Luciana Bruno

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.

Quantitative single particle tracking of NGF–receptor complexes: Transport is bidirectional but biased by longer retrograde run lengths

The retrograde transport of nerve growth factor (NGF) in neurite‐like processes of living differentiated PC12 cells was studied using streptavidin‐quantum dots (QDs) coupled to monobiotin‐NGF. These reagents were active in differentiation, binding, internalization, and transport. Ten‐35% of the QD–NGF–receptor complexes were mobile. Quantitative single particle tracking revealed a bidirectional step‐like motion, requiring intact microtubules, with a net retrograde velocity of 0.054 ± 0.020 μm/s. Individual runs had a mean velocity of ∼0.15 μm/s at room temperature, and the run times were exponentially distributed. The photostability and brightness of QDs permit extended real‐time analysis of individual QDbNGF– receptor complexes trafficking within neurites.

Mechanical Properties of Organelles Driven by Microtubule-Dependent Molecular Motors in Living Cells

The organization of the cytoplasm is regulated by molecular motors which transport organelles and other cargoes along cytoskeleton tracks. Melanophores have pigment organelles or melanosomes that move along microtubules toward their minus and plus end by the action of cytoplasmic dynein and kinesin-2, respectively. In this work, we used single particle tracking to characterize the mechanical properties of motor-driven organelles during transport along microtubules. We tracked organelles with high temporal and spatial resolutions and characterized their dynamics perpendicular to the cytoskeleton track. The quantitative analysis of these data showed that the dynamics is due to a spring-like interaction between melanosomes and microtubules in a viscoelastic microenvironment. A model based on a generalized Langevin equation explained these observations and predicted that the stiffness measured for the motor complex acting as a linker between organelles and microtubules is ∼ one order smaller than that determined for motor proteins in vitro. This result suggests that other biomolecules involved in the interaction between motors and organelles contribute to the mechanical properties of the motor complex. We hypothesise that the high flexibility observed for the motor linker may be required to improve the efficiency of the transport driven by multiple copies of motor molecules.

Fast axonal transport of the proteasome complex depends on membrane interaction and molecular motor function

ABSTRACT Protein degradation by the ubiquitin-proteasome system in neurons depends on the correct delivery of the proteasome complex. In neurodegenerative diseases, aggregation and accumulation of proteins in axons link transport defects with degradation impairments; however, the transport properties of proteasomes remain unknown. Here, using in vivo experiments, we reveal the fast anterograde transport of assembled and functional 26S proteasome complexes. A high-resolution tracking system to follow fluorescent proteasomes revealed three types of motion: actively driven proteasome axonal transport, diffusive behavior in a viscoelastic axonema and proteasome-confined motion. We show that active proteasome transport depends on motor function because knockdown of the KIF5B motor subunit resulted in impairment of the anterograde proteasome flux and the density of segmental velocities. Finally, we reveal that neuronal proteasomes interact with intracellular membranes and identify the coordinated transport of fluorescent proteasomes with synaptic precursor vesicles, Golgi-derived vesicles, lysosomes and mitochondria. Taken together, our results reveal fast axonal transport as a new mechanism of proteasome delivery that depends on membrane cargo ‘hitch-hiking’ and the function of molecular motors. We further hypothesize that defects in proteasome transport could promote abnormal protein clearance in neurodegenerative diseases.

Tracer dispersion in packings of porous activated carbon grains

Abstract A new method based on radioactive dispersion is reported which permits the accurate determination of the parameters which describe the transport and exchange mechanisms for packed beds of porous particles: characteristic exchange time between the internal and external pores (including adsorption effects), mean tracer velocity, mobile fluid fraction and longitudinal hydrodynamic dispersion coefficient. The method relies upon the fitting of the Coats–Smith model to radioactive tracer transit time distributions at regularly spaced intervals along a packed column for low Peclet numbers. Relationships between the transport and exchange mechanism parameters and relatively easily measured properties of the porous particles (size, porosity, intraparticle diffusion coefficient, adsorption isotherm) and packed bed (packing density) have been determined. These relationships may be used to estimate the hydrodynamic parameters. It is shown that the transport and exchange mechanism parameters determined at low Pe numbers using the new method can be used to accurately predict behavior of the packed bed at high Pe numbers provided transport within the porous particles is explicitly included within the model.

Anomalous Dynamics of Melanosomes Driven by Myosin-V in Xenopus laevis Melanophores

The organization of the cytoplasm is regulated by molecular motors, which transport organelles and other cargoes along cytoskeleton tracks. In this work, we use single particle tracking to study the in vivo regulation of the transport driven by myosin-V along actin filaments in Xenopus laevis melanophores. Melanophores have pigment organelles or melanosomes, which, in response to hormones, disperse in the cytoplasm or aggregate in the perinuclear region. We followed the motion of melanosomes in cells treated to depolymerize microtubules during aggregation and dispersion, focusing the analysis on the dynamics of these organelles in a time window not explored before to our knowledge. These data could not be explained by previous models that only consider active transport. We proposed a transport-diffusion model in which melanosomes may detach from actin tracks and reattach to nearby filaments to resume the active motion after a given time of diffusion. This model predicts that organelles spend approximately 70% and 10% of the total time in active transport during dispersion and aggregation, respectively. Our results suggest that the transport along actin filaments and the switching from actin to microtubule networks are regulated by changes in the diffusion time between periods of active motion driven by myosin-V.

Concentration dependence of diffusion–adsorption rate in activated carbon

A new method based on transfer rate measurements of a radioactive solute inside activated carbon grains is presented which allows to isolate the relative contributions of the chemical (reversible adsorption) and geometrical (tortuous di!usion paths) e!ects on the overall e!ective di!usion coe

Transport Properties of Melanosomes along Microtubules Interpreted by a Tug-of-War Model with Loose Mechanical Coupling

cient. Transfer rate measurements were performed with two di!erent initial conditions: carbon grains either initially saturated with a solution of the same concentration as the bath (self-di+usion) or free of solute. In the self-di!usion case the adsorption equilibrium for the solute is undisturbed during the process and the adsorption dynamics for tracer particles becomes linear. These processes can be analyzed in terms of an e!ective self-di!usion coe

Lateral Motion and Bending of Microtubules Studied with a New Single-Filament Tracking Routine in Living Cells

cient (D %&& ). The concentration dependence of the transfer rate is shown to be controlled by nonlinearities of the adsorption isotherm. We also show that the self-di!usion coe

Mapping the Dynamics of the Glucocorticoid Receptor within the Nuclear Landscape

cient (D s ) is one order of magnitude lower than in free bulk di!usion. Finally, in the second case (grains initially free of solute) we predict satisfactorily sorption rate curves, as well as the dependence of the transfer rate on the initial concentration, using results obtained in the self-di!usion case. ( 1999 Elsevier Science Ltd. All rights reserved.