Scott Forth

Nanofabricated quartz cylinders for angular trapping: DNA supercoiling torque detection.

We designed and created nanofabricated quartz cylinders well suited for torque application and detection in an angular optical trap. We made the cylinder axis perpendicular to the extraordinary axis of the quartz crystal and chemically functionalized only one end of each cylinder for attachment to a DNA molecule. We directly measured the torque on a single DNA molecule as it underwent a phase transition from B-form to supercoiled P-form.

Dynamic regulation of transcription factors by nucleosome remodeling

The chromatin landscape and promoter architecture are dominated by the interplay of nucleosome and transcription factor (TF) binding to crucial DNA sequence elements. However, it remains unclear whether nucleosomes mobilized by chromatin remodelers can influence TFs that are already present on the DNA template. In this study, we investigated the interplay between nucleosome remodeling, by either yeast ISW1a or SWI/SNF, and a bound TF. We found that a TF serves as a major barrier to ISW1a remodeling, and acts as a boundary for nucleosome repositioning. In contrast, SWI/SNF was able to slide a nucleosome past a TF, with concurrent eviction of the TF from the DNA, and the TF did not significantly impact the nucleosome positioning. Our results provide direct evidence for a novel mechanism for both nucleosome positioning regulation by bound TFs and TF regulation via dynamic repositioning of nucleosomes. DOI: http://dx.doi.org/10.7554/eLife.06249.001

Torque Measurement at the Single-Molecule Level

Methods for exerting and measuring forces on single molecules have revolutionized the study of the physics of biology. However, it is often the case that biological processes involve rotation or torque generation, and these parameters have been more difficult to access experimentally. Recent advances in the single-molecule field have led to the development of techniques that add the capability of torque measurement. By combining force, displacement, torque, and rotational data, a more comprehensive description of the mechanics of a biomolecule can be achieved. In this review, we highlight a number of biological processes for which torque plays a key mechanical role. We describe the various techniques that have been developed to directly probe the torque experienced by a single molecule, and detail a variety of measurements made to date using these new technologies. We conclude by discussing a number of open questions and propose systems of study that would be well suited for analysis with torsional measurement techniques.

Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends.

The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. Here, we purify and characterize tubulin heterodimers that have human β-tubulin isotype III (TUBB3), as well as heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments. Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.

Reconstitution of the augmin complex provides insights into its architecture and function

Proper microtubule nucleation during cell division requires augmin, a microtubule-associated hetero-octameric protein complex. In current models, augmin recruits γ-tubulin, through the carboxyl terminus of its hDgt6 subunit to nucleate microtubules within spindles. However, augmin’s biochemical complexity has restricted analysis of its structural organization and function. Here, we reconstitute human augmin and show that it is a Y-shaped complex that can adopt multiple conformations. Further, we find that a dimeric sub-complex retains in vitro microtubule-binding properties of octameric complexes, but not proper metaphase spindle localization. Addition of octameric augmin complexes to Xenopus egg extracts promotes microtubule aster formation, an activity enhanced by Ran–GTP. This activity requires microtubule binding, but not the characterized hDgt6 γ-tubulin-recruitment domain. Tetrameric sub-complexes induce asters, but activity and microtubule bundling within asters are reduced compared with octameric complexes. Together, our findings shed light on augmin’s structural organization and microtubule-binding properties, and define subunits required for its function in organizing microtubule-based structures.

Discontinuities at the DNA supercoiling transition.

While slowly turning the ends of a single molecule of DNA at constant applied force, a discontinuity was recently observed at the supercoiling transition when a small plectoneme is suddenly formed. This can be understood as an abrupt transition into a state in which stretched and plectonemic DNA coexist. We argue that there should be discontinuities in both the extension and the torque at the transition and provide experimental evidence for both. To predict the sizes of these discontinuities and how they change with the overall length of DNA, we organize a phenomenological theory for the coexisting plectonemic state in terms of four parameters. We also test supercoiling theories, including our own elastic rod simulation, finding discrepancies with experiment that can be understood in terms of the four coexisting state parameters.

The mechanics of microtubule networks in cell division

The primary goal of a dividing somatic cell is to accurately and equally segregate its genome into two new daughter cells. In eukaryotes, this process is performed by a self-organized structure called the mitotic spindle. It has long been appreciated that mechanical forces must be applied to chromosomes. At the same time, the network of microtubules in the spindle must be able to apply and sustain large forces to maintain spindle integrity. Here we consider recent efforts to measure forces generated within microtubule networks by ensembles of key proteins. New findings, such as length-dependent force generation, protein clustering by asymmetric friction, and entropic expansion forces will help advance models of force generation needed for spindle function and maintaining integrity.

Direct Measurements of Torque During Holliday Junction Migration

DNA experiences torsional stress resulting from the activities of motor enzymes and bound proteins. The mechanisms by which this torsional stress is dissipated to maintain DNA structural integrity are not fully known. Here, we show that a Holliday junction can limit torsion by coupling rotation to translocation and torque to force. The torque required to mechanically migrate through individual junctions was found to be an order of magnitude smaller than that required to melt DNA. We also directly show that substantially more torque was required to migrate through even a single-base sequence heterology, which has important implications for the activity of junction-migrating enzymes.

Asymmetric Friction of Non-Motor Maps can lead to their Directional Motion in Active Microtubule Networks

Cells utilize dynamic biopolymer networks to carry out mechanical tasks during diverse processes. These cellular processes require microtubules to be organized into distinct structures, such as asters or bundles. Within these dynamic motifs, microtubule-associated proteins (MAPs) are frequently under load, but how force modulates these proteins’ function is poorly understood. Here, we combine optical-trapping with TIRF-based microscopy to measure the force-dependence of microtubule interaction for three non-motor MAPs (NuMA, PRC1, and EB1) required for cell division. We find that frictional forces increase non-linearly with the velocity of MAP motion across microtubules and depend on filament polarity, with NuMAs friction being lower when moving towards minus-ends, EB1s lower towards plus-ends, and PRC1 exhibiting no directional preference. Mathematical models predict, and experiments confirm, that MAPs with asymmetric friction can move directionally within actively fluctuating microtubule pairs that they crosslink. Our findings reveal how non-motor MAPs can generate frictional resistance in dynamic cytoskeletal networks via micromechanical adaptations whose anisotropy may be optimized for MAP localization and function within cellular structures.

Dynamic Regulation of Transcription Factors by Nucleosome Remodeling

In eukaryotic cells, transcription activation and repression are regulated by transcription factors (TFs) and nucleosomes. Although it has been demonstrated that a TF and a nucleosome can directly compete for binding to the same region of DNA, it is unclear whether and how such a competition may continue after a TF has been bound and a nucleosome has been assembled. In this study, we have addressed an aspect of this question by investigating whether a nucleosome under remodeling can displace a bound TF. We determined the locations of both a nucleosome and a TF on DNA by mechanically unzipping the DNA molecule. We found that under the action of ISWI remodeling, a nucleosome was not repositioned past a TF. In contrast, under the action of SWI/SNF remodeling, a nucleosome was repositioned past a TF with concurrent eviction of the TF from the DNA. Our results demonstrate a novel mechanism for how TFs may be regulated via dynamic repositioning of nucleosomes.