Organization and Regulation of Chromatin by Liquid-Liquid Phase Separation, bioRxiv, 2019-01-18
Genomic DNA is highly compacted in the nucleus of eukaryotic cells as a nucleoprotein assembly called chromatin. The basic unit of chromatin is the nucleosome, where ~146 base pair increments of the genome are wrapped and compacted around the core histone proteins. Further genomic organization and compaction occur through higher order assembly of nucleosomes. This organization regulates many nuclear processes, and is controlled in part by histone post-transtranslational modifications and chromatin-binding proteins. Mechanisms that regulate the assembly and compaction of the genome remain unclear. Here we show that in the presence of physiologic concentrations of mono- and divalent salts, histone tail-driven interactions drive liquid-liquid phase separation (LLPS) of nucleosome arrays, resulting in substantial condensation. Phase separation of nucleosomal arrays is inhibited by histone acetylation, whereas histone H1 promotes phase separation, further compaction, and decreased dynamics within droplets, mirroring the relationship between these modulators and the accessibility of the genome in cells. These results indicate that under physiologically relevant conditions, LLPS is an intrinsic behavior of the chromatin polymer, and suggest a model in which the condensed phase reflects a genomic 'ground state' that can produce chromatin organization and compaction in vivo. The dynamic nature of this state could enable known modulators of chromatin structure, such as post-translational modifications and chromatin binding proteins, to act upon it and consequently control nuclear processes such as transcription and DNA repair. Our data suggest an important role for LLPS of chromatin in the organization of the eukaryotic genome.
biorxiv biophysics 100-200-users 2019A DNA-based voltmeter for organelles Supplementary information, bioRxiv, 2019-01-17
The role of membrane potential in most intracellular organelles remains unexplored because of the lack of suitable probes. We describe a DNA-based fluorescent reporter that quantitates membrane potential and can be targeted to specific organelles in live cells. It is equipped with a voltage sensitive fluorophore, a reference fluorophore for ratiometric quantification, and acts as an endocytic tracer. We could thereby measure the membrane potential of different intracellular organelles in living cells, which has not been possible previously. Our understanding of how membrane potential regulates organelle biology is poised to expand through the use of these new sensors.
biorxiv biophysics 0-100-users 2019Recording of sonic attacks on U.S. diplomats in Cuba spectrally matches the echoing call of a Caribbean cricket, bioRxiv, 2019-01-04
Beginning in late 2016, diplomats posted to the United States embassy in Cuba began to experience unexplained health problems including ear pain, tinnitus, vertigo, and cognitive difficulties which reportedly began after they heard strange noises in their homes or hotel rooms. In response, the U.S. government dramatically reduced the number of diplomats posted at the U.S. embassy in Havana. U.S. officials initially believed a sonic attack might be responsible for their ailments. The sound linked to these attacks, which has been described as a high-pitched beam of sound, was recorded by U.S. personnel in Cuba and released by the Associated Press (AP). Because these recordings are the only available non-medical evidence of the sonic attacks, much attention has focused on identifying health problems and the origin of the acoustic signal. As shown here, the calling song of the Indies short-tailed cricket (Anurogryllus celerinictus) matches, in nuanced detail, the AP recording in duration, pulse repetition rate, power spectrum, pulse rate stability, and oscillations per pulse. The AP recording also exhibits frequency decay in individual pulses, a distinct acoustic signature of cricket sound production. While the temporal pulse structure in the recording is unlike any natural insect source, when the cricket call is played on a loudspeaker and recorded indoors, the interaction of reflected sound pulses yields a sound virtually indistinguishable from the AP sample. This provides strong evidence that an echoing cricket call, rather than a sonic attack or other technological device, is responsible for the sound in the released recording. Although the causes of the health problems reported by embassy personnel are beyond the scope of this paper, our findings highlight the need for more rigorous research into the source of these ailments, including the potential psychogenic effects, as well as possible physiological explanations unrelated to sonic attacks.
biorxiv biophysics 200-500-users 2019An RNA-binding region regulates CTCF clustering and chromatin looping, bioRxiv, 2018-12-13
Mammalian genomes are folded into Topologically Associating Domains (TADs), consisting of cell-type specific chromatin loops anchored by CTCF and cohesin. Since CTCF and cohesin are expressed ubiquitously, how cell-type specific CTCF-mediated loops are formed poses a paradox. Here we show RNase-sensitive CTCF self-association in vitro and that an RNA-binding region (RBR) mediates CTCF clustering in vivo. Intriguingly, deleting the RBR abolishes or impairs almost half of all chromatin loops in mouse embryonic stem cells. Disrupted loop formation correlates with abrogated clustering and diminished chromatin binding of the RBR mutant CTCF protein, which in turn results in a failure to halt cohesin-mediated extrusion. Thus, CTCF loops fall into at least 2 classes RBR-independent and RBR-dependent loops. We suggest that evidence for distinct classes of RBR-dependent loops may provide a mechanism for establishing cell-specific CTCF loops regulated by RNAs and other RBR partner.
biorxiv biophysics 200-500-users 2018Structure of a cytochrome-based bacterial nanowire, bioRxiv, 2018-12-11
AbstractElectrically conductive pili from Geobacter species, termed bacterial “nanowires”, are intensely studied for their biological significance and potential in the development of new materials. We have characterized a unique nanowire from conductive G. sulfurreducens pili preparations by cryo-electron microscopy composed solely of the c-type cytochrome OmcS. We present here, at 3.4 Å resolution, a novel structure of a cytochrome-based filament and discuss its possible role in long-range biological electron transport.Summary sentenceCryo-electron microscopy reveals the remarkable assembly of a c-type cytochrome into filaments comprising a heme-based bacterial nanowire.
biorxiv biophysics 0-100-users 2018Atomic Level Characterisation of Millisecond-Time Scale Protein Motions through a Combined Molecular Simulations and NMR Approach, bioRxiv, 2018-12-09
AbstractAdvances in biomolecular sciences are closely linked to our ability to chart the energy landscapes of biomolecules with atomic details. Here we validate a new paradigm to characterise thermodynamics and kinetics of millisecond timescale conformational transitions between ground state and transient excited states in the enzyme cyclophilin A (CypA). We describe a novel methodology that combines molecular dynamics simulations and Markov State modelling with NMR measurements to provide atomic-level insights into the nature of CypA transient conformational states. The computed conformational ensembles also enabled the predictive design and experimental validation of a single-site mutant that dramatically perturbs millisecond timescale loop motions, converting a CypA excited state into the ground state. The resulting models open up new horizons for targeting CypA with inhibitors and pave the way towards rational design of protein energy landscapes for protein engineering and drug discovery purposes.
biorxiv biophysics 0-100-users 2018