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Guanylate-Binding Proteins (GBPs) are interferon-inducible guanosine triphosphate hydrolases (GTPases) that mediate immune effector functions against intracellular pathogens. A key step for the antimicrobial activity of GBPs is the formation of homo- and heterooligomeric complexes on the membrane of pathogen-associated compartments or cytosolinvasive bacteria. Similar to other large GTPases of the dynamin family, oligomerisation and membrane association of GBPs depend on their GTPase activity. How nucleotide binding and hydrolysis prime GBPs for membrane targeting and coatomer formation remains unclear. Here, we report the cryo-EM structure of the full-length human GBP1 dimer in its guanine nucleotide-bound state and resolve the molecular ultrastructure of GBP1 coatomer assemblies on liposomes and bacterial lipopolysaccharide membranes. We show how nucleotide binding promotes large-scale conformational changes of the middle and GTPase effector domains that expose the isoprenylated carboxyl-terminus for association with lipid membranes. Our structure reveals how the α-helical stalks of the middle domain form a parallel arrangement firmly held in a unique cross-over arrangement by intermolecular contacts between adjacent monomers. This conformation is critical for GBP1 dimers to assemble into densely packed coatomers on target membranes. The extended α-helix of the effector domain is flexible and permits intercalation into the dense lipopolysaccharide layer on the outer membrane of gram-negative bacterial pathogens. We show that nucleotide-dependent oligomerisation and GTP hydrolysis yield GBP1 membrane scaffolds with contractile abilities that promote the formation of tubular membrane protrusions and membrane fragmentation. Collectively, our data provide a structural and mechanistic framework for interrogating the molecular basis for GBP1 effector functions in intracellular immunity.

Stem-cell derived in vitro systems, such as organoids or embryoids, hold great potential for modeling in vivo development. Full control over their initial composition, scalability, and easily measurable dynamics make those systems useful for studying specific developmental processes in isolation. Here we report the formation of gastruloids consisting of mouse embryonic stem cells (mESCs) and extraembryonic endoderm (XEN) cells. These XEN-enhanced gastruloids (XEGs) exhibit the formation of neural epithelia, which are absent in gastruloids derived from mESCs only. By single-cell RNA-seq, imaging, and differentiation experiments, we demonstrate the neural characteristics of the epithelial tissue. We further show that the mESCs induce the differentiation of the XEN cells to a visceral endoderm-like state. Finally, we demonstrate that local inhibition of WNT signaling and production of a basement membrane by the XEN cells underlie the formation of the neuroepithelial tissue. In summary, we establish XEGs to explore heterotypic cellular interactions and their developmental consequences in vitro.

Elucidating the dynamics of cellular differentiation in space and time is key to advancing organoid biology and technology. Direct visualization of differentiation patterns is challenging, however, in part because of the difficulty of simultaneously detecting all relevant cell types by fluorescence imaging. Here we present TypeTracker, which determines the differentiation trajectories of all cells within a region of interest, including their type transitions, growth, divisions, and changes in spatial organization. We show how the lineage tree topology, as determined by automated cell tracking, allows for the retrospective identification of cell types across multiple generations. The data revealed various surprising aspects of intestinal organoid differentiation, including symmetric fate adoption by sister cells, cell type abundance regulated by division, and type commitment of cells occurring prior to their spatial reorganization. Our method can be applied broadly to study other organoid systems and to screen for compounds that affect differentiation programs.

The limits of evolution have long fascinated biologists. However, the causes of evolutionary constraint have remained elusive due to a poor mechanistic understanding of studied phenotypes. Recently, a range of innovative approaches have leveraged mechanistic information on regulatory networks and cellular biology. These methods combine systems biology models with population and single-cell quantification and with new genetic tools, and they have been applied to a range of complex cellular functions and engineered networks. In this article, we review these developments, which are revealing the mechanistic causes of epistasis at different levels of biological organization¤mdash¤in molecular recognition, within a single regulatory network, and between different networks¤mdash¤providing first indications of predictable features of evolutionary constraint.

While CRISPR-Cas defence mechanisms have been studied on a population level, their temporal dynamics and variability in indi- vidual 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 distri- bution of clearance times. In contrast, for invaders with escaping PAM mutations we found large cell-to-cell variability, which origi- nates from primed CRISPR adaptation. Faster growth and cell divi- sion 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 Cas- cade binding to the mutated invader DNA, rather than spacer inte- gration, 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 heterogene- ity at the cellular level are crucial to understanding the strategy of bacteria in their competition with other species and phages.

During renewal of the intestine, cells are continuously generated by proliferation. Proliferation and differentiation must be tightly balanced, as any bias towards proliferation results in uncontrolled exponential growth. Yet, the inherently stochastic nature of cells raises the question how such fluctuations are limited. We used time-lapse microscopy to track all cells in crypts of growing mouse intestinal organoids for multiple generations, allowing full reconstruction of the underlying lineage dynamics in space and time. Proliferative behavior was highly symmetric between sister cells, with both sisters either jointly ceasing or continuing proliferation. Simulations revealed that such symmetric proliferative behavior minimizes cell number fluctuations, explaining our observation that proliferating cell number remained constant even as crypts increased in size considerably. Proliferative symmetry did not reflect positional symmetry, but rather lineage control through the mother cell. Our results indicate a concrete mechanism to balance proliferation and differentiation with minimal fluctuations, that may be broadly relevant for other tissues.

Self-renewing tissues require that a constant number of proliferating cells is maintained over time. This maintenance can be ensured at the single-cell level or the population level. Maintenance at the population level leads to fluctuations in the number of proliferating cells over time. Often, it is assumed that those fluctuations can be reduced by increasing the number of asymmetric divisions, i.e. divisions where only one of the daughter cells remains proliferative. Here, we study a model of cell proliferation that incorporates a stem cell niche of fixed size, and explicitly model the cells inside and outside the niche. We find that in this model fluctuations are minimized when the difference in growth rate between the niche and the rest of the tissue is maximized and all divisions are symmetric divisions, producing either two proliferating or two non-proliferating daughters. We show that this optimal state leaves visible signatures in clone size distributions and could thus be detected experimentally.

The chaperone heat shock protein 90 (Hsp90) is well known to undergo important conformational changes, which depend on nucleotide and substrate interactions. Conversely, how the conformations of its unstable and disordered substrates are affected by Hsp90 is difficult to address experimentally yet is central to its function. Here, using optical tweezers, we find that Hsp90 promotes local contractions in unfolded chains that drive their global compaction down to dimensions of folded states. This compaction has a gradual nature while showing small steps, is stimulated by ATP, and performs mechanical work against counteracting forces that expand the chain dimensions. The Hsp90 interactions suppress the formation of larger-scale folded, misfolded, and aggregated structures. The observations support a model in which Hsp90 alters client conformations directly by promoting local intra-chain interactions while suppressing distant ones. We conjecture that chain compaction may be central to how Hsp90 protects unstable clients and cooperates with Hsp70.

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.

SUMMARYThe chaperone Hsp90 is well known to undergo important conformational changes, which depend on nucleotide, co-chaperones, substrate interactions and post-translational modifications. Conversely, how the conformations of its unstable and disordered substrates are affected by Hsp90 is difficult to address experimentally, yet central to its function. Here, using optical tweezers and luciferase and glucocorticoid receptor substrates, we find that Hsp90 promotes local contractions in unfolded chains that drive their global compaction down to dimensions of folded states. This compaction has a gradual nature while showing small steps, is stimulated by ATP, and performs mechanical work against counteracting forces that expand the chain dimensions. The Hsp90 interactions suppress the formation of larger-scale folded, misfolded and aggregated structures. The observations support a model in which Hsp90 alters client conformations directly by promoting local intra-chain interactions while suppressing distant ones. We conjecture that chain compaction may be central to how Hsp90 protects unstable kinases and receptor clients, regulates their activity, and how Hsp90 cooperates with Hsp70.

Funder: Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Netherlands Organisation for Scientific Research); doi: https://doi.org/10.13039/501100003246, Many proteins form dynamic complexes with DNA, RNA, and other proteins, which often involves protein conformational changes that are key to function. Yet, methods to probe these critical dynamics are scarce. Here we combine optical tweezers with fluorescence imaging to simultaneously monitor the conformation of individual proteins and their binding to partner proteins. Central is a protein–DNA coupling strategy, which uses exonuclease digestion and partial re-synthesis to generate DNA overhangs of different lengths, and ligation to oligo-labeled proteins. It provides up to 40 times higher coupling yields than existing protocols and enables new fluorescence-tweezers assays, which require particularly long and strong DNA handles. We demonstrate the approach by detecting the emission of a tethered fluorescent protein and of a molecular chaperone (trigger factor) complexed with its client. We conjecture that our strategy will be an important tool to study conformational dynamics within larger biomolecular complexes.

Co-co assembly for oligomers Most of the human proteome forms oligomeric protein complexes, but how they assemble is poorly understood. Bertolini et al. used a ribosome-profiling approach to explore the existence of a cotranslational assembly mode based on the interaction of two nascent polypeptides, which they call the “co-co” assembly. Proteome-wide data were used to show whether, when, and how efficiently nascent complex subunits interact. The findings also show that human cells use co-co assembly to produce hundreds of different homo-oligomers. Co-co assembly involving ribosomes translating one messenger RNA may resolve the longstanding question of how cells prevent unwanted interactions between different protein isoforms to efficiently produce functional homo-oligomers. Science , this issue p. 57

Molecular catch bonds are ubiquitous in biology and well-studied in the context of leukocyte extravasion1, cellular mechanosensing2,3, and urinary tract infection4. Unlike normal (slip) bonds, catch bonds strengthen under tension. The current paradigm is that this remarkable ability enables cells to increase their adhesion in fast fluid flows1,4, and hence provides ‘strength-on-demand’. Recently, cytoskeletal crosslinkers have been discovered that also display catch bonding5–8. It has been suggested that they strengthen cells, following the strength-on-demand paradigm9,10. However, catch bonds tend to be weaker compared to regular (slip) bonds because they have cryptic binding sites that are often inactive11–13. Therefore, the role of catch bonding in the cytoskeleton remains unclear. Here we reconstitute cytoskeletal actin networks to show that catch bonds render them both stronger and more deformable than slip bonds, even though the bonds themselves are weaker. We develop a model to show that weak binding allows the catch bonds to mitigate crack initiation by moving from low- to high-tension areas in response to mechanical loading. By contrast, slip bonds remain trapped in stress-free areas. We therefore propose that the mechanism of catch bonding is typified by dissociation-on-demand rather than strength-on-demand. Dissociation-on-demand can explain how both cytolinkers5–8,10,14,15 and adhesins1,2,4,12,16–20 exploit continuous redistribution to combine mechanical strength with the adaptability required for movement and proliferation21. Our findings provide a new perspective on diseases where catch bonding is compromised11,12 such as kidney focal segmental glomerulosclerosis22,23, caused by the α-actinin-4 mutant studied here. Moreover, catch bonds provide a route towards creating life-like materials that combine strength with deformability24.

Proteins commonly fold cotranslationally on the ribosome, while the nascent chain emerges from the ribosomal tunnel. Protein domains that are sufficiently small can even fold while still located inside the tunnel. However, the effect of the tunnel on the folding dynamics of these domains is still not well understood. Here, we combine optical tweezers with single-molecule FRET and molecular dynamics simulations to investigate folding of the small zinc-finger domain ADR1a inside and at the vestibule of the ribosomal tunnel. The tunnel is found to accelerate folding and stabilize the folded state, reminiscent of the effects of chaperonins. However, a simple mechanism involving stabilization by confinement does not reproduce the results. Instead, it appears that electrostatic interactions between the protein and ribosome contribute to the observed folding acceleration and stabilization of ADR1a.

Unfolded proteins ubiquitously collapse into a compact yet dynamic state1,2. While this compaction is pivotal to protein folding3, aggregation4,5, intrinsic disorder6, and phase separation7, its role in protein quality control mechanisms remains obscure8. Collapse has been characterized mainly for polypeptides that are free in solution, in terms of kinetics, chain expansion, and effect on folding9,10. Yet, theory suggests that the solvent-mediated forces driving collapse can be altered near hydrophobic and charged surfaces, which are observed for many proteins including GroEL-ES11,12. Notably, while GroEL-ES is the archetypal protein-folding chaperone, its folding mechanism remains unresolved13,14. GroEL-ES is proposed to sterically confine polypeptides within its closed chamber15, unfold misfolded states16,17, or promote folding indirectly by suppressing aggregation18,19. Here, using integrated protein manipulation and imaging, we show that GroEL-ES can strengthen the collapse of polypeptide substrates, and hence stimulate folding directly. Strikingly, attractive forces pull substrate chains into the open GroEL cavity -unclosed by GroES-, and hence trigger a gradual compaction and discrete folding transitions, even for slow-folding proteins. This collapse enhancement is strongest in the nucleotide-bound states of GroEL, and is aided by GroES binding to the cavity rim, and by the amphiphilic C-terminal tails at the cavity bottom. Peptides corresponding to these C-termini alone are sufficient to strengthen the collapse. The results show a mechanism that allows folding to be stimulated: by strengthening the collapse, residues are brought together that must contact to fold. The notion that one protein can modulate the collapse of another may be generally important in protein conformation and coacervation control, for systems ranging from the GroEL-ES homologue TRiC/CCT20, to the oncogenic c-Myc/Max complex21, and the nuclear pore transporter transportin22.

SummaryGrowth and division are central to cell size. Bacteria achieve size homeostasis by dividing when growth has added a constant size since birth, termed the “adder” principle, by unknown mechanisms [1–4]. Growth is well known to be regulated by ppGpp, which controls anything from ribosome production to metabolic enzyme activity and replication initiation, and whose absence or excess can induce the stress response, filamentation, and yield growth-arrested miniature cells [5–8]. These observations raise unresolved questions about the relation between ppGpp and size homeostasis mechanisms during normal exponential growth. Here, to untangle effects of ppGpp and nutrients, we gained control of cellular ppGpp by inducing the synthesis and hydrolysis enzymes RelA and Mesh1. We found that ppGpp not only exerts control over the growth rate, but also over cell division and hence the steady state cell size. The added size responds rapidly to changes in the ppGpp level, aided by transiently accelerated or delayed divisions, and establishes its new constant value while the growth rate still adjusts. Moreover, the magnitude of the added size and resulting steady-state birth size correlate consistently with the ppGpp level, rather than with the growth rate, which results in cells of different size that grow equally fast. Our findings suggest that ppGpp serves as a critical regulator that coordinates cell size and growth control.

CRISPR-Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences into human organoids is lacking robust knock-in approaches. Here, we describe CRISPR-Cas9-mediated homology-independent organoid transgenesis (CRISPR-HOT), which enables efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences into desired loci, without the necessity to inactivate TP53 in untransformed cells, which was previously used to increase HDR-mediated knock-in. CRISPR-HOT was used to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter-in which the mitotic spindle was labelled by endogenously tagged tubulin and the cell membrane by endogenously tagged E-cadherin-uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed that TP53 is involved in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.Artegiani, Hendriks et al. describe a CRISPR-Cas9-based method to efficiently generate human knock-in organoids using non-homologous end joining to study rare intestinal cell types and human hepatocyte division.

Advances in our ability to zoom in on single cells have revealed striking heterogeneity within isogenic populations. Attention has so far focussed predominantly on underlying stochastic variability in regulatory pathways and downstream differentiation events. In contrast, the role of stochasticity in metabolic processes and networks has long remained unaddressed. Here we review recent studies that have begun to overcome key technical challenges in addressing this issue. First findings have already demonstrated that metabolic networks are stochastic in nature, and highlight the plethora of cellular processes that are critically affected by it.

Protein folding is well known to be supervised by a dedicated class of proteins called chaperones. However, the core mode of action of these molecular machines has remained elusive due to several reasons including the promiscuous nature of the interactions between chaperones and their many clients, as well as the dynamics and heterogeneity of chaperone conformations and the folding process itself. While troublesome for traditional bulk techniques, these properties make an excellent case for the use of single-molecule approaches. In this review, we will discuss how force spectroscopy, fluorescence microscopy, FCS, and FRET methods are starting to zoom in on this intriguing and diverse molecular toolbox that is of direct importance for protein quality control in cells, as well as numerous degenerative conditions that depend on it.

SUMMARYStem-cell derived in vitro systems, such as organoids or embryoids, hold great potential for modeling in vivo biology and engineering living systems with novel functions. To unlock that potential, we need new ways to elicit higher-level organization. Here we show that adding extraembryonic endoderm (XEN) cells to mouse gastruloids leads to the formation of neural epithelia. By single-cell RNA-seq, imaging and differentiation experiments, we demonstrate the neural characteristics and spatial patterning of the epithelial tissue. We further show that the XEN cells differentiate reciprocally to a visceral endoderm-like state. Finally, we demonstrate that local inhibition of WNT signaling and production of a basement membrane by the XEN cells underlie the formation of the neuroepithelial tissue. In summary, we establish “XEN Enhanced Gastruloids” (XEGs) to explore heterotypic cellular interactions as a means to achieve complex, tissue-level organization in vitro.

Gene regulation networks allow organisms to adapt to diverse environmental niches. However, the constraints underlying the evolution of regulatory phenotypes remain ill-defined both theoretically and experimentally. Here, we show that the concept of partial order identifies such constraints, and test the predictions by experimentally evolving an engineered signal-integrating network in multiple environments. We find that populations: 1) expand in fitness space along the Pareto-optimal front predicted by conflicts in regulatory demands, by fine-tuning binding affinities within the network, 2) expand beyond this constraint by changes in the network structure, thus allowing access to new fitness domains. Strikingly, the constraint predictions are based on whether the network output increases or decreases in response to the different signals, and do not require information on the network architecture or underlying genetics. Overall, our findings show that limited knowledge on current regulatory phenotypes can provide predictions on future evolutionary constraints.

The ability to reverse protein aggregation is vital to cells

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.

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.

Small heat shock proteins (sHsp) constitute an evolutionary conserved yet diverse family of chaperones acting as first line of defence against proteotoxic stress. sHsps coaggregate with misfolded proteins but the molecular basis and functional implications of these interactions, as well as potential sHsp specific differences, are poorly explored. In a comparative analysis of the two yeast sHsps, Hsp26 and Hsp42, we show in vitro that model substrates retain near-native state and are kept physically separated when complexed with either sHsp, while being completely unfolded when aggregated without sHsps. Hsp42 acts as aggregase to promote protein aggregation and specifically ensures cellular fitness during heat stress. Hsp26 in contrast lacks aggregase function but is superior in facilitating Hsp70/Hsp100-dependent post-stress refolding. Our findings indicate the sHsps of a cell functionally diversify in stress defence, but share the working principle to promote sequestration of misfolding proteins for storage in native-like conformation., Small heat shock proteins (sHsps) contribute to cellular recovery and survival following stress causing elevated levels of misfolded or unfolded proteins. Here the authors demonstrate that sHsps function by maintaining aggregating proteins in close-to-native conformations to facilitate chaperone-mediated refolding.

Hsp70 binds unfolded protein segments in its groove, but can also bind and stabilize folded protein structures, owing to its moveable lid, with ATP hydrolysis and co-chaperones allowing control of these contrasting effects. The protein-chaperone system centred on Hsp70 performs a variety of cellular control tasks, including folding assistance, protection against aggregation, trafficking and regulation of enzyme activity, a versatility that has been hard to reconcile with structural data, which suggest that Hsp70 only binds extended polypeptide segments. Now, using laser molecular tweezers, Sander Tans and colleagues show that the bacterial homolog of Hsp70, known as DnaK, relies on its 'groove' to bind unfolded proteins, but can also bind folded structures, thanks to its 'lid', with control of ATP hydrolysis by co-chaperones allowing regulation of such contrasting effects. Contrary to known stabilization mechanisms, through precise structural fit, Hsp70 can stabilize a vast repertoire of client proteins, through a clamp-like, ATP-driven conformational change. 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 regulation1,2,3,4,5, and interacts with non-folded chains, as well as near-native, misfolded, and aggregated proteins6,7,8,9,10. 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 lid11,12,13,14,15. 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 mechanisms15,16,17,18. 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 cycle19. 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.

Time-lapse microscopy is routinely used to follow cells within organoids, allowing direct study of division and differentiation patterns. There is an increasing interest in cell tracking in organoids, which makes it possible to study their growth and homeostasis at the single-cell level. As tracking these cells by hand is prohibitively time consuming, automation using a computer program is required. Unfortunately, organoids have a high cell density and fast cell movement, which makes automated cell tracking difficult. In this work, a semi-automated cell tracker has been developed. To detect the nuclei, we use a machine learning approach based on a convolutional neural network. To form cell trajectories, we link detections at different time points together using a min-cost flow solver. The tracker raises warnings for situations with likely errors. Rapid changes in nucleus volume and position are reported for manual review, as well as cases where nuclei divide, appear and disappear. When the warning system is adjusted such that virtually error-free lineage trees can be obtained, still less than 2% of all detected nuclei positions are marked for manual analysis. This provides an enormous speed boost over manual cell tracking, while still providing tracking data of the same quality as manual tracking.

Summary Gene regulation networks allow organisms to adapt to diverse environmental niches. However, the constraints underlying the evolution of gene regulation remain ill defined. Here, we show that partial order—a concept that ranks network output levels as a function of different input signals—identifies such constraints. We tested our predictions by experimentally evolving an engineered signal-integrating network in multiple environments. We find that populations: (1) expand in fitness space along the Pareto-optimal front associated with conflicts in regulatory demands, by fine-tuning binding affinities within the network, and (2) expand beyond the Pareto-optimal front through changes in the network structure. Our constraint predictions are based only on partial order and do not require information on the network architecture or underlying genetics. Overall, our findings show that limited knowledge of current regulatory phenotypes can provide predictions on future evolutionary constraints.

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.

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.

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 biopsies or lavage material. The pseudostratified airway organoid epithelium consists of basal cells, functional multi-ciliated cells, mucus-producing goblet cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoid culture conditions also allow gene editing as well as the derivation of various types of lung cancer organoids. Respiratory syncytial virus (RSV) infection recapitulated central disease features and dramatically increases organoid cell motility, found to be driven by the non-structural viral NS2 protein. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.

Chaperones assist in protein folding, but what this common phrase means in concrete terms has remained surprisingly poorly understood. We can readily measure chaperone binding to unfolded proteins, but how they bind and affect proteins along folding trajectories has remained obscure. Here we review recent efforts by our labs and others that are beginning to pry into this issue, with a focus on the chaperones trigger factor and Hsp70. Single-molecule methods are central, as they allow the stepwise process of folding to be followed directly. First results have already revealed contrasts with long-standing paradigms: rather than acting only "early" by stabilizing unfolded chain segments, these chaperones can bind and stabilize partially folded structures as they grow to their native state. The findings suggest a fundamental redefinition of the protein folding problem and a more extensive functional repertoire of chaperones than previously assumed.

Optical tweezers allow the detection of unfolding and refolding transitions in individual proteins, and how interacting molecules such as chaperones affect these transitions. Typical methods that tether individual proteins are based on cysteine chemistry, which is less suitable for proteins with essential cysteines. Here we describe a cysteine-independent tethering protocol that can be performed in situ.

Optical tweezers allow the detection of unfolding and refolding transitions in individual proteins, and how interacting molecules such as chaperones affect these transitions. Typical methods that tether individual proteins are based on cysteine chemistry, which is less suitable for proteins with essential cysteines. Here we describe a cysteine-independent tethering protocol that can be performed in situ.

Spatial confinement limits the conformational space accessible to biomolecules but the implications for bimolecular topology are not yet known. Folded linear biopolymers can be seen as molecular circuits formed by intramolecular contacts. The pairwise arrangement of intra-chain contacts can be categorized as parallel, series or cross, and has been identified as a topological property. Using molecular dynamics simulations, we determine the contact order distributions and topological circuits of short semi-flexible linear and ring polymer chains with a persistence length of lp under a spherical confinement of radius Rc. At low values of lp/Rc, the entropy of the linear chain leads to the formation of independent contacts along the chain and accordingly, increases the fraction of series topology with respect to other topologies. However, at high lp/Rc, the fraction of cross and parallel topologies are enhanced in the chain topological circuits with cross becoming predominant. At an intermediate confining regime, we identify a critical value of lp/Rc, at which all topological states have equal probability. Confinement thus equalizes the probability of more complex cross and parallel topologies to the level of the more simple, non-cooperative series topology. Moreover, our topology analysis reveals distinct behaviours for ring- and linear polymers under weak confinement; however, we find no difference between ring- and linear polymers under strong confinement. Under weak confinement, ring polymers adopt parallel and series topologies with equal likelihood, while linear polymers show a higher tendency for series arrangement. The radial distribution analysis of the topology reveals a non-uniform effect of confinement on the topology of polymer chains, thereby imposing more pronounced effects on the core region than on the confinement surface. Additionally, our results reveal that over a wide range of confining radii, loops arranged in parallel and cross topologies have nearly the same contact orders. Such degeneracy implies that the kinetics and transition rates between the topological states cannot be solely explained by contact order. We expect these findings to be of general importance in understanding chaperone assisted protein folding, chromosome architecture, and the evolution of molecular folds.