In mammals, HP1-mediated heterochromatin forms positionally and mechanically stable genomic domains even though the component HP1 paralogs, HP1α, HP1β, and HP1γ, display rapid on-off dynamics. Here, ...we investigate whether phase-separation by HP1 proteins can explain these biological observations. Using bulk and single-molecule methods, we show that, within phase-separated HP1α-DNA condensates, HP1α acts as a dynamic liquid, while compacted DNA molecules are constrained in local territories. These condensates are resistant to large forces yet can be readily dissolved by HP1β. Finally, we find that differences in each HP1 paralog's DNA compaction and phase-separation properties arise from their respective disordered regions. Our findings suggest a generalizable model for genome organization in which a pool of weakly bound proteins collectively capitalize on the polymer properties of DNA to produce self-organizing domains that are simultaneously resistant to large forces at the mesoscale and susceptible to competition at the molecular scale.
Within the mitotic spindle, kinesin motors cross-link and slide overlapping microtubules. Some of these motors exhibit off-axis power strokes, but their impact on motility and force generation in ...microtubule overlaps has not been investigated. Here, we develop and utilize a three-dimensional in vitro motility assay to explore kinesin-14, Ncd, driven sliding of cross-linked microtubules. We observe that free microtubules, sliding on suspended microtubules, not only rotate around their own axis but also move around the suspended microtubules with right-handed helical trajectories. Importantly, the associated torque is large enough to cause microtubule twisting and coiling. Further, our technique allows us to measure the in situ spatial extension of the motors between cross-linked microtubules to be about 20 nm. We argue that the capability of microtubule-crosslinking kinesins to cause helical motion of overlapping microtubules around each other allows for flexible filament organization, roadblock circumvention and torque generation in the mitotic spindle.
Single crystals of the charge-transfer salt picene/2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane have been grown using physical vapor transport. The crystal structure was determined using ...single-crystal X-ray diffraction. It was found that the crystals grow in a 1:1 molecular ratio and adopt a monoclinic structure with alternate stacking. Both X-ray data and Raman measurements show that the grown crystals are of good quality. From structure and infrared data, the charge transfer between acceptor and donor molecules was estimated to be approximately 0.14–0.19 electron. Transport measurements indicate a nonmetallic ground state with an activation energy of 0.6 eV. The supporting density functional theory calculations on molecular model systems as well as on crystalline structures confirm the amount of charge transfer and provide first insights into the electronic structure of the new material.
SignificanceBiomolecular condensates are intracellular organelles that are not bounded by membranes and often show liquid-like, dynamic material properties. They typically contain various types of ...proteins and nucleic acids. How the interaction of proteins and nucleic acids finally results in dynamic condensates is not fully understood. Here we use optical tweezers and fluorescence microscopy to study how the prototypical prion-like protein Fused-in-Sarcoma (FUS) condenses with individual molecules of single- and double-stranded DNA. We find that FUS adsorbs on DNA in a monolayer and hence generates an effectively sticky FUS-DNA polymer that collapses and finally forms a dynamic, reversible FUS-DNA co-condensate. We speculate that protein monolayer-based protein-nucleic acid co-condensation is a general mechanism for forming intracellular membraneless organelles.
Biomolecular condensates are dynamic intracellular structural units or distinct reaction spaces that can form by condensation of their constituents from the cytoplasm or the nucleoplasm. It is ...generally not clear yet, how dynamic, continuum-like condensate properties relevant for large-scale intracellular organisation emerge from the interplay of proteins and nucleic acids on the level of few individual molecules. With this work, we expand the portfolio of methods to investigate the role of protein-nucleic acid interactions in biomolecular condensates by introducing optical tweezers-based mechanical micromanipulation of single DNA molecules combined with confocal fluorescence microscopy to the field. We used this approach to characterise how the two landmark proteins1 Fused in Sarcoma and Heterochromatin Protein 1 form condensates with single DNA molecules. Fused in Sarcoma (FUS) is a key protein for various aspects of the nucleic acid metabolism and evidence is accumulating that biomolecular condensation is crucial for both, its physiological functions and its role in pathological aggregate formation. In this thesis, we directly visualised the formation of FUS condensates with single molecules of ssDNA and dsDNA. We showed that the formation of these microcondensates is based on nucleic acid scaffolding. We explored their mechanical properties and found that the mechanical tension that (FUS dsDNA) condensates can withstand or exert is in the range below 2 pN. We further demonstrated that already on this fundamental scale and with limited amounts of constituent molecules, dynamic properties like shape relaxations, reminiscent of viscoelastic materials, can emerge. Heterochromatin Protein 1 (HP1) is a prototypic chromatin organising factor that is in particular involved in the formation of dynamically compacted heterochromatin domains. HP1 forms biomolecular condensates and compacts DNA strands in vitro. In this work, we measured the influence of HP1 on the
mechanical properties of individual DNA molecules and demonstrated the response of HP1-DNA condensates to different environmental conditions. We contributed a methodological framework to characterise viscoelastic-like systems on the single molecule level.
Taken together, our optical tweezers-based approach revealed structural and mechanical properties of prototypic protein-DNA condensates and hence helped to elucidate mechanisms underlying their formation in unprecedented spatiotemporal and mechanical detail. We anticipate that this method can become a valuable tool to investigate how large-scale intracellular organisation based on protein-nucleic acid condensation emerges from interactions between individual building blocks.
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