Long-Lived Binding of Sox2 to DNA Predicts Cell Fate in the Four-Cell Mouse Embryo.

Transcription factor (TF) binding to DNA is fundamental for gene regulation. However, it remains unknown how the dynamics of TF-DNA interactions change during cell-fate determination in vivo. Here, we use photo-activatable FCS to quantify TF-DNA binding in single cells of developing mouse embryos. In blastocysts, the TFs Oct4 and Sox2, which control pluripotency, bind DNA more stably in pluripotent than in extraembryonic cells. By contrast, in the four-cell embryo, Sox2 engages in more long-lived interactions than does Oct4. Sox2 long-lived binding varies between blastomeres and is regulated by H3R26 methylation. Live-cell tracking demonstrates that those blastomeres with more long-lived binding contribute more pluripotent progeny, and reducing H3R26 methylation decreases long-lived binding, Sox2 target expression, and pluripotent cell numbers. Therefore, Sox2-DNA binding predicts mammalian cell fate as early as the four-cell stage. More generally, we reveal the dynamic repartitioning of TFs between DNA sites driven by physiological epigenetic changes. VIDEO ABSTRACT.

Diffusion of single dye molecules in hydrated TiO2 mesoporous films.

Mesoporous oxide films are attractive frameworks in technological areas such as catalysis, sensing, environmental protection, and photovoltaics. Herein, we used fluorescence correlation spectroscopy to explore how the pore dimensions of hydrated TiO2 mesoporous calcined films modulate the molecular diffusion. Rhodamine B molecules in mesoporous films follow a Fickian process 2-3 orders slower compared to the probe in water. The mobility increases with the pore and neck radii reaching an approximately constant value for a neck radius >2.8 nm. However, the pore size does not control the dye diffusion at low ionic strength emphasizing the relevance of the probe interactions with the pore walls on dye mobility. In conclusion, our results show that the thermal conditioning of TiO2 mesoporous films provides an exceptional tool for controlling the pore and neck radii on the nanometer scale and has a major impact on molecular diffusion within the mesoporous network.

Lateral motion and bending of microtubules studied with a new single-filament tracking routine in living cells.

The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with ∼9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of ∼20 µm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells.