhance the gene expression of not only insulin 1 but also glucose transporter-2 (Glut2; Slc2a2) and glucokinase (Gck), two molecules important for glucose sensing. Mifepristone, a pharmacological inhibitor of glucocorticoid receptor (GR) signaling, reduced the effects of glucocorticoids on Glut2 and Gck expression. The effects of glucocorticoids on ES-derived cells were further validated in immature primary islets. Isolated islets from 1-week-old mice had an increased Glut2 and Gck expression in response to a 4-day treatment of exogenous hydrocortisone in vitro. Gene deletion of GR in beta cells using rat insulin 2 promoter-driven Cre crossed with GR(flox/flox) mice resulted in a reduced gene expression of Glut2, but not Gck, and an abrogation of insulin secretion when islets were incubated in 0.5 mM d-glucose and stimulated by 17 mM d-glucose in vitro. These results demonstrate that glucocorticoids positively regulate glucose sensors in immature murine beta-like cells.

Non-canonical amino acids are finding increasing use in basic and applied research. Proteins that evolved naturally for biological function did so by exploiting the chemistries of the canonical amino acids; however, when proteins are repurposed for biomedical and pharmacological applications, they are often subject to conditions different from those characteristic of their original biological environments. Non-canonical amino acids can impart properties that are inaccessible within canonical protein sequence space, and can thereby lead to improved or new functionality. We describe simple methods for global replacement of canonical amino acids by their non-canonical counterparts in recombinant proteins made in high yield in bacterial expression hosts. These methods can be used to engineer both chemical and physical properties of recombinant proteins.

Protein synthesis is a dynamic process that tunes the cellular proteome in response to internal and external demands. Metabolic labeling approaches identify the general proteomic response but cannot visualize specific newly synthesized proteins within cells. Here we describe a technique that couples noncanonical amino acid tagging or puromycylation with the proximity ligation assay to visualize specific newly synthesized proteins and monitor their origin, redistribution and turnover in situ.

Non-canonical amino acids are finding increasing use in basic and applied research. Proteins that evolved naturally for biological function did so by exploiting the chemistries of the canonical amino acids; however, when proteins are repurposed for biomedical and pharmacological applications, they are often subject to conditions different from those characteristic of their original biological environments. Non-canonical amino acids can impart properties that are inaccessible within canonical protein sequence space, and can thereby lead to improved or new functionality. We describe simple methods for global replacement of canonical amino acids by their non-canonical counterparts in recombinant proteins made in high yield in bacterial expression hosts. These methods can be used to engineer both chemical and physical properties of recombinant proteins.

SummaryQuorum sensing is a cell-cell communication process that bacteria use to transition between individual and social lifestyles. In vibrios, homologous small RNAs called the Qrr sRNAs function at the center of quorum-sensing pathways. The Qrr sRNAs regulate multiple mRNA targets including those encoding the quorum-sensing regulatory components luxR, luxO, luxM, and aphA. We show that a representative Qrr, Qrr3, uses four distinct mechanisms to control its particular targets: the Qrr3 sRNA represses luxR through catalytic degradation, represses luxM through coupled degradation, represses luxO through sequestration, and activates aphA by revealing the ribosome binding site while the sRNA itself is degraded. Qrr3 forms different base-pairing interactions with each mRNA target, and the particular pairing strategy determines which regulatory mechanism occurs. Combined mathematical modeling and experiments show that the specific Qrr regulatory mechanism employed governs the potency, dynamics, and competition of target mRNA regulation, which in turn, defines the overall quorum-sensing response.

Programmable colloidal assembly enables the creation of mesoscale materials in a bottom-up manner. Although DNA oligonucleotides have been used extensively as the programmable units in this paradigm, proteins, which exhibit more diverse modes of association and function, have not been widely used to direct colloidal assembly. Here we use protein–protein interactions to drive controlled aggregation of polystyrene microparticles, either through reversible coiled-coil interactions or through intermolecular isopeptide linkages. The sizes of the resulting aggregates are tunable and can be controlled by the concentration of immobilized surface proteins. Moreover, particles coated with different protein pairs undergo orthogonal assembly. We demonstrate that aggregates formed by association of coiled-coil proteins, in contrast to those linked by isopeptide bonds, are dispersed by treatment with chemical denaturants or soluble competing proteins. Finally, we show that protein–protein interactions can be used to assemble complex core–shell aggregates. This work illustrates a versatile strategy for engineering colloidal systems for use in materials science and biotechnology.

Daily injections of insulin provide lifesaving benefits to millions of diabetics. But currently available prandial insulins are suboptimal: The onset of action is delayed by slow dissociation of the insulin hexamer in the subcutaneous space, and insulin forms amyloid fibrils upon storage in solution. Here we show, through the use of non-canonical amino acid mutagenesis, that replacement of the proline residue at position 28 of the insulin B-chain (ProB28) by (4S)-hydroxyproline (Hzp) yields an active form of insulin that dissociates more rapidly, and fibrillates more slowly, than the wild-type protein. Crystal structures of dimeric and hexameric insulin preparations suggest that a hydrogen bond between the hydroxyl group of Hzp and a backbone amide carbonyl positioned across the dimer interface may be responsible for the altered behavior. The effects of hydroxylation are stereospecific; replacement of ProB28 by (4R)-hydroxyproline (Hyp) causes little change in the rates of fibrillation and hexamer disassociation. These results demonstrate a new approach that fuses the concepts of medicinal chemistry and protein design, and paves the way to further engineering of insulin and other therapeutic proteins.

Single-molecule fluorescence in situ hybridization (smFISH) is a simple and widely used method to measure mRNA transcript abundance and localization in single cells. A comparable single-molecule in situ method to measure mRNA translation would enable a more complete understanding of gene regulation. Here we describe a fluorescence assay to detect ribosome interactions with mRNA (FLARIM). The method adapts smFISH to visualize and characterize translation of single molecules of mRNA in fixed cells. To visualize ribosome–mRNA interactions, we use pairs of oligonucleotide probes that bind separately to ribosomes (via rRNA) and to the mRNA of interest, and that produce strong fluorescence signals via the hybridization chain reaction (HCR) when the probes are in close proximity. FLARIM does not require genetic manipulation, is applicable to practically any endogenous mRNA transcript, and provides both spatial and temporal information. We demonstrate that FLARIM is sensitive to changes in ribosome association with mRNA upon inhibition of global translation with puromycin. We also show that FLARIM detects changes in ribosome association with an mRNA whose translation is upregulated in response to increased concentrations of iron., We developed a method to image and quantify changes in mRNA translation in fixed cells. The method utilizes RNA in situ hybridization and the hybridization chain reaction to produce bright fluorescence when ribosomes are bound to an mRNA of interest.

Bio-orthogonal non-canonical amino acid tagging allows time-resolved proteomic analysis of quorum sensing in Vibrio harveyi., Bacteria use a process of chemical communication called quorum sensing to assess their population density and to change their behavior in response to fluctuations in the cell number and species composition of the community. In this work, we identified the quorum-sensing-regulated proteome in the model organism Vibrio harveyi by bio-orthogonal non-canonical amino acid tagging (BONCAT). BONCAT enables measurement of proteome dynamics with temporal resolution on the order of minutes. We deployed BONCAT to characterize the time-dependent transition of V. harveyi from individual- to group-behaviors. We identified 176 quorum-sensing-regulated proteins at early, intermediate, and late stages of the transition, and we mapped the temporal changes in quorum-sensing proteins controlled by both transcriptional and post-transcriptional mechanisms. Analysis of the identified proteins revealed 86 known and 90 new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.

Non-canonical amino acids (ncAAs) provide powerful tools for engineering the chemical and physical properties of proteins. However, introducing ncAAs into proteins can affect protein properties in unpredictable ways, thus necessitating screening efforts to identify mutants with desirable properties. In this work, we describe an Escherichia coli cell surface display platform for the directed evolution of clickable antibody fragments. This platform enabled isolation of antibody fragments with improved digoxigenin binding and modest affinity maturation in several different ncAA contexts. Azide-functionalized fragments exhibited improved binding kinetics relative to their methionine counterparts, facile chemical modification through azide–alkyne cycloaddition, and retention of binding properties after modification. The results described here suggest new possibilities for protein engineering, including modulation of molecular recognition events by ncAAs and direct screening of libraries of chemically modified proteins.

Live-cell imaging, combined with mapping of 3D matrix displacements, identifies sites at which cells apply contractile forces to the matrix and reveals roles for physical forces in cell division., Physical forces direct the orientation of the cell division axis for cells cultured on rigid, two-dimensional (2D) substrates. The extent to which physical forces regulate cell division in three-dimensional (3D) environments is not known. Here, we combine live-cell imaging with digital volume correlation to map 3D matrix displacements and identify sites at which cells apply contractile force to the matrix as they divide. Dividing cells embedded in fibrous matrices remained anchored to the matrix by long, thin protrusions. During cell rounding, the cells released adhesive contacts near the cell body while applying tensile forces at the tips of the protrusions to direct the orientation of the cell division axis. After cytokinesis, the daughter cells respread into matrix voids and invaded the matrix while maintaining traction forces at the tips of persistent and newly formed protrusions. Mechanical interactions between cells and the extracellular matrix constitute an important mechanism for regulation of cell division in 3D environments.

Tumor suppressor microRNA-126 (miR-126) is often down-regulated in cancer cells, and its over-expression is found to inhibit cancer metastasis. To elucidate the mechanism of tumor suppression by miR-126, we analyzed the proteomic response to miR-126 over-expression in the human metastatic breast cancer cell line MDA-MB-231. To acquire quantitative, time-resolved information, we combined two complementary proteomic methods, BONCAT and SILAC. We discovered a new direct target of miR-126: CD97, a pro-metastatic G-protein-coupled receptor (GPCR) that has been reported to promote tumor cell invasion, endothelial cell migration and tumor angiogenesis. This discovery establishes a link between down-regulation of miR-126 and over-expression of CD97 in cancer, and provides new mechanistic insight into the role of miR-126 in inhibiting both cell-autonomous and non-cell-autonomous cancer progression.

Control of molecular topology constitutes a fundamental challenge in macromolecular chemistry. Here we describe the synthesis and characterization of artificial elastin-like proteins (ELPs) with unconventional nonlinear topologies including circular, tadpole, star, and H-shaped proteins using genetically encoded SpyTag–SpyCatcher chemistry. SpyTag is a short polypeptide that binds its protein partner SpyCatcher and forms isopeptide bonds under physiological conditions. Sequences encoding SpyTag and SpyCatcher can be strategically placed into ELP genes to direct post-translational topological modification in situ. Placement of SpyTag at the N-terminus and SpyCatcher at the C-terminus directs formation of circular ELPs. Induction of expression at 16 °C with 10 μM IPTG yields 80% monomeric cyclic protein. When SpyTag is placed in the middle of the chain, it exhibits an even stronger tendency toward cyclization, yielding up to 94% monomeric tadpole proteins. Telechelic ELPs containing either SpyTag or SpyCatcher can be expressed, purified, and then coupled spontaneously upon mixing in vitro. Block proteins, 3-arm or 4-arm star proteins, and H-shaped proteins have been prepared, with the folded CnaB2 domain that results from the SpyTag–SpyCatcher reaction as the molecular core or branch junction. The modular character of the SpyTag–SpyCatcher strategy should make it useful for preparing nonlinear macromolecules of diverse sequence and structure.

Reliable methods to determine the subcellular localization of bacterial proteins are needed for the study of prokaryotic cell biology. We describe here a simple and general technique for imaging of bacterial proteins in situ by fluorescence microscopy. The method uses the eukaryotic enzyme N-myristoyltransferase to modify the N-terminus of the protein of interest with an azido fatty acid. Subsequent strain-promoted azide–alkyne cycloaddition allows conjugation of dyes and imaging of tagged proteins by confocal fluorescence microscopy. We demonstrate the method by labeling the chemotaxis proteins Tar and CheA and the cell division proteins FtsZ and FtsA in Escherichia coli. We observe distinct spatial patterns for each of these proteins in both fixed and live cells. The method should prove broadly useful for protein imaging in bacteria.

Cells alter the proteome to respond to environmental and developmental cues. Global analysis of proteomic responses is of limited value in heterogeneous environments, where there is no ‘average’ cell. Advances in sequencing, protein labeling, mass spectrometry, and data analysis have fueled recent progress in the investigation of specific subpopulations of cells in complex systems. Here we highlight recently developed chemical tools that enable cell-selective proteomic analysis of complex biological systems, from bacterial pathogens to whole animals.

When biological cells migrate, divide, and invade, they push and pull on individual fibers of the matrix surrounding them. The resulting fiber displacements are neither uniform nor smooth; rather, displacements localize to form dense fibrous bands that span from one cell to another. It is thought that these bands may be a mechanism by which cells can sense their neighbors, but this hypothesis remains untested, because the mechanism for band formation remains unknown. Using digital volume correlation, we measure the displacements induced by contractile cells embedded in a fibrous matrix. We find that cell-induced displacements propagate over a longer range than predicted by linear elasticity. To explain the long-range propagation of displacements, we consider the effect of buckling of individual matrix fibers, which generates a nonlinear stress-strain relationship. We show that fiber buckling is the mechanism that causes the displacements to propagate over a long range and the bands to form between nearby cells. The results thus show that buckling of individual fibers provides a mechanism by which cells may sense their distant neighbors mechanically.

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133(+)Sox9/EGFP(+) ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133(+)Sox9/EGFP(+) ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

When several ACS Central Science Editors met for dinner at the 2015 Pacifichem meeting, conversation turned to the grand challenges facing science and society, and those we felt chemistry was in a unique position to solve. With the New Year, we thought we would share our ideas in our first editorial of 2016. The arenas in which we see chemistry having the largest influence are the molecular bases of disease and aging, alternative energy advancement, and the conservation of our elemental resources.

The interactions between biochemical and mechanical signals during cell adhesion, migration, spreading and other processes influence cellular behavior. Three-dimensional measurement techniques are needed to investigate the effect of mechanical properties of the substrate on cellular behavior. This paper discusses a three-dimensional full-field measurement technique that has been developed for measuring large deformations in soft materials. The technique utilizes a digital volume correlation (DVC) algorithm to track motions of sub-volumes within 3-D images obtained using laser scanning confocal microscopy. The technique is well-suited for investigating 3-D mechanical interactions between cells and the extracellular matrix and for obtaining local constitutive properties of soft biomaterials. Results from the migration of single fibroblast cells on polyacrylamide gels and their implications for cell motility models are discussed. The implications that the traction distributions of epithelial cell clusters have on the inhibition of proliferation due to cell contact and scattering of cells in a cluster are discussed. These results provide insights on force fields generated by cells and the role of the mechanical properties of the substrate on cellular interactions and mechanotransduction. Analytical solutions and finite element simulations are used to elucidate the mechanics of cellular forces exerted on the extracellular matrix.

Graft-through ring-opening metathesis polymerization (ROMP) using ruthenium N-heterocyclic carbene catalysts has enabled the synthesis of bottle-brush polymers with unprecedented ease and control. Here we report the first bivalent-brush polymers; these materials were prepared by graft-through ROMP of drug-loaded polyethylene-glycol (PEG) based macromonomers (MMs). Anticancer drugs doxorubicin (DOX) and camptothecin (CT) were attached to a norbornene-alkyne-PEG MM via a photocleavable linker. ROMP of either or both drug-loaded MMs generated brush homo- and co-polymers with low polydispersities and defined molecular weights. Release of free DOX and CT from these materials was initiated by exposure to 365 nm light. All of the CT and DOX polymers were at least 10-fold more toxic to human cancer cells after photoinitiated drug release while a copolymer carrying both CT and DOX displayed 30-fold increased toxicity upon irradiation. Graft-through ROMP of drug-loaded macromonomers provides a general method for the systematic study of structure-function relationships for stimuli-responsive polymers in biological systems.

In 2009, the National Academy for Engineering issued the Grand Challenges for Engineering in the 21st Century comprised of 14 technical challenges that must be addressed to build a healthy, profitable, sustainable, and secure global community (http://www.engineeringchallenges.org). Although crucial, none of the NEA Grand Challenges adequately addressed the challenges that face the biomaterials community. In response to the NAE Grand Challenges, Monty Reichert of Duke University organized a panel entitled Grand Challenges in Biomaterials at the at the 2010 Society for Biomaterials Annual Meeting in Seattle. Six members of the National Academies—Buddy Ratner, James Anderson, Allan Hoffman, Art Coury, Cato Laurencin, and David Tirrell—were asked to propose a grand challenge to the audience that, if met, would significantly impact the future of biomaterials and medical devices. Successfully meeting these challenges will speed the 60-plus year transition from commodity, off-the-shelf biomaterials to bioengineered chemistries, and biomaterial devices that will significantly advance our ability to address patient needs and also to create new market opportunities.

The processes of wound healing and collective cell migration have been studied for decades. Intensive research has been devoted to understanding the mechanisms involved in wound healing, but the role of cell-substrate interactions is still not thoroughly understood. Here we probe the role of cell-substrate interactions by examining in vitro the healing of mono layers of human corneal epithelial (HCE) cells cultured on artificial extra cellular matrix (aECM) proteins. We find that the rate of wound healing is dependent on the concentration off ibronectin-derived (RGD) cell adhesion lig and sin the aECM substrate. The wound closure rate varies nearly six fold on the substrates examined, despite the fact that the rates of migration and proliferation of individual cells show little sensitivity to the RGD concentration (which varies 40 fold). To explain this apparent contradiction, we study collective migration by means of a dynamic Monte Carlo simulation. The cells in the simulation spread, retract, and proliferate with probabilities obtained from a simple phenomenon logical model. The results indicate that the overall wound closure rate is determined primarily by the rate at which cells cross the boundary between the Aecm protein and the matrix deposited under the cell sheet. Accepted version

Protein translation has been implicated in different forms of synaptic plasticity, but direct in situ visualization of new proteins is limited to one or two proteins at a time. Here we describe a metabolic labeling approach based on incorporation of noncanonical amino acids into proteins followed by chemoselective fluorescence tagging by means of 'click chemistry'. After a brief incubation with azidohomoalanine or homopropargylglycine, a robust fluorescent signal was detected in somata and dendrites. Pulse-chase application of azidohomoalanine and homopropargylglycine allowed visualization of proteins synthesized in two sequential time periods. This technique can be used to detect changes in protein synthesis and to evaluate the fate of proteins synthesized in different cellular compartments. Moreover, using strain-promoted cycloaddition, we explored the dynamics of newly synthesized membrane proteins using single-particle tracking and quantum dots. The newly synthesized proteins showed a broad range of diffusive behaviors, as would be expected for a pool of labeled proteins that is heterogeneous.

Cells engage in mechanical force exchange with their extracellular environment through tension generated by the cytoskeleton. A method combining laser scanning confocal microscopy (LSCM) and digital volume correlation (DVC) enables tracking and quantification of cell-mediated deformation of the extracellular matrix in all three spatial dimensions. Time-lapse confocal imaging of migrating 3T3 fibroblasts on fibronectin (FN)-modified polyacrylamide gels of varying thickness reveals significant in-plane (x, y) and normal (z) displacements, and illustrates the extent to which cells, even in nominally two-dimensional (2-D) environments, explore their surroundings in all three dimensions. The magnitudes of the measured displacements are independent of the elastic moduli of the gels. Analysis of the normal displacement profiles suggests that normal forces play important roles even in 2-D cell migration.