The Ndc80 complex is a key site of regulated kinetochore-microtubule attachment, but the molecular mechanism underlying its function remains unknown. Here we present a subnanometer resolution cryo-electron microscopy reconstruction of the human Ndc80 complex bound to microtubules, sufficient for precise docking of crystal structures of the component proteins. We find that Ndc80 binds the microtubule with a tubulin monomer repeat, recognizing α- and β-tubulin at both intra- and inter-dimer interfaces in a manner that is sensitive to tubulin conformation. Furthermore, Ndc80 complexes self-associate along protofilaments via interactions mediated by the amino-terminal tail of the Ndc80 protein, the site of phospho-regulation by the Aurora B kinase. Ndc80's mode of interaction with the microtubule and its oligomerization suggest a mechanism by which Aurora B could regulate the stability of load-bearing Ndc80-microtubule attachments.

Summary The estrogen receptor (ER), glucocorticoid receptor (GR), and forkhead box protein 1 (FoxA1) are significant factors in breast cancer progression. FoxA1 has been implicated in establishing ER binding patterns though its unique ability to serve as a pioneer factor. However, the molecular interplay between ER, GR, and FoxA1 requires further investigation. Here we show that ER and GR both have the ability to alter the genomic distribution of the FoxA1 pioneer factor. Single-molecule tracking experiments in live cells reveal a highly dynamic interaction of FoxA1 with chromatin in vivo. Furthermore, the FoxA1 factor is not associated with detectable footprints at its binding sites throughout the genome. These findings support a model wherein interactions between transcription factors and pioneer factors are highly dynamic. Furthermore, at a subset of genomic sites, the role of pioneer can be reversed, with the steroid receptors serving to enhance binding of FoxA1.

Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single‐molecule imaging approaches, we directly correlated binding of the Gal4 transcription factor with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell time sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform called orbital tracking, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model in which multiple RNA polymerases initiate transcription during one burst as long as the transcription factor is bound to DNA, and bursts terminate upon transcription factor dissociation.

Population-based assays have been employed extensively to investigate the interactions of transcription factors (TFs) with chromatin and are often interpreted in terms of static and sequential binding. However, fluorescence microscopy techniques reveal a more dynamic binding behaviour of TFs in live cells. Here we analyse the strengths and limitations of in vivo single-molecule tracking and performed a comprehensive analysis on the intranuclear dwell times of four steroid receptors and a number of known cofactors. While the absolute residence times estimates can depend on imaging acquisition parameters due to sampling bias, our results indicate that only a small proportion of factors are specifically bound to chromatin at any given time. Interestingly, the glucocorticoid receptor and its cofactors affect each other’s dwell times in an asymmetric manner. Overall, our data indicate transient rather than stable TF-cofactors chromatin interactions at response elements at the single-molecule level.