Tradeoffs and optimality in the evolution of gene regulation.

Cellular regulation is believed to evolve in response to environmental variability. However, this has been difficult to test directly. Here, we show that a gene regulation system evolves to the optimal regulatory response when challenged with variable environments. We engineered a genetic module subject to regulation by the lac repressor (LacI) in E. coli, whose expression is beneficial in one environmental condition and detrimental in another. Measured tradeoffs in fitness between environments predict the competition between regulatory phenotypes. We show that regulatory evolution in adverse environments is delayed at specific boundaries in the phenotype space of the regulatory LacI protein. Once this constraint is relieved by mutation, adaptation proceeds toward the optimum, yielding LacI with an altered allosteric mechanism that enables an opposite response to its regulatory ligand IPTG. Our results indicate that regulatory evolution can be understood in terms of tradeoff optimization theory.

Publisher Correction: Processive extrusion of polypeptide loops by a Hsp100 disaggregase.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Processive extrusion of polypeptide loops by a Hsp100 disaggregase.

The ability to reverse protein aggregation is vital to cells1,2. Hsp100 disaggregases such as ClpB and Hsp104 are proposed to catalyse this reaction by translocating polypeptide loops through their central pore3,4. This model of disaggregation is appealing, as it could explain how polypeptides entangled within aggregates can be extracted and subsequently refolded with the assistance of Hsp704,5. However, the model is also controversial, as the necessary motor activity has not been identified6-8 and recent findings indicate non-processive mechanisms such as entropic pulling or Brownian ratcheting9,10. How loop formation would be accomplished is also obscure. Indeed, cryo-electron microscopy studies consistently show single polypeptide strands in the Hsp100 pore11,12. Here, by following individual ClpB-substrate complexes in real time, we unambiguously demonstrate processive translocation of looped polypeptides. We integrate optical tweezers with fluorescent-particle tracking to show that ClpB translocates both arms of the loop simultaneously and switches to single-arm translocation when encountering obstacles. ClpB is notably powerful and rapid; it exerts forces of more than 50 pN at speeds of more than 500 residues per second in bursts of up to 28 residues. Remarkably, substrates refold while exiting the pore, analogous to co-translational folding. Our findings have implications for protein-processing phenomena including ubiquitin-mediated remodelling by Cdc48 (or its mammalian orthologue p97)13 and degradation by the 26S proteasome14.

Bacterial coexistence driven by motility and spatial competition.

Elucidating elementary mechanisms that underlie bacterial diversity is central to ecology1,2 and microbiome research3. Bacteria are known to coexist by metabolic specialization4, cooperation5 and cyclic warfare6-8. Many species are also motile9, which is studied in terms of mechanism10,11, benefit12,13, strategy14,15, evolution16,17 and ecology18,19. Indeed, bacteria often compete for nutrient patches that become available periodically or by random disturbances2,20,21. However, the role of bacterial motility in coexistence remains unexplored experimentally. Here we show that-for mixed bacterial populations that colonize nutrient patches-either population outcompetes the other when low in relative abundance. This inversion of the competitive hierarchy is caused by active segregation and spatial exclusion within the patch: a small fast-moving population can outcompete a large fast-growing population by impeding its migration into the patch, while a small fast-growing population can outcompete a large fast-moving population by expelling it from the initial contact area. The resulting spatial segregation is lost for weak growth-migration trade-offs and a lack of virgin space, but is robust to population ratio, density and chemotactic ability, and is observed in both laboratory and wild strains. These findings show that motility differences and their trade-offs with growth are sufficient to promote diversity, and suggest previously undescribed roles for motility in niche formation and collective expulsion-containment strategies beyond individual search and survival.

Human Small Heat Shock Protein B8 Inhibits Protein Aggregation without Affecting the Native Folding Process.

Small Heat Shock Proteins (sHSPs) are key components of our Protein Quality Control system and are thought to act as reservoirs that neutralize irreversible protein aggregation. Yet, sHSPs can also act as sequestrases, promoting protein sequestration into aggregates, thus challenging our understanding of their exact mechanisms of action. Here, we employ optical tweezers to explore the mechanisms of action of the human small heat shock protein HSPB8 and its pathogenic mutant K141E, which is associated with neuromuscular disease. Through single-molecule manipulation experiments, we studied how HSPB8 and its K141E mutant affect the refolding and aggregation processes of the maltose binding protein. Our data show that HSPB8 selectively suppresses protein aggregation without affecting the native folding process. This anti-aggregation mechanism is distinct from previous models that rely on the stabilization of unfolded polypeptide chains or partially folded structures, as has been reported for other chaperones. Rather, it appears that HSPB8 selectively recognizes and binds to aggregated species formed at the early stages of aggregation, preventing them from growing into larger aggregated structures. Consistently, the K141E mutation specifically targets the affinity for aggregated structures without impacting native folding, and hence impairs its anti-aggregation activity.

Long-term expanding human airway organoids for disease modeling.

Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.

Weak catch bonds make strong networks.

Molecular catch bonds are ubiquitous in biology and essential for processes like leucocyte extravasion1 and cellular mechanosensing2. Unlike normal (slip) bonds, catch bonds strengthen under tension. The current paradigm is that this feature provides 'strength on demand3', thus enabling cells to increase rigidity under stress1,4-6. However, catch bonds are often weaker than slip bonds because they have cryptic binding sites that are usually buried7,8. Here we show that catch bonds render reconstituted cytoskeletal actin networks stronger than slip bonds, even though the individual bonds are weaker. Simulations show that slip bonds remain trapped in stress-free areas, whereas weak binding allows catch bonds to mitigate crack initiation by moving to high-tension areas. This 'dissociation on demand' explains how cells combine mechanical strength with the adaptability required for shape change, and is relevant to diseases where catch bonding is compromised7,9, including focal segmental glomerulosclerosis10 caused by the α-actinin-4 mutant studied here. We surmise that catch bonds are the key to create life-like materials.

Single cell variability of CRISPR-Cas interference and adaptation.

While CRISPR-Cas defence mechanisms have been studied on a population level, their temporal dynamics and variability in individual cells have remained unknown. Using a microfluidic device, time-lapse microscopy and mathematical modelling, we studied invader clearance in Escherichia coli across multiple generations. We observed that CRISPR interference is fast with a narrow distribution of clearance times. In contrast, for invaders with escaping PAM mutations we found large cell-to-cell variability, which originates from primed CRISPR adaptation. Faster growth and cell division and higher levels of Cascade increase the chance of clearance by interference, while slower growth is associated with increased chances of clearance by priming. Our findings suggest that Cascade binding to the mutated invader DNA, rather than spacer integration, is the main source of priming heterogeneity. The highly stochastic nature of primed CRISPR adaptation implies that only subpopulations of bacteria are able to respond quickly to invading threats. We conjecture that CRISPR-Cas dynamics and heterogeneity at the cellular level are crucial to understanding the strategy of bacteria in their competition with other species and phages.

Alternative modes of client binding enable functional plasticity of Hsp70.

The Hsp70 system is a central hub of chaperone activity in all domains of life. Hsp70 performs a plethora of tasks, including folding assistance, protection against aggregation, protein trafficking, and enzyme activity regulation, and interacts with non-folded chains, as well as near-native, misfolded, and aggregated proteins. Hsp70 is thought to achieve its many physiological roles by binding peptide segments that extend from these different protein conformers within a groove that can be covered by an ATP-driven helical lid. However, it has been difficult to test directly how Hsp70 interacts with protein substrates in different stages of folding and how it affects their structure. Moreover, recent indications of diverse lid conformations in Hsp70-substrate complexes raise the possibility of additional interaction mechanisms. Addressing these issues is technically challenging, given the conformational dynamics of both chaperone and client, the transient nature of their interaction, and the involvement of co-chaperones and the ATP hydrolysis cycle. Here, using optical tweezers, we show that the bacterial Hsp70 homologue (DnaK) binds and stabilizes not only extended peptide segments, but also partially folded and near-native protein structures. The Hsp70 lid and groove act synergistically when stabilizing folded structures: stabilization is abolished when the lid is truncated and less efficient when the groove is mutated. The diversity of binding modes has important consequences: Hsp70 can both stabilize and destabilize folded structures, in a nucleotide-regulated manner; like Hsp90 and GroEL, Hsp70 can affect the late stages of protein folding; and Hsp70 can suppress aggregation by protecting partially folded structures as well as unfolded protein chains. Overall, these findings in the DnaK system indicate an extension of the Hsp70 canonical model that potentially affects a wide range of physiological roles of the Hsp70 system.

The Anti-Aggregation Holdase Hsp33 Promotes the Formation of Folded Protein Structures.

Holdase chaperones are known to be central to suppressing aggregation, but how they affect substrate conformations remains poorly understood. Here, we use optical tweezers to study how the holdase Hsp33 alters folding transitions within single maltose binding proteins and aggregation transitions between maltose binding protein substrates. Surprisingly, we find that Hsp33 not only suppresses aggregation but also guides the folding process. Two modes of action underlie these effects. First, Hsp33 binds unfolded chains, which suppresses aggregation between substrates and folding transitions within substrates. Second, Hsp33 binding promotes substrate states in which most of the chain is folded and modifies their structure, possibly by intercalating its intrinsically disordered regions. A statistical ensemble model shows how Hsp33 function results from the competition between these two contrasting effects. Our findings reveal an unexpectedly comprehensive functional repertoire for Hsp33 that may be more prevalent among holdases and dispels the notion of a strict chaperone hierarchy.

Apical constriction is necessary for crypt formation in small intestinal organoids.

Small intestinal organoids have become an important tool to study crypt homeostasis, cell fate dynamics and tissue biomechanics. Yet, the mechanisms that drive the budding of crypts from the smooth organoid epithelium remain incompletely understood. Locally enhanced proliferation has been suggested to induce tissue buckling and crypt initiation. Here we report that changes in cell morphology play a crucial role in crypt formation. Crypt formation is preceded by local epithelial thickening, apicobasal elongation, and apical narrowing, resulting in a wedge-like cell-shape, followed by apical evagination and crypt outgrowth. Myosin II activity is found to coincide with apical constriction of cells, while inhibition of myosin suppresses apical constriction and bud formation. The data suggest that myosin-driven apical constriction is a key driving force of bud initiation in small intestinal organoids.

Stochasticity of metabolism and growth at the single-cell level.

Elucidating the role of molecular stochasticity in cellular growth is central to understanding phenotypic heterogeneity and the stability of cellular proliferation. The inherent stochasticity of metabolic reaction events should have negligible effect, because of averaging over the many reaction events contributing to growth. Indeed, metabolism and growth are often considered to be constant for fixed conditions. Stochastic fluctuations in the expression level of metabolic enzymes could produce variations in the reactions they catalyse. However, whether such molecular fluctuations can affect growth is unclear, given the various stabilizing regulatory mechanisms, the slow adjustment of key cellular components such as ribosomes, and the secretion and buffering of excess metabolites. Here we use time-lapse microscopy to measure fluctuations in the instantaneous growth rate of single cells of Escherichia coli, and quantify time-resolved cross-correlations with the expression of lac genes and enzymes in central metabolism. We show that expression fluctuations of catabolically active enzymes can propagate and cause growth fluctuations, with transmission depending on the limitation of the enzyme to growth. Conversely, growth fluctuations propagate back to perturb expression. Accordingly, enzymes were found to transmit noise to other unrelated genes via growth. Homeostasis is promoted by a noise-cancelling mechanism that exploits fluctuations in the dilution of proteins by cell-volume expansion. The results indicate that molecular noise is propagated not only by regulatory proteins but also by metabolic reactions. They also suggest that cellular metabolism is inherently stochastic, and a generic source of phenotypic heterogeneity.

Deciphering Metabolic Heterogeneity by Single-Cell Analysis.

Single-cell analysis provides insights into cellular heterogeneity and dynamics of individual cells. This Feature highlights recent developments in key analytical techniques suited for single-cell metabolic analysis with a special focus on mass spectrometry-based analytical platforms and RNA-seq as well as imaging techniques that reveal stochasticity in metabolism.

Reshaping of the conformational search of a protein by the chaperone trigger factor.

Protein folding is often described as a search process, in which polypeptides explore different conformations to find their native structure. Molecular chaperones are known to improve folding yields by suppressing aggregation between polypeptides before this conformational search starts, as well as by rescuing misfolds after it ends. Although chaperones have long been speculated to also affect the conformational search itself--by reshaping the underlying folding landscape along the folding trajectory--direct experimental evidence has been scarce so far. In Escherichia coli, the general chaperone trigger factor (TF) could play such a role. TF has been shown to interact with nascent chains at the ribosome, with polypeptides released from the ribosome into the cytosol, and with fully folded proteins before their assembly into larger complexes. To investigate the effect of TF from E. coli on the conformational search of polypeptides to their native state, we investigated individual maltose binding protein (MBP) molecules using optical tweezers. Here we show that TF binds folded structures smaller than one domain, which are then stable for seconds and ultimately convert to the native state. Moreover, TF stimulates native folding in constructs of repeated MBP domains. The results indicate that TF promotes correct folding by protecting partially folded states from distant interactions that produce stable misfolded states. As TF interacts with most newly synthesized proteins in E. coli, we expect these findings to be of general importance in understanding protein folding pathways.

Breaking evolutionary constraint with a tradeoff ratchet.

Epistatic interactions can frustrate and shape evolutionary change. Indeed, phenotypes may fail to evolve when essential mutations are only accessible through positive selection if they are fixed simultaneously. How environmental variability affects such constraints is poorly understood. Here, we studied genetic constraints in fixed and fluctuating environments using the Escherichia coli lac operon as a model system for genotype-environment interactions. We found that, in different fixed environments, all trajectories that were reconstructed by applying point mutations within the transcription factor-operator interface became trapped at suboptima, where no additional improvements were possible. Paradoxically, repeated switching between these same environments allows unconstrained adaptation by continuous improvements. This evolutionary mode is explained by pervasive cross-environmental tradeoffs that reposition the peaks in such a way that trapped genotypes can repeatedly climb ascending slopes and hence, escape adaptive stasis. Using a Markov approach, we developed a mathematical framework to quantify the landscape-crossing rates and show that this ratchet-like adaptive mechanism is robust in a wide spectrum of fluctuating environments. Overall, this study shows that genetic constraints can be overcome by environmental change and that cross-environmental tradeoffs do not necessarily impede but also, can facilitate adaptive evolution. Because tradeoffs and environmental variability are ubiquitous in nature, we speculate this evolutionary mode to be of general relevance.

Evolutionary constraints in variable environments, from proteins to networks.

Environmental changes can not only trigger a regulatory response, but also impose evolutionary pressures that can modify the underlying regulatory network. Here, we review recent approaches that are beginning to disentangle this complex interplay between regulatory and evolutionary responses. Systematic genetic reconstructions have shown how evolutionary constraints arise from epistatic interactions between mutations in fixed environments. This approach is now being extended to more complex environments and systems. The first results suggest that epistasis is affected dramatically by environmental changes and, hence, can profoundly affect the course of evolution. Thus, external environments not only define the selection of favored phenotypes, but also affect the internal constraints that can limit the evolution of these phenotypes. These findings also raise new questions relating to the conditions for evolutionary transitions and the evolutionary potential of regulatory networks.

Topology of internally constrained polymer chains.

Linear chains with intra-chain contacts can adopt different topologies and allow transitions between them, but it remains unclear how this process can be controlled. This question is important to systems ranging from proteins to chromosomes, which can adopt different conformations that are key to their function and toxicity. Here, we investigate how the topological dynamics of a simple linear chain is affected by interactions with a binding partner, using Monte Carlo and Molecular Dynamics simulations. We show that two point contacts with a binding partner are sufficient to accelerate or decelerate the formation of particular topologies within linear chains. Computed ''folding-time landscapes" that detail the folding time within the topology space show that such contacts deform these landscapes and hence alter the occupation probability of topological states. The results provide a mechanism by which chain topologies can be controlled externally, which opens up the possibility of regulating topological dynamics and the formation of more complex topologies. The findings may have important implications for understanding the mechanism of chaperone action as well as genome architecture and evolution.

Generation and filtering of gene expression noise by the bacterial cell cycle.

BACKGROUND: Gene expression within cells is known to fluctuate stochastically in time. However, the origins of gene expression noise remain incompletely understood. The bacterial cell cycle has been suggested as one source, involving chromosome replication, exponential volume growth, and various other changes in cellular composition. Elucidating how these factors give rise to expression variations is important to models of cellular homeostasis, fidelity of signal transmission, and cell-fate decisions. RESULTS: Using single-cell time-lapse microscopy, we measured cellular growth as well as fluctuations in the expression rate of a fluorescent protein and its concentration. We found that, within the population, the mean expression rate doubles throughout the cell cycle with a characteristic cell cycle phase dependent shape which is different for slow and fast growth rates. At low growth rate, we find the mean expression rate was initially flat, and then rose approximately linearly by a factor two until the end of the cell cycle. The mean concentration fluctuated at low amplitude with sinusoidal-like dependence on cell cycle phase. Traces of individual cells were consistent with a sudden two-fold increase in expression rate, together with other non-cell cycle noise. A model was used to relate the findings and to explain the cell cycle-induced variations for different chromosomal positions. CONCLUSIONS: We found that the bacterial cell cycle contribution to expression noise consists of two parts: a deterministic oscillation in synchrony with the cell cycle and a stochastic component caused by variable timing of gene replication. Together, they cause half of the expression rate noise. Concentration fluctuations are partially suppressed by a noise cancelling mechanism that involves the exponential growth of cellular volume. A model explains how the functional form of the concentration oscillations depends on chromosome position.

Single-Cell Analysis of the Dps Response to Oxidative Stress.

UNLABELLED: Microorganisms have developed an elaborate spectrum of mechanisms to respond and adapt to environmental stress conditions. Among these is the expression of dps, coding for the DNA-binding protein from starved cells. Dps becomes the dominant nucleoid-organizing protein in stationary-phase Escherichia coli cells and is required for robust survival under stress conditions, including carbon or nitrogen starvation, oxidative stress, metal exposure, and irradiation. To study the complex regulation of Dps in E. coli, we utilized time-lapse fluorescence microscopy imaging to examine the kinetics, input encoding, and variability of the Dps response in single cells. In the presence of an oxidative stressor, we observed a single pulse of activation of Dps production. Increased concentrations of H2O2 led to increased intensity and duration of the pulse. While lower concentrations of H2O2 robustly activated the Dps response with little effect on the growth rate, higher concentrations of H2O2 resulted in dramatically lower and highly varied growth rates. A comparison of cells exposed to the same concentration of H2O2 revealed that increased levels of Dps expression did not confer a growth advantage, indicating that recovery from stress may rely primarily upon variation in the amount of damage caused to individual cells. IMPORTANCE: We show for the first time the response of the DNA-binding protein from starved cells (Dps) to oxidative stress in single cells of E. coli Through time-lapse fluorescence microscopy, a single pulse of Dps production is observed in cells exposed to H2O2, with a duration and intensity of induction proportional to the concentration of the applied stress. More intense Dps expression did not provide a growth benefit to the bacteria, suggesting that healing from oxidative stress may largely depend upon the amount of damage in each individual cell.