Lennart Hilbert

Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos

Upon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA-bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo. DOI: http://dx.doi.org/10.7554/eLife.23326.001

Pre-power-stroke cross-bridges contribute to force transients during imposed shortening in isolated muscle fibers.

When skeletal muscles are activated and mechanically shortened, the force that is produced by the muscle fibers decreases in two phases, marked by two changes in slope (P1 and P2) that happen at specific lengths (L1 and L2). We tested the hypothesis that these force transients are determined by the amount of myosin cross-bridges attached to actin and by changes in cross-bridge strain due to a changing fraction of cross-bridges in the pre-power-stroke state. Three separate experiments were performed, using skinned muscle fibers that were isolated and subsequently (i) activated at different Ca2+ concentrations (pCa2+ 4.5, 5.0, 5.5, 6.0) (n = 13), (ii) activated in the presence of blebbistatin (n = 16), and (iii) activated in the presence of blebbistatin at varying velocities (n = 5). In all experiments, a ramp shortening was imposed (amplitude 10%Lo, velocity 1 Lo•sarcomere length (SL)•s−1), from an initial SL of 2.5 µm (except by the third group, in which velocities ranged from 0.125 to 2.0 Lo•s−1). The values of P1, P2, L1, and L2 did not change with Ca2+ concentrations. Blebbistatin decreased P1, and it did not alter P2, L1, and L2. We developed a mathematical cross-bridge model comprising a load-dependent power-stroke transition and a pre-power-stroke cross-bridge state. The P1 and P2 critical points as well as the critical lengths L1 and L2 were explained qualitatively by the model, and the effects of blebbistatin inhibition on P1 were also predicted. Furthermore, the results of the model suggest that the mechanism by which blebbistatin inhibits force is by interfering with the closing of the myosin upper binding cleft, biasing cross-bridges into a pre-power-stroke state.

Unphosphorylated calponin enhances the binding force of unphosphorylated myosin to actin.

BACKGROUND Smooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin. METHODS To measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (Funb) in the absence and presence of calponin or phosphorylated calponin. RESULTS Funb from F-actin alone (0.12±0.01pN; mean±SE) was significantly increased in the presence of calponin (0.20±0.02pN). This enhancement was lost when calponin was phosphorylated (0.12±0.01pN). To further verify that this enhancement of Funb was due to the cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the Funb obtained at a [KCl] of 25mM (0.21±0.02pN; mean±SE) was significantly decreased at a [KCl] of 150mM, (0.13±0.01pN). CONCLUSIONS This study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state. GENERAL SIGNIFICANCE This study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved.

A tunable refractive index matching medium for live imaging cells, tissues and model organisms

In light microscopy, refractive index mismatches between media and sample cause spherical aberrations that often limit penetration depth and resolution. Optical clearing techniques can alleviate these mismatches, but they are so far limited to fixed samples. We present Iodixanol as a non-toxic medium supplement that allows refractive index matching in live specimens and thus substantially improves image quality in live-imaged primary cell cultures, planarians, zebrafish and human cerebral organoids. DOI: http://dx.doi.org/10.7554/eLife.27240.001

Transcription establishes microenvironments that organize euchromatin

Chromatin is organized into heterochromatin, which is transcriptionally inactive, and euchromatin, which can switch between transcriptionally active and inactive states. This switch in euchromatin activity is accompanied by changes in its spatial distribution. How euchromatin rearrangements are established is unknown. Here we use super-resolution and live-cell microscopy to show that transcriptionally inactive euchromatin moves away from transcriptionally active euchromatin. This movement is driven by the formation of RNA-enriched microenvironments that exclude inactive euchromatin. Using theory, we show that the segregation into RNA-enriched microenvironments and euchromatin domains can be considered an active microemulsion. The tethering of transcripts to chromatin via RNA polymerase II forms effective amphiphiles that intersperse the two segregated phases. Taken together with previous experiments, our data suggest that chromatin is organized in the following way: heterochromatin segregates from euchromatin by phase separation, while transcription organizes euchromatin similar to an active microemulsion.The three-dimensional organization of the genome is essential for development and health. Although the organization of euchromatin (transcriptionally permissive chromatin) dynamically adjusts to changes in transcription, the underlying mechanisms remain elusive. Here, we studied how transcription organizes euchromatin, using experiments in zebrafish embryonic cells and theory. We show that transcription establishes an interspersed pattern of chromatin domains and RNA-enriched microenvironments. Specifically, accumulation of RNA in the vicinity of transcription sites creates microenvironments that locally remodel chromatin by displacing not transcribed chromatin. Ongoing transcriptional activity stabilizes the interspersed pattern of chromatin domains and RNA-enriched microenvironments by establishing contacts between chromatin and RNA. We explain our observations with an active microemulsion model based on two macromolecular mechanisms: RNA/RNA-binding protein complexes generally segregate from chromatin, while transcribed chromatin is retained among RNA/RNA-binding protein accumulations. We propose that microenvironments might be central to genome architecture and serve as gene regulatory hubs.

Molecular Mechanical Differences between Isoforms of Contractile Actin in the Presence of Isoforms of Smooth Muscle Tropomyosin

The proteins involved in smooth muscles molecular contractile mechanism – the anti-parallel motion of actin and myosin filaments driven by myosin heads interacting with actin – are found as different isoforms. While their expression levels are altered in disease states, their relevance to the mechanical interaction of myosin with actin is not sufficiently understood. Here, we analyzed in vitro actin filament propulsion by smooth muscle myosin for -actin (A), -actin-tropomyosin- (A-Tm), -actin-tropomyosin- (A-Tm), -actin (A), -actin-tropomyosin- (A-Tm), and -actin-tropomoysin- (A-Tm). Actin sliding analysis with our specifically developed video analysis software followed by statistical assessment (Bootstrapped Principal Component Analysis) indicated that the in vitro motility of A, A, and A-Tm is not distinguishable. Compared to these three ‘baseline conditions’, statistically significant differences () were: A-Tm – actin sliding velocity increased 1.12-fold, A-Tm – motile fraction decreased to 0.96-fold, stop time elevated 1.6-fold, A-Tm – run time elevated 1.7-fold. We constructed a mathematical model, simulated actin sliding data, and adjusted the kinetic parameters so as to mimic the experimentally observed differences: A-Tm – myosin binding to actin, the main, and the secondary myosin power stroke are accelerated, A-Tm – mechanical coupling between myosins is stronger, A-Tm – the secondary power stroke is decelerated and mechanical coupling between myosins is weaker. In summary, our results explain the different regulatory effects that specific combinations of actin and smooth muscle tropomyosin have on smooth muscle actin-myosin interaction kinetics.

Computational study of synchrony in fields and microclusters of ephaptically coupled neurons

Neuronal hypersynchrony is implicated in epilepsy and other diseases. The low-frequency, spatially averaged electric fields from many thousands of neurons have been shown to promote synchrony. It remains unclear whether highly transient, spatially localized electric fields from single action potentials (ephaptic coupling) significantly affect spike timing of neighboring cells and in consequence, population synchrony. In this study, we simulated the extracellular potentials and the resulting coupling between neurons in the NEURON environment and generalized their connection rules to create an oscillator network model of a sheet of ephaptically coupled neurons. With the use of both models, we explained several aspects of epileptiform behavior not previously modeled by synaptically coupled networks. Importantly, reduction of neuron spacing induced synchronization via single-spike ephaptic coupling, agreeing with seizure suppression seen clinically and in vitro via extracellular volume adjustment. Further reduction of neuron spacing yielded locally synchronized clusters, providing a mechanism for recent in vitro observations of localized neuronal synchrony in the absence of synaptic and gap-junction coupling.

7 Amino Acids can Make the Difference: Chemical Cycle Kinetics Connect Smooth Muscle Myosin's Loop1 Alterations to its Dynamical Differences

Background: The (+) and (-)insert myosin isoforms present in smooth muscle differ by a seven amino acid sequence. The sequence, which is present only in the (+)insert isoform, is part of surface loop1, which is involved in ADP/ATP binding to and release from myosins catalytic pocket.We use a chemical cycle kinetics model of the smooth muscle actomyosin interaction to explore the effects of the loop1 difference on actomyosin kinetics. We connect differences in the nucleotide binding/release energy barriers with differences in cycling rate and on-time. The model also provides a framework for testing other molecular hypotheses about actin-myosin interaction kinetics of single binding sites.Methods: 1.We constructed a kinetic cycle model, that accommodates the essential aspects of the interactions occurring at one actin-myosin binding site. The effective transition rates between the actomyosin cycle states incorporate the free energy differences, energy barriers, substrate concentrations and load on the filament. The diagram method gives an analytic expression for the actomyosin on-time (τon) and probabilities of occupation of a tight-bound state.2.We adjusted the model parameters, so the model results match motility assay filament velocities (V) for (-)insert isoform from literature(V=d/τon, where d is the myosin step size).[Baker et al. J Biol Chem 278(31):28533-28539]3.We decreased the energy barrier for nucleotide binding/release till a two-fold increase in V was achieved. This two-fold increase of V is one generally accepted feature of the (+)insert isoform.Results/Discussion: A 0.9kBT loop1 energy barrier decrease can account for a two-fold increase in V. It also results in 87% actin binding sites tightly bound for (-)insert, compared to 76% for (+)insert. This might contribute to latching behavior in smooth muscle with high (-)insert content.NIH R01-HL 103405-01.