Explore the vast range of titles available.

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.

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.

The inherent stochasticity in metabolic reactions is a potent source of phenotypic heterogeneity in cell populations, with potentially fundamental implications for cancer research. Natural instability in cellular metabolism Molecular fluctuations in individual metabolic reactions are widely thought to have little effect on cell growth, because of averaging over the many biochemical reactions involved. Now Sander Tans and colleagues use time-lapse microscopy to accurately determine the growth rate of single bacterial cells while also monitoring individual enzyme levels, and find that elemental molecular noise does propagate and cause variation in growth. Conversely, they observe that growth fluctuations propagate back to perturb gene expression, so that molecular noise in one gene can influence unrelated genes via growth. The results demonstrate that the inherent variability in metabolic reactions is a potent source of phenotypic heterogeneity in cell populations, with fundamental implications for cancer research. Elucidating the role of molecular stochasticity 1 in cellular growth is central to understanding phenotypic heterogeneity 2 and the stability of cellular proliferation 3 . The inherent stochasticity of metabolic reaction events 4 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 5 , 6 . Stochastic fluctuations in the expression level 1 , 7 , 8 , 9 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 10 , 11 , 12 , the slow adjustment of key cellular components such as ribosomes 13 , 14 , and the secretion 15 and buffering 16 , 17 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 18 , 19 but also by metabolic reactions. They also suggest that cellular metabolism is inherently stochastic, and a generic source of phenotypic heterogeneity.

Just how chaperones clear protein aggregates is a notoriously impenetrable problem. A new study now shows how single-molecule movies of Hsp104 and Hsp70 chaperones acting on amyloid fibers are the key to revealing their underlying cooperation in time and space.

The ability to reverse protein aggregation is vital to cells 1 , 2 . Hsp100 disaggregases such as ClpB and Hsp104 are proposed to catalyse this reaction by translocating polypeptide loops through their central pore 3 , 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 Hsp70 4 , 5 . However, the model is also controversial, as the necessary motor activity has not been identified 6 – 8 and recent findings indicate non-processive mechanisms such as entropic pulling or Brownian ratcheting 9 , 10 . How loop formation would be accomplished is also obscure. Indeed, cryo-electron microscopy studies consistently show single polypeptide strands in the Hsp100 pore 11 , 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 proteasome 14 . A combination of optical tweezers and fluorescent-particle tracking is used to dissect the dynamics of the Hsp100 disaggregase ClpB, and show that the processive extrusion of polypeptide loops is the mechanistic basis of its activity.

Inferring the directionality of interactions between cellular processes is a major challenge in systems biology. Time-lagged correlations allow to discriminate between alternative models, but they still rely on assumed underlying interactions. Here, we use the transfer entropy (TE), an information-theoretic quantity that quantifies the directional influence between fluctuating variables in a model-free way. We present a theoretical approach to compute the transfer entropy, even when the noise has an extrinsic component or in the presence of feedback. We re-analyze the experimental data from Kiviet et al. (2014) where fluctuations in gene expression of metabolic enzymes and growth rate have been measured in single cells of E. coli. We confirm the formerly detected modes between growth and gene expression, while prescribing more stringent conditions on the structure of noise sources. We furthermore point out practical requirements in terms of length of time series and sampling time which must be satisfied in order to infer optimally transfer entropy from times series of fluctuations.

Elucidating elementary mechanisms that underlie bacterial diversity is central to ecology 1 , 2 and microbiome research 3 . Bacteria are known to coexist by metabolic specialization 4 , cooperation 5 and cyclic warfare 6 – 8 . Many species are also motile 9 , which is studied in terms of mechanism 10 , 11 , benefit 12 , 13 , strategy 14 , 15 , evolution 16 , 17 and ecology 18 , 19 . Indeed, bacteria often compete for nutrient patches that become available periodically or by random disturbances 2 , 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. In mixed bacterial populations that colonize nutrient patches, a growth–migration trade-off can lead to spatial exclusion that provides an advantage to populations that become rare, thereby stabilizing the community.

Molecular catch bonds are ubiquitous in biology and essential for processes like leucocyte extravasion 1 and cellular mechanosensing 2 . Unlike normal (slip) bonds, catch bonds strengthen under tension. The current paradigm is that this feature provides ‘strength on demand 3 ’, thus enabling cells to increase rigidity under stress 1 , 4 – 6 . However, catch bonds are often weaker than slip bonds because they have cryptic binding sites that are usually buried 7 , 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 compromised 7 , 9 , including focal segmental glomerulosclerosis 10 caused by the α-actinin-4 mutant studied here. We surmise that catch bonds are the key to create life-like materials. Reconstituted cytoskeleton networks linked with catch bonds display increased mechanical strength and crack resistance than those containing slip bonds, and simultaneously being more deformable, which allows for better adaptability to new mechanical environments.

Stochasticity in gene regulation has been characterized extensively, but how it affects cellular growth and fitness is less clear. We study the growth of E. coli cells as they shift from glucose to lactose metabolism, which is characterized by an obligatory growth arrest in bulk experiments that is termed the lag phase. Here, we follow the growth dynamics of individual cells at minute-resolution using a single-cell assay in a microfluidic device during this shift, while also monitoring lac expression. Mirroring the bulk results, the majority of cells displays a growth arrest upon glucose exhaustion, and resume when triggered by stochastic lac expression events. However, a significant fraction of cells maintains a high rate of elongation and displays no detectable growth lag during the shift. This ability to suppress the growth lag should provide important selective advantages when nutrients are scarce. Trajectories of individual cells display a highly non-linear relation between lac expression and growth, with only a fraction of fully induced levels being sufficient for achieving near maximal growth. A stochastic molecular model together with measured dependencies between nutrient concentration, lac expression level, and growth accurately reproduces the observed switching distributions. The results show that a growth arrest is not obligatory in the classic diauxic shift, and underscore that regulatory stochasticity ought to be considered in terms of its impact on growth and survival.

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. A novel mechanism for Hsp70 action 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 regulation 1 , 2 , 3 , 4 , 5 , and interacts with non-folded chains, as well as near-native, misfolded, and aggregated proteins 6 , 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 lid 11 , 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 mechanisms 15 , 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 cycle 19 . 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.